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Notes: Objective To investigate lifetime prevalence of infertility, the seeking of infertility treatment and outcomes of treatment.Design Cross-sectional postal questionnaire study.Setting County of Copenhagen, Denmark.Subjects Three thousand, seven hundred and forty-three women, 15 to 44 years old, selected at random were asked about infertility, their seeking of infertility treatment, diagnoses provided by their doctors and subsequent parenthood. Response rate was 78%, n= 2865. A random sample of non-responders was interviewed by telephone.Main outcome measures Fertility status, seeking of infertility treatment, subsequent deliveries and adoptions.Results Of the women who had attempted to have a child, 26.2% had experienced infertility; 4.1% of the women aged 25 to 44 years were currently primarily infertile and 8.6% had involuntarily not delivered a first child; 47.4% of the infertile women had sought infertility treatment. Significant predictors for seeking infertility treatment were school education 〉 9 years and not having delivered a child. Of the infertile women 54.9% subsequently had a child. Only 30% of these reported that the successful delivery was treatment-related.Conclusions The health care system should see to it that infertile couples from lower social classes are offered information on the possibility of infertility treatment. High quality infertility treatment has to include both the “supply” of taking care of the infertile couple's psychosocial strain and the goal of ensuring successful pregnancies.

Notes: Objective To ascertain whether there is variation in obstetric practice within a defined subgroup of primiparous women.Design Analysis of routinely collected data.Subjects Ten thousand two hundred and ninety-five primiparous deliveries defined as ‘normal’ in Scotland during 1990 and 44,820 primiparous deliveries defined as ‘normal’ in England between April 1990 and April 1991.Results Little variation was found in the distribution of mothers' ages and gestational ages at delivery, and babies' birthweights. In both England and Scotland there was considerable variation between regions in instrumental delivery rates and caesarean section rates. There were many deficiencies in the quality of the data provided by the English Maternity Hospital Episode System.Conclusions There is regional variation in instrumental delivery rates and caesarean section rates in England and Scotland. The poor quality of data for England makes interpretation of the cause of variation difficult because the extent to which variation may reflect deficiencies in the data, rather than differences in practice, is unknown. Improvements need to be made in Maternity Hospital Episode system data, increasing both coverage and data quality. Nevertheless, similar variations in instrumental delivery and caesarean section rates may be associated with differences in population characteristics not measured in these data sets of differences in obstetric practice.

Notes: Objective To examine the effect of topical oestradiol on skin collagen and elastin.Subjects Twelve postmenopausal women, aged 52 to 76 years.Interventions Topical oestradiol treatment on the skin of lower abdomen and the vehicle only on the contralateral site; once a day for three months.Main outcome measures The content of skin hydroxyproline; the levels of the carboxyterminal propeptide of human type I procollagen (PICP) and of the aminoterminal propetide of human type III procollagen (PIIINP).The number and the quality of collagen and elastic microfibrils.Results The amount of hydroxyproline in the skin significantly (P= 0.012) increased from 11.8 to 16.3 pgμ(38 %) during oestradiol treatment. After treatment, the PICP level in the blister fluid was significantly (P= 0.024) higher on the treated site than on the control site. Also the level of PIIINP increased, but the change was not statistically significant. Electron microscopy showed morphologic improvement of elastic and collagen fibres, while the number of oxytalan and elaunin fibres was unchanged in light microscopy.Conclusions Topical oestradiol treatment increases the amount of skin collagen. The increase in the level of PICP and PIIINP in skin blister fluid indicates that oestradiol treatment stimulates collagen synthesis. Furthermore, our results show that topical ostradiol treatment has a greater influence on the amount than on the quality of skin collagen. On the contrary, in elastic tissue the oestradiol treatment will only result in morphologic improvement.

Notes: Objective To evaluate pre-operatively the sonographic morphology and colour Doppler findings of borderline ovarian tumours and to compare these findings to those of benign and malignant tumours.Methods Pre-operative transvaginal and colour Doppler ultrasound examinations were performed on 150 women with adnexal tumours. Pulsatility index, resistance index, peak systolic velocity, site, number and confluence of vessels were recorded.Results Fifty-six women had malignant ovarian tumours, 74 had benign and 20 had borderline tumours. No biological, morphological or demographic parameters were specifically predictive of borderline tumours. Intratumoral vessels with a pulsatility index of below 1.0 were observed in 19 of the 20 borderline tumours; a morphological score suggested malignancy in 15 women whereas the CA125 exceeded 30 u/ml in 10 cases. Confluence of blood vessels was observed only in three cases. A model including intracystic complexity (either vegetations or septa), pulsatility index of less than 1.0, absence of confluence of vessels, CA125 of less than 150 u/L, in a woman under 60 years of age allowed borderline tumours to be detected with 85% sensitivity, 92 % specificity and 91 % accuracy.Conclusion Borderline tumours have haemodynamics resembling those of malignant tumours but the distribution of vessels is often similar to that observed in benign tumours; this observation should be considered when proposing multiparameter scoring systems including colour Doppler ultrasound to identify malignancies of the ovary. Colour Doppler findings may be of assistance in the follow up of women after conservative surgery for ovarian malignancies.

Notes: Objective To describe the changing clinical and immunological characteristics and timing of diagnosis of HIV-infected pregnant women enrolled in the European Collaborative Study.Design A prospective study of the mothers of children enrolled in the European Collaborative Study on children born to HIV-infected women.Setting Twenty-one European centres in seven countries.Subjects One thousand six hundred and ninety HIV-infected women and their 1754 deliveries.Results The proportion of women in whom HIV infection had been diagnosed before pregnancy increased significantly over time, from 7% in 1984–1985 to 65% in 1994 (P 〈 0.001). The prevalence of breastfeeding, which was related to the timing of diagnosis, significantly declined over the study period. The mean CD4 count was 510 cells/mm3, and there was a significant decline in average CD4 count over the study period. Black women had a significantly lower CD4 count than white women. From survival analysis it is estimated that five years after delivery 14% of women will have died and 24% will have developed CDC stage IV disease.Conclusions Timing of diagnosis is of critical importance if mother-to-child transmission is to be reduced through avoidance of breastfeeding and zidovudine therapy and effective antenatal screening policies have become increasingly important. The rate of progression of maternal disease highlights the implications of HIV infection for their children, both infected and uninfected.

Notes: Objective To assess longitudinally fetal cerebral vasodilatation in small-for-gestational age fetuses to investigate whether intrauterine death might be predictable.Design Prospective observational study.Setting Ultrasound department in a university hospital.Subjects Five pregnancies with ultrasonographically confirmed small fetuses (abdominal circumference less than the 3rd centile) monitored longitudinally until time of intrauterine death.Main outcome measure Time between last ultrasound examination and diagnosis of intrauterine death, and variation in middle cerebral artery pulsitility index prior to death.Results Two of the five fetuses showed a reversal of adaptation (as indicated by an elevation of the middle cerebral artery pulsitility index) within 48 hours of intrauterine death. The other three had their final ultrasound examination 3 to 7 days before death and showed no such reversal of adaptation.Conclusion Reversal of adaptation in fetal hypoxaemia as indicated by a rise in the middle cerebral artery pulsitility index may be a predictor of intrauterine death within 48 hours. Whether delivery after reversal of adaptation would result in salvage of neurologically intact babies needs to be investigated.

Notes: Objective To determine fetal scalp blood lactate with a new test strip method in parturients with normal and abnormal cardiotocograms during labour and to describe the relation to maternal lactate, fetal scalp blood pH, cord artery lactate and acid-base balance.Setting Labour wards at the University Hospitals of Huddinge and Lund and at the County Hospital of Östersund, Sweden.Materials and method Fetal scalp blood was sampled for lactate (n= 269) and pH (n= 285) determination in 177 parturients with abnormal intrapartum CTG. Lactate and pH were also analysed in a group of 64 women with normal pregnancies and with a reactive fetal heart rate tracing prior to sampling of fetal scalp blood. At fetal blood sampling lactate was also determined in maternal capillary blood, while at birth lactate and acid-base balance in cord artery blood was performed in almost all cases.Main outcome measurements Medians and percentiles (lactate and acid-base balance). Correlation between fetal scalp blood lactate (dependent) and scalp blood pH, cord artery blood lactate and acid-base parameters and labour time prior to fetal blood sampling.Results In the group with abnormal cardiotocograms, fetal scalp and umbilical artery blood lactate and acid-base parameters differed significantly from the same parameters in the normal group. The fetal-maternal lactate gradient changed from negative in the normal group to positive in the fetal distress group. Multiple regression analysis, with scalp lactate as the dependent parameter, revealed a significant correlation with fetal scalp blood pH (P 〈 0.001) and umbilical artery lactate (P 〈 0.01).Conclusions Intrapartum scalp blood lactate was significantly correlated with pH and cord artery lactate. The results indicate that increased lactate levels in fetal blood sampling describes fetal lactacidosis. The new disposable test strip requiring only 5 μl of blood for lactate determination may be better than traditional methods for monitoring fetal wellbeing in labour.

Notes: Objective To determine the causes and management of chronic vulval symptoms and to compare the findings in patients first presenting to a gynaecologist with those in patients first presenting to a dermatologist.Design A prospective study of 144 patients, approximately half each being referred to a gynaecologist and a dermatologist. Diagnosis was based on clinical history, vulvoscopy, vulval biopsy and bacteriology. Biopsies were examined by a histopathologist experienced in dermatopathology and gynaecological pathology.Results The two patient groups were similar in both range and frequency of conditions. The commonest cause of chronic vulval symptoms was dermatitis, which was found in 64% of our patients. Dermatitis occurred alone in 55% and was found in association with histological evidence of human papilloma virus (HPV) in a further 9%. These patients responded to simple dermatological methods, mainly topical corticosteroids. Histopathological evidence of HPV was encountered in only 23% of our patients, and of these 36% also demonstrated dermatitis on biopsy. Most responded to topical corticosteroids. Another 7% had lichen sclerosus, and all responded to potent topical corticosteroid. The remaining 15% demonstrated a range of diagnoses, including psoriasis, dysaesthetic vulvodynia, vulval intraepithelial neoplasia (VIN) and chronic candidiasis. The majority of patients had a corticosteroid responsive dermatosis rather than a gynaecological condition.Conclusions The majority of patients with a chronically symptomatic vulva who present to either a gynaecologist or a dermatologist have a dermatological condition that responds to simple dermatological treatments. We believe that the presence or absence of the human papilloma virus is not relevant to most patients with a chronically symptomatic vulva and treatments should not be aimed at eradicating this virus. Histopathologists and gynaecologists who have focused on gynaecological disorders have often missed simple dermatological conditions that are easily treatable.

Notes: Objective The purpose of this study was to show the benefits and limitations of vulvar biopsy in the setting of a multidisciplinary clinic specialising in non-neoplastic diseases of the vagina and vulva.Design One hundred and fourteen vulvar biopsies were reviewed and classified according to the classification of the International Society for the Study of Vulvar Diseases.Results The histological diagnoses were lichen sclerosus 25 YO, nonlichen simplex chronicus 35%, erosive inflammatory dermatoses comprising psoriasis, spongiotic dermatitis, dermatophytosis and psoriasiform dermatitis 13%, erosive vulvitis and lichen planus 9%, nonspecific inflammation 6%, miscellaneous 9% and normal 4%.Conclusions Biopsies in cases of lichen sclerosus were useful for confirmation of clinical diagnosis and to exclude early invasive malignancy. In lichen simplex chronicus, biopsies helped exclude an underlying dermatosis requiring specific treatment. Psoriasis, spongiotic dermatitis, dermatophytosis and excoriated lichen simplex chronicus posed a common clinical differential diagnosis of the reddened vulva. The eroded vulva often proved a diagnostic problem clinically and histologically. The clinical syndrome of vestibulitis did not have a specific histological picture, and biopsies showed nonspecific inflammation, mild hyperplasia or were normal. No case of squamous cell hyperplasia was diagnosed and the place of this diagnosis in the ISSVD classification needs review.

Notes: Objective To outline the problems associated with female genital mutilation and to highlight the need for deinfibulation before delivery.Design A review of women attending a newly established African Well Woman Clinic. Age at infibulation, gravidity of clinic attenders and adequacy of introitus for management of labour were assessed.Setting Northwick Park Hospital, Harrow, Middlesex.Subjects Fifty women attending a newly established African Well Woman Clinic, of whom 13 were nulliparous, 14 were primigravid and 23 were multigravid.Results The average age at which infibulation had occurred was 6–7 years. At the time of clinic attendance the mean age of pregnant and nonpregnant patients was 26 and 23.3 years, respectively. Of the 14 primigravid patients, only 50% had an adequate introitus to allow management of the first and second stages of labour. Five had deinfibulation performed antenatally or at delivery. Ninety-three percent of the primigravid patients and 74% of the multigravid patients had a vaginal delivery.Conclusions We believe that the Northwick Park Hospital management policy for intibulated women closely mirrors the cultural practices in Somalia. The policy also improves obstetric management of infibulated patients. Twenty-six percent of referrals were of nonpregnant women, and this practice is to be encouraged.

Notes: Objective To determine clinical practice amongst bstetricians in the UK in the antepartum and intrapartum management of pregnant women with recurrent genital herpes infection.Methods All Members and Fellows of the Royal College of Obstetricians and Gynaecologists resident in the UK were sent a questionnaire requesting information concerning their management of pregnant women with recurrent genital herpes infection.Results There was a 76% response rate to the questionnaire. Of the 1201 obstetricians who responded, only 369 (31%) admitted to having a formal policy governing the management of herpes in pregnancy within their unit. However, regular screening was advocated by 718 (60%), of whom 463 (64%) performed regular antenatal swabs for viral culture. At the time of presentation in labour 974 obstetricians (81)routinely examined the genitals for evidence of a recurrence.When asked in what circumstances caesarean section would be considered an appropriate method of delivery in women with genital herpes infection, 1107 (92%) felt that visible active lesions at the time of labour was sufficient. However, when the membranes had been ruptured for more than four hours in the presence of genital lesions, only 678 (56%) considered this an indication for caesarean section. Caesarean section was more likely to be considered appropriate in this situation by obstetricians who performed antenatal screening (χ2= 30.38, P 〈 0.0001). Five hundred and ninety-six obstetricians (50%) felt that a positive viral culture obtained at antenatal screening from the most recent occasion prior to presentation in labour was an indication for caesarean section, although of this group 192 (32%) said they did not perform antenatal screening by viral culture.The reporting of a recurrence by the patient without visible evidence of disease was considered an appropriate indication for caesarean section by 438 respondents (36%). Maternal request for caesarean section regardless of recurrences at delivery was onsidered an acceptable indication for operative delivery by 745 obstetricians (62%).Conclusions 1. There seems to be little agreement amongst obstetricians in the UK regarding the management of recurrent genital herpes infection in pregnancy. 2. The management possibilities are reviewed and suggestions are made for a more cohesive approach to the problem.

Notes: Objective To evaluate the role of transabdominal cervicoisthmic cerclage (TCC) in the management of recurrent mid-trimester pregnancy miscarriage and preterm delivery.Design An observational study of 50 women with history of recurrent second trimester miscarriage and preterm delivery in whom TCC was performed.Setting A tertiary referral centre for high risk obstetrics.Results TCC was performed on 51 occasions in 50 women. One had TCC performed in two successive pregnancies. Prior to the procedure, they had experienced 36 first trimester miscarriages and 163 later pregnancy losses. These included 152 midtrimester miscarriages and 11 neonatal deaths after preterm delivery. There had been 4 survivors after preterm delivery. The total number of pregnancies after TCC was 61 with three women losing one pregnancy and subsequently having a successful pregnancy. Of these women 44 now have had a successful outcome. Six have not had a successful outcome. Eight women have had two successful consecutive pregnancies after TCC. The fetal survival was 85.2% post-procedure.Conclusions TCC is a procedure that may be used in a highly selected group of women with anatomical defects of the cervix and previous failed transvaginal cerclage resulting in recurrent second trimester pregnancy loss and preterm birth. Strict selection criteria were applied as TCC is an invasive procedure. Although TCC is not widely used our study suggests it may be the procedure to consider in women who have had recurrent second trimester pregnancy losses and preterm births associated with cervical damage.

Notes: Objective To determine whether the anatomy of an obstetric brachial plexus lesion (OBPL) is causally related to the preceding obstetric history.Design Anatomical classification of the OBPL during reconstructive neurosurgical treatment in consecutive infants who had surgery for OBPL between 1986 and 1994 and relating these findings with the characteristics of the preceding birth.Setting De Wever Hospital, Heerlen, The Netherlands.Subjects All infants who had surgical treatment for OBPL between 1 April 1986 and 1 January 1994 (n= 130).Results An Erb's C546 injury was preceded more frequently by a difficult breech birth (19/26 cases or 73 YO). In contrast, the more extensive forms of Erb's palsy classified as a C547 injury or a total palsy with a C5–Tl injury were observed more frequently after complicated cephalic birth (52/59 or 88%, and 43/45 or 96%, respectively). The extent of anatomical damage as expressed by the incidence of an avulsion of one or more spinal nerves was 18/26 (69 %) in Erb C5–C6, 13/59 (22%) in Erb C547 and 21/45 (47%) in total C5–T1 palsy.Conclusion The Erb's CS-C6 palsy, occasionally bilateral and/or complicated by phrenic nerve injury, was the most frequent form of OBPL after a breech birth. The more extensive form of Erb's palsy and the total palsy were observed more frequently after delivery in a cephalic presentation. The pure form of Erb's palsy and the total palsy were characterised by a higher incidence of nerve avulsions than the extensive form of Erb's palsy.

Notes: Objective To examine the management of cord prolapse and its morbidity and mortality.Design Retrospective study of consecutive babies born after cord prolapse, identified using the Oxford Obstetric Data System, and those with registered handicap, identified by the Oxford Region Register of Early Childhood Impairments.Setting District maternity hospital managing more than 6000 deliveries annually.Subjects One hundred and thirty–two babies born after the identification of cord prolapse in the John Radcliffe Hospital between January 1984 and December 1992.Main outcome measures Survival rates, condition at birth assessed by Apgar scores at 1 and 5 minutes and blood gas values on cord blood samples, and incidence of major handicap at three years of age.Results The incidence of cord prolapse was 1 in 426 total births. There were six stillbirths and six neonatal deaths. One baby died as a result of birth asphyxia. The uncorrected perinatal mortality rate was 91 per 1000. Of 120 survivors, only one baby was known to suffer a major neurological handicap. Electronic cardiotocographs aided the diagnosis of cord prolapse in 41 % of cases. Apgar scores were better with a shorter diagnosis to delivery interval, but cord gas results did not correlate well with Apgar scores or the diagnosis to delivery interval.Conclusions Cord prolapse occurs with a relatively stable incidence in this population irrespective of changes in obstetric practices. Despite the high incidence of ominous cardiotocographs, low Apgar scores and acidaemia on blood gas analysis, the fetal outcome is not as poor as might be expected and mortality is predominantly attributable to congenital anomalies and prematurity rather than birth asphyxia.

Notes: Objective To examine prospectively the effects of maternal betamethasone administration on fetal heart rate variation, body, breathing and eye movements and the rest-activity cycle.Design Thirty-one women on 38 occasions were at risk of premature delivery and received two doses of betamethasone 24 h apart. Gestational age ranged between 26 and 32 weeks. Fetal heart rate was monitored on each of five successive days (0–4) and fetal body, breathing and eye movements were recorded on days 0, 2 and 4.Results Compared with the control day before steroid administration (day 0), both long term and short term fetal heart rate variation were reduced on days 2 and 3 (P 〈 0.01). In one-third of the cases, fetal heart rate variation fell transiently below the lower normal range for gestational age. Body movements were reduced on day 2 by 50% (P 0.01) due to prolonged periods of inactivity (P 〈 0.01). Breathing movements were largely absent on day 2 (P 〈 0.01), but the occurrence of eye movements remained unchanged after betamethasone administration. All values returned to baseline on day 4, indicating that no fetal deterioration had occurred during the course of the study period. Similar responses to betamethasone were observed in five fetuses when studied at re-presentation two weeks later.Conclusions Maternal betamethasone administration causes a considerable but transient reduction in fetal body movements and activity periods, breathing and heart rate variation, without affecting fetal eye movements. Knowledge of this phenomenon is important when assessing the fetal condition. The effect may be due to a glucocorticoid receptor mediated process in the fetal brain.

Notes: Objective To introduce a simple, well-standardised vaginal ultrasound technique and to compare the position and mobility of the bladder neck in continent and stress incontinent women using this technique.Design A single-centre prospective case-control study.Setting Ikazia Hospital, Rotterdam, The Netherlands.Subjects One hundred and sixty women; sixty randomly chosen women referred to our outpatient department who volunteered for the study to develop a standardised technique, fifty stress-incontinent women and fifty controls who volunteered for the study for comparison using the standardised technique.Main outcome measures Standardisation with regard to bladder volume, horizontal axis and Valsalva force. The position of the bladder neck at rest, during straining and during squeezing.Results The probe we use does not alter bladder neck mobility. A standardised bladder volume of 250 ml was used rather than maximum bladder capacity. A Foley catheter introduced into the bladder, with the balloon half-filled with soapy water and half with air gives an easily recognisable fluid level, which is parallel to the horizontal axis of the patient. A standardised Valsalva force of 30 cm H2O can exclude differences in bladder neck mobility due to spontaneous and uncontrolled abdominal force. Measurements by two independently working investigators showed good conformity. The position of the bladder neck in the stress incontinent women was significantly lower and significantly more posterior at rest, during straining and during squeezing. At the same time in stress incontinent women there was significantly more descent during straining and less elevation during squeezing. However, there was a considerable overlap between the two groups for all parameters.Conclusions This standardised vaginal ultrasound technique is a feasible, acceptable and reproducible technique for the study of female bladder neck mobility. The position and mobility of the bladder neck is significantly different in stress incontinent women as compared to continent controls. The great overlap between the two groups still limits the clinical relevance.

Notes: Objective To study the current in-hospital, 30–day and 42–day mortality after conventional gynaecologic procedures in Finland, with special reference to hysterectomy.Design Nationwide six-year annual study.Setting Data were from the Finnish Population Register Centre, the Finnish Cause-of-Death and Hospital Discharge Register, and the Register for Legal Abortions and Sterilisations.Subjects Gynaecologic operations (n= 299257) performed between January 1986 and December 1991.Main outcome measures The overall and age-adjusted mortality rates during the initial hospitalisation, as well as 30 and 42 days after the operations. Age-adjusted probability of dying withn 42 days after hysterectomy compared with the overall probability of age-matched Finnish female control population.Results Overall mortality rates per 10000 hysterectomies increased gradually from 6.0 during initial hospitalisation to 9.1 and 12.9 when calculated 30 and 42 days post-operatively. The overall 42–day mortality rates of radical hysterectomy, curettage and laparoscopy (other than sterilisation) exceeded the post-hysterectomy mortality rate, while the rates after caesarean section, legal abortion and laparoscopic sterilisation did not. No deaths occurred after laparoscopic sterilisation (n= 40346). The patients who died after radical hysterectomy, curettage and for other laparoscopy than sterilisation were old, and the great majority of them died of cancer.Conclusions The mortality rates after gynaecologic procedures in Finland are currently very low and have clearly decreased in recent decades. Patients may be reassured that conventional gynaecologic operations are safe.

Notes: Objective To evaluate the policy of an annual smear to screen renal transplant recipients for cervical intraepithelial neoplasia and invasive carcinoma and to determine the incidence of abnormal smears and CIN before and after the introduction of cyclosporine (1983).Design A retrospective study over the period 1971 to 1992.Subjects Postmenarchial women who received renal transplants and who were on immuno-suppressive treatment for at least one month.Mean outcome measures Cytology and histology results.Results A total of 144 women who received renal transplantation were eligible for our study. Observation time varied from 1 to 227 months (median 59 months) with a mean for the group transplanted before 1983 (Group A) of 103 months, and for the group transplanted after 1983 (Group B) of 46 months. Of these women, 25 had an abnormal smear. Of these, 14 were confirmed by histology and repeated smears of the other 11 patients were negative. Within the 60 women in Group A with an abnormal smear, six had CIN I or CIN II, three had CIN III and one showed adenocarcinoma of the endometrium. Among the 84 women in Group B, four had CIN I or CIN II and none had CIN III. The overall incidence of abnormal cytology was 17.3%, with no invasive cervical carcinoma in this group.Conclusions Our policy of screening is adequate. With the introduction of cyclosporine the incidence of abnormal cytology and histology has a tendency to decrease. However, the duration of risk is not comparable yet.

Notes: Abstract: A partial cDNA encoding most of the third intracellular loop of the mouse α1d-adrenergic receptor subtype was amplified from hippocampus by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligodeoxynucleotide primers. This DNA fragment was used as a probe to isolate an α1d-adrenergic receptor cDNA from a mouse brain cDNA library. The deduced amino acid sequence encodes a potential protein of 562 amino acids, and northern hybridization of poly(A)+ RNA isolated from mouse brain detected a single 3.0-kb transcript. Partial cDNA fragments of the α1b- and α1a-adrenergic receptor subtypes were also amplified from mouse brain and sequenced. Analysis of the mRNA expression by RT-PCR indicated that the α1-adrenergic receptors are widely distributed in mouse tissues. The α1d subtype is expressed in brain areas such as hippocampus, striatum, and brainstem and also in many extracerebral tissues, such as lung, liver, heart, kidney, and spleen. The α1a subtype is also expressed in many tissues, whereas the α1b subtype has a more restricted expression, with high levels in striatum, brainstem, and diencephalus.

Notes: Abstract: Among various tissues of mouse, β1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.

Notes: Abstract: To elucidate the significance of branched-chain amino acids (BCAAs) for brain energy metabolism, the capacity to use BCAAs for oxidative metabolism was investigated in astroglia-rich primary cultures derived from newborn rat brain. The cells selectively removed BCAAs from the culture medium, the disappearance following first-order kinetics. The BCAAs disappeared rapidly in spite of the presence of sufficient glucose as substrate for the generation of energy. Taking into consideration that the ketogenic amino acid leucine could be degraded only to acetyl-CoA and acetoacetate, and with the knowledge that astroglial cells have the capacity to secrete ketone bodies, this amino acid was chosen for further metabolic studies. After incubation of the cells with leucine, acetoacetate, d-β-hydroxybutyrate, and α-ketoisocaproate were found to have accumulated in the culture medium. Identification of the radioactive metabolites generated from [4,5-3H]leucine established that the source of the substances released was indeed leucine. These results indicate that, at least in culture, astroglial cells degrade leucine via the known metabolite α-ketoisocaproate, to acetoacetate, which can be further reduced to d-β-hydroxybutyrate. It is hypothesized that upon release from brain astrocytes, the ketone bodies could serve as fuel molecules for neighboring cells such as neurons and oligodendrocytes. In view of these and other results, astrocytes may be considered the brain's fuel processing plants.

Notes: Abstract: VGF is a neuroendocrine-specific gene product that is up-regulated by nerve growth factor in the PC12 cell line. In rat neuroendocrine tissues two polypeptides of 90 and 80 kDa were detected by an antiserum to an N-terminal domain of VGF (from residues 4 to 240). In parallel, an antiserum directed against the C-terminal nonapeptide of VGF (from residues 609 to 617) revealed several additional posttranslational products. Peptides of apparent molecular sizes of 20, 18, and 10 kDa were prominent in nerve tissues and the hypophysis but absent in the adrenal medulla, and their relative abundance varied in distinct regions of the CNS. In PC12 cells VGF was proteolytically processed only after nerve growth factor treatment, and primary cultures of rat cerebellar granule cells accumulated the low-molecular-weight forms of VGF during in vitro maturation. In these cells the specific cleavages of VGF occurred in a postendoplasmic reticulum compartment; the processed forms were enriched in the secretory vesicles and were preferentially secreted upon cell membrane depolarization. Distinct differential distribution in the CNS and in vitro release of such posttranslational products indicate that these species may represent biologically relevant forms of VGF that play a role in neuronal communication.

Notes: Abstract: The excitatory neurotransmitter glutamate is believed to play important roles in development, synaptic plasticity, and neurodegenerative conditions. Recent studies have shown that neurotrophic factors can modulate neuronal excitability and survival and neurite outgrowth responses to glutamate, but the mechanisms are unknown. The present study tested the hypothesis that neurotrophic factors modulate responses to glutamate by affecting the expression of specific glutamate-receptor proteins. Exposure of cultured embryonic rat hippocampal cells to basic fibroblast growth factor (bFGF) resulted in a concentration-dependent increase in levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor subunit GluR1 protein as determined by western blot, dot-blot, and immunocytochemical analyses. In contrast, bFGF did not alter levels of GluP2/3, GluR4, or the NMDA-receptor subunit NR1. Nerve growth factor did not affect GluR1 levels. Calcium-imaging studies revealed that elevation of [Ca2+]i, resulting from selective AMPA-receptor activation, was enhanced in bFGF-pretreated neurons. On the other hand, [Ca2+]i responses to NMDA-receptor activation were suppressed in bFGF-treated neurons, consistent with previous studies showing that bFGF can protect neurons against NMDA toxicity. Moreover, neurons pretreated with bFGF were relatively resistant to the toxicities of glutamate and AMPA, both of which were shown to be mediated by NMDA receptors. These data suggest that differential regulation of the expression of specific glutamate-receptor subunits may be an important mechanism whereby neurotrophic factors modulate activity-dependent neuronal plasticity and vulnerability to excitotoxicity.

Notes: Abstract: In an attempt to elucidate the role of docosahexaenoic acid (DHA; 22:6n-3) in the developing brain, a method was devised whereby rapid enrichment of fetal brain and liver lipid with DHA was achieved. Fetal rats at 17 days of gestation were injected intraamniotically with ethyl docosahexaenoate (EtDHA). Control fetuses were administered ethyl oleate (EtOle). Brain lipid DHA content increased by almost 21% (p = 0.02) 3 days after EtDHA administration as compared with EtOle-injected fetuses, whereas liver lipid DHA content increased by almost 60% (p = 0.0002). At this time brain phosphatidylinositol content doubled, whereas phosphatidylserine (PS) content increased by 〉50% (p = 0.03). Increases in liver PS (+25.8%; p = 0.015) and sphingomyelin (+43.6%; p = 0.01) content were observed. A redistribution of total brain phospholipid (PL) DHA was observed following Et-DHA administration, resulting in a 56.4% increase in PS-DHA abundance (p 〈 0.05) and an 8.8% decrease in phosphatidylethanolamine-DHA abundance (p = 0.05). These results suggest modulation of fetal brain and liver PL and provide a method for enrichment of DHA content in discrete PLs during intrauterine life.

Notes: Abstract: β-Amyloid cores contain considerable amounts of d-Ser and d-Asp residues in Alzheimer's disease. We investigated the cytotoxic effects of various synthetic β-amyloids, including d-Ser-substituted derivatives, on primary cultured neurons and nonneuronal HeLa cells. β25–35, its d-Ser26-substituted derivative, and β1–40 in 10–100 nM specifically suppressed mitochondrial succinate dehydrogenase activity [MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction] in HeLa cells, which are dependent on ATP production mainly from glycolysis, but did not exert detectable cytotoxicity, assessed by dye exclusion test, NADH levels, and uptake of [3H]Leu and [3H]Tdr. The β-amyloids, on the other hand, did exert neurodegenerative effects on rat hippocampal cultured neurons in which ATP is mostly synthesized by the mitochondrion. The activities of β25–35 and [d-Ser26]β25–35 are dependent on their having β-structures and not random forms. Although β25–35 was degraded rapidly by proteinase(s) in brain extract or leucine aminopeptidase, [d-Ser26]β25–35 is fairly resistant. These results indicate that one of the primary targets of β-amyloids is suppression of mitochondrial succinate dehydrogenase, and the vulnerability of the brain to β-amyloids can be explained by its large dependence on mitochondrial energy production. Moreover, racemization of serine residues of β-amyloids may be involved in neurodegeneration and formation of senile plaques through escaping from the degradation process by brain proteinases.

Notes: Abstract: The incorporation of tritium label into quinolinic acid (QUIN), kynurenic acid (KYNA), and other kynurenine (KYN) pathway metabolites was studied in normal and QUIN-lesioned rat striata after a focal injection of [5-3H]KYN in vivo. The time course of metabolite accumulation was examined 15 min to 4 h after injection of [5-3H]KYN, and the concentration dependence of KYN metabolism was studied in rats killed 2 h after injection of 1.5–1,500 µM [5-3H]KYN. Labeled QUIN, KYNA, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and xanthurenic acid (XA) were recovered from the striatum in every experiment. Following injection of 15 µM [5-3H]KYN, a lesion-induced increase in KYN metabolism was noted. Thus, the proportional recoveries of [3H]KYNA (5.0 vs. 1.8%), [3H]3-HK (20.9 vs. 4.5%), [3H]XA (1.5 vs. 0.4%), and [3H]QUIN (3.6 vs. 0.6%) were markedly elevated in the lesioned striatum. Increases in KYN metabolism in lesioned tissue were evident at all time points and KYN concentrations used. Lesion-induced increases of the activities of kynurenine-3-hydroxylase (3.6-fold), kynureninase (7.6-fold), kynurenine aminotransferase (1.8-fold), and 3-hydroxyanthranilic acid oxygenase (4.2-fold) likely contributed to the enhanced flux through the pathway in the lesioned striatum. These data provide evidence for the existence of a functional KYN pathway in the normal rat brain and for a substantial increase in flux after neuronal ablation. This method should be of value for in vivo studies of cerebral KYN pathway function and dysfunction.

Notes: Abstract: HPLC and gas chromatography-mass spectrometry analyses of 18 amino acids, N-acetylaspartate, N-acetylaspartyglutamate, and 5-hydroxyindoleacetic acid, derived from serotonin, and homovanillic acid, derived from dopamine, were performed in CSF collected from a group of patients with schizophrenia who either had been drug free for at least 1 year (n = 5) or were drug naive for psychotropic drugs (n = 21) and in 15 control subjects. Significant differences were found only for taurine (15% lower in the patients) and isoleucine (7% higher). A number of unidentified substances were detected, one of which proved to be markedly reduced (16%) among the schizophrenic patients. Liquid chromatography-mass spectrometry with continuous flow-fast atom bombardment interface allowed us to identify this substance as γ-glutamyglutamine. The decreased level of γ-glutamylglutamine may reflect a deficiency in the γ-glutamyltransferase system, a system probably involved in glutamate uptake, or a deficiency in glutamine, an important precursor of releasable glutamate. Although glutamate was nonsignificantly reduced in the patients, it was one of the five substances (including γ-glutamylglutamine) that were necessary for the best discrimination between the schizophrenic patients and the controls. These findings support the notion that the glutamatergic system is affected in schizophrenic disorders. In addition, they underscore the need to apply rigid bioanalytical techniques and use drug-naive patients to gain in-depth information on the pathophysiology of brain disorders such as schizophrenia.

Notes: Abstract: The precursor for rat vasoactive intestinal polypeptide (preproVIP) is processed by proteolytic cleavage into a signal peptide and five further functional domains: preproVIP 22–79, peptide histidine isoleucine (PHI), preproVIP 111–122, VIP, and preproVIP 156–170. To investigate the biosynthetic processing of preproVIP in peripheral parasympathetic neurons, the sphenopalatine ganglion and one of its projection areas, the nasal mucosa, were used. By immunohistochemistry it was shown that in the sphenopalatine ganglion, preproVIP-derived peptides are localized mainly in neuronal cell bodies, whereas in the nasal mucosa immunoreactivity was found only in nerve fibers and terminals. The peptides were quantified and characterized by radioimmunoassay, HPLC, and gel chromatography using antisera specific for the different precursor products. In the rat sphenopalatine ganglion, the different peptides were found in approximately equimolar amounts, with the exception of PHI and its C-terminally extended variant, PHV, which were present at considerably lower concentrations. However, in the nasal mucosa there was a preferential accumulation of VIP to at least three times the concentration of any of the other peptides. Our results suggest that all preproVIP-derived peptides are present and processed in the sphenopalatine ganglion but that there is a selective accumulation of VIP in the nerve terminals. This indicates that VIP is physiologically the most important transmitter among the preproVIP-derived peptides in parasympathetic nerves originating in the sphenopalatine ganglion.

Notes: Abstract: We have used purified microglial cultures obtained from neonatal rat cerebral cortex to investigate the ability of microglia to release prostanoids after exposure to bacterial lipopolysaccharide, a classic macrophage activator. Release of prostaglandin E2, prostaglandin D2, and thromboxane A2 was low in basal conditions and increased in a dose- and time-dependent way upon lipopolysaccharide treatment (1–100 ng/ml), by a mechanism requiring de novo protein synthesis. When compared with astrocytes, microglial cells appeared to respond more effectively to lipopolysaccharide, being able to release prostanoids after exposure to a 100-fold lower concentration of lipopolysaccharide. In addition to prostanoids, we also measured the release of leukotriene B4; although lipopolysaccharide failed to stimulate leukotriene B4 release by microglial cells, it doubled the basal production in astrocytes. Lipopolysaccharide enhanced the release of preloaded [3H]arachidonic acid from microglial membrane phospholipids by a mechanism inhibited by the protein synthesis inhibitor cycloheximide, which suggests that the increased availability of arachidonic acid contributed to the enhanced prostanoid production. Lipopolysaccharide, however, also stimulated prostanoid synthesis by inducing cyclooxygenase activity, as shown by determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin and by assessing the level of the inducible form of cyclooxygenase by western blot analysis. Among the mechanisms potentially involved in the regulation of microglial prostanoid production, we studied the effect of β-adrenergic receptor activation. The β-agonist isoproterenol was inactive by itself but doubled the effect of lipopolysaccharide. The drug appeared to act mainly through the inducible cyclooxygenase; because it did not stimulate arachidonic acid release, it enhanced the lipopolysaccharide-evoked prostanoid production observed after aspirin pretreatment and induced de novo synthesis of cyclooxygenase detectable by western blot analysis. We suggest that during cerebral inflammatory processes microglia can contribute to the establishment of high prostanoid levels, which can be further elevated by β-adrenergic activation.

Notes: Abstract: Excessive free radical formation or antioxidant enzyme deficiency can result in oxidative stress, a mechanism proposed in the toxicity of MPTP and in the etiology of Parkinson's disease (PD). However, it is unclear if altered antioxidant enzyme activity is sufficient to increase lipid peroxidation in PD. We therefore investigated if MPTP can alter the activity of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) and the level of lipid peroxidation. l-Deprenyl, prior to MPTP administration, is used to inhibit MPP+ formation and its subsequent effect on antioxidant enzymes. MPTP induced a threefold increase in SOD activity in the striatum of C57BL/6 mice. No parallel increase in GSH-PX or CAT activities was observed, while striatal lipid peroxidation decreased. At the level of the substantia nigra (SN), even though increases in CAT activity and reduction in SOD and GSH-PX activities were detected, lipid peroxidation was not altered. Interestingly, l-deprenyl induced similar changes in antioxidant enzymes and lipid peroxidation levels, as did MPTP. Taken together, these results suggest that an alteration in SOD activity, without compensatory increases in CAT or GSH-PX activities, is not sufficient to induce lipid peroxidation.

Notes: Abstract: Possible roles of prostaglandins (PGs) in interleukin-1 (IL-1)-induced activation of noradrenergic neurons were examined by assessing norepinephrine (NE) turnover in the brain and peripheral organs of rats. An intraperitoneal injection of human recombinant IL-1β accelerated NE turnover in the hypothalamus, spleen, lung, diaphragm, and pancreas. A similar increase in NE turnover was also observed after intracerebroventricular injection of corticotropin-releasing hormone (CRH). Pretreatment with indomethacin (cyclooxygenase inhibitor) abolished the IL-1-induced, but not the CRH-induced, increase in hypothalamic and splenic NE turnover. To elucidate which eicosanoid-cyclooxygenase product(s) is responsible for accelerating NE turnover, PGD2, PGE2, PGF2α, U-46619 (stable thromboxane A2 analogue), or carbacyclin (stable prostacyclin analogue) was administered intracerebroventricularly. Among them, PGE2 was the only eicosanoid effective in increasing NE turnover in spleen, whereas PGD2 was effective in the hypothalamus. The stimulative effect of PGD2 was abolished by pretreatment with intracerebroventricular injection of a CRH antiserum. These results suggest that the action of IL-1 is mediated through PGD2 production to activate the noradrenergic neurons in the hypothalamus, and through PGE2 production to increase sympathetic nerve activity in spleen.

Notes: Abstract: The metabolism of Met-enkephalin and cholecystokinin (CCK) 8-(sulfated) by intact microslices was studied in rat brain regions. Incubation of brain slices with Met-enkephalin (400 µM) resulted in a linear rate of disappearance of parent peptide and appearance of metabolic fragments whose rate of accumulation was specific to brain region. The degradative rate (pmol/min/mg of protein) of Met-enkephalin was high in caudate-putamen (5,160 ± 120) and lower in nucleus accumbens (3,630 ± 110) and frontal cortex (3,180 ± 120). Inhibition of aminopeptidases decreased Met-enkephalin degradation (50–97% vs. control) in frontal cortex but was less effective in caudate-putamen (20–34%). Tyr-Gly-Gly and Phe-Met were recovered in caudate-putamen and nucleus accumbens, whereas negligible quantities of these fragments were recovered from frontal cortex. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11, decreased Met-enkephalin degradation in caudate-putamen (14%) but had no effect on that in frontal cortex. A cocktail of bestatin or leuhistin (inhibitors of aminopeptidases), phosphoramidon, and captopril (an inhibitor of angiotensin converting enzyme) protected Met-enkephalin from degradation (recovery 〉95%) in caudate-putamen. CCK 8-(sulfated) degradation on slices from caudate-putamen, nucleus accumbens, and frontal cortex was not altered by inhibitors of neutral endopeptidase 24.11, metalloendopeptidase 24.15, angiotensin converting enzyme, or thiol proteases. Inhibitors of either aminopeptidases or serine proteases produced small reductions (13–30%) in CCK degradation in each region. These data provide evidence for regional and structural specificity in terminating the actions of neuropeptides.

Notes: Abstract: Animals trained in a passive avoidance task exhibit a transient time-dependent increase in hippocampal neural cell adhesion molecule (NCAM) polysialylation at 12–24 h following the initial learning trial. Using immunocytochemical techniques with a monoclonal antibody that specifically recognises NCAM-polysialic acid homopolymers, a distinct population of granule-like cells, at the border of the granule cell layer and the hilus in the dentate gyrus of the adult rat hippocampus, has been demonstrated to exhibit time-dependent change in frequency at 10–12 h following the initial learning of a one-trial, step-through, passive avoidance response. These changes were paradigm specific as they failed to occur in those animals rendered amnesic with scopolamine. These polysialylated dentate neurons are not de novo granule cell precursors as administration of 5-bromo-2′-deoxyuridine every 2 h from the point of learning to the 12-h posttraining time showed no significant difference between trained and passive animals in the small number of heterogeneously distributed, labelled cells. These findings directly identify a morphological substrate of memory, implied by previous correlative and interventive studies on NCAM function.

Notes: Abstract: During transient cerebral ischemia, there is a temporary and robust accumulation of extracellular GABA in the hippocampus. We examined whether the acute exposure of GABAA/benzodiazepine receptors to high concentrations of GABA early after ischemia results in receptor down-regulation as observed in vitro. Gerbils were killed 30 and 60 min following a 5-min bilateral carotid occlusion, and their brains were prepared for receptor autoradiography. The hydrophilic GABAA receptor antagonist [3H]SR-95531 and the hydrophobic benzodiazepine agonist [3H]flunitrazepam were used to distinguish between cell surface and internalized receptors. Ischemia significantly decreased [3H]SR-95531 binding in hippocampal areas CA1 and CA3 and in the dentate gyrus 30 min after ischemia. Scatchard analysis in area CA1 revealed that ischemia decreased the Bmax as low as 44%. The affinity of the remaining sites was increased substantially (72% decrease in KD). As expected, there were no changes in the binding of [3H]flunitrazepam to hippocampus in the early postischemic period because the benzodiazepine could bind to both internalized receptors and those on the cell surface. We hypothesize that prolonged exposure (∼30–45 min) of GABAA receptors to high concentrations of synaptic GABA in vivo causes receptor down-regulation, perhaps via receptor internalization.

Notes: Abstract: We studied effects of Ca2+ in the incubation medium on [3H]dopamine ([3H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca2+ (0–30 min) and Ca2+ concentration (0–10 mM) in Krebs-Ringer medium affected [3H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 mM Ca2+ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in Vmax of the [3H]DA uptake process. On the other hand, [3H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 mM Ca2+. The Ca2+-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca2+-free medium following preincubation with 1 mM Ca2+. Protein kinase C inhibitors did not affect apparently Ca2+-dependent enhancement of the uptake, whereas 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62; a Ca2+/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca2+-dependent enhancement of [3H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.

Notes: Abstract: The δ subunit of F1F0 ATPase (ATP synthase complex) is part of the stalk connecting the F1 and F0 moieties. Studies in Escherichia coli suggest that the analogous bacterial subunit, called ε, is essential for the ATPase assembly energy coupling. Platelet-derived growth factor (PDGF) is an important growth factor for various cell types, including neurons of the CNS. Using two-dimensional gel electrophoresis, microsequencing, western blot analysis, and immunoprecipitation techniques, we have found that PDGF induces phosphorylation of the δ subunit or a closely related peptide in cultured mouse cortical neurons.

Notes: Abstract: Metallothionein (MT) protein and mRNA levels were monitored following exposure of rat neonatal primary astrocyte cultures to methylmercury (MeHg). MT-I and MT-II mRNAs were probed on northern blots with an [α-32P]dCTP-labeled synthetic cDNA probe specific for rat MT mRNA. MT-I and MT-II mRNAs were detected in untreated cells, suggesting constitutive MT expression in these cells. The probes hybridize to a single mRNA with a size appropriate for MT, ∼550 and 350 bp for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 × 10−6M MeHg for 6 h was increased over MT-I and MT-II mRNA levels in controls. Western blot analysis revealed a time-dependent increase in MT protein synthesis through 96 h of exposure to MeHg. Consistent with the constitutive expression of MTs at both the mRNA level and the protein level, we have also demonstrated a time-dependent increase in MT immunoreactivity in astrocytes exposed to MeHg. The cytotoxic effects of MeHg were measured by the rate of astrocytic d-[3H]aspartate uptake. Preexposure of astrocytes to CdCl2, a potent inducer of MTs, completely reversed the inhibitory effect of MeHg on d-[3H]aspartate uptake that occurs in MeHg-treated astrocytes with constitutive MT levels. Associated with CdCl2 treatment was a time-dependent increase in astrocytic MT levels. In summary, astrocytes constitutively express MTs; treatment with MeHg increases astrocytic MT expression, and increased MT levels (by means of CdCl2 pretreatment) attenuate MeHg-induced toxicity. Increased MT expression may represent a generalized response to heavy metal exposure, thus protecting astrocytes and perhaps also, indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.

Notes: Abstract: TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Here, we report that both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-γ (PLC-γ) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-γ, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival-promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c-ras in a concentration-dependent manner. These results suggest that two different pathways, the c-ras and the PLC-γ pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.

Notes: Abstract: The basal and K+-induced release of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were measured in microdialysate samples obtained in vivo from the nucleus accumbens region of rats subchronically exposed to 50 ppm lead for 90 days. The basal and stimulus-induced release of dopamine and the metabolites were significantly reduced in the lead-exposed rats as compared with the controls. These reductions in dopamine and its metabolites are consistent with the reports of decreased dopamine availability associated with lead-induced changes in certain behavioral indices (fixed-interval performance) in rats. Furthermore, these changes were observed at blood lead levels similar to those considered to cause impairment in cognitive functions in children.

Notes: Abstract: Glycated residues of τ protein from paired helical filaments isolated from the brains of Alzheimer's disease patients were localized by doing a proteolytic cleavage of the protein, fractionation of the resulting peptides, and identification of those peptides using specific antibodies. The most suitable residues for glycation, lysines, present at the tubulin-binding motif of τ protein, seem to be preferentially modified compared with those lysines present at other regions. Among these modified lysines, those located in the sequence comprising residues 318–336 (in the largest human τ isoform) were found to be glycated, as determined by the reaction with an antibody that recognizes a glycated peptide containing this sequence. Because those lysines are present in a tubulin binding motif of τ protein, its modification could result in a decrease in the interaction of τ with tubulin.

Notes: Abstract: To elucidate whether the high sensitivity of gerbil compared with rat hippocampus to metabolic stress results from tissue-specific or hemodynamic factors, ischemia-induced metabolic disturbances [energy metabolism and protein synthesis rate (PSR)] were studied using the in vitro model of the hippocampal slice preparation. At the end of in vitro ischemia, ATP content was measured in individual slices with HPLC. In other groups of slices, PSR was measured after 120 min of recovery after in vitro ischemia. ATP breakdown was almost identical in rat and gerbil slices at all temperatures (37°C, 34°C, or 31°C) and periods of ischemia (5, 10, or 15 min) studied. In contrast to the identical rate of ATP depletion during ischemia, however, postischemic disturbances in PSR were significantly increased in gerbil slices compared with rat slices and this relationship was stable after different periods of ischemia and at different incubation temperatures. The results illustrate that the pattern of ischemia-induced disturbances observed in vivo can also be reproduced using the in vitro model of hippocampal slice preparation, as evidenced by the postischemic disturbance in PSR. It is concluded that comparison of the extent of metabolic disturbances in gerbil and rat hippocampal slices after transient in vitro ischemia may help to elucidate the mechanisms of ischemic cell damage.

Notes: Abstract: VAMP/synaptobrevin (SYB), an integral membrane protein of small synaptic vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G and is thought to play an important role in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca2+/calmodulin-dependent protein kinase II (CaMKII) associated with synaptic vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with synaptic vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaMKII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane.

Notes: Abstract: Tissue transglutaminase (EC 2.3.2.13) is a calcium-activated enzyme that cross-links specific substrate proteins into insoluble, protease-resistant, high molecular weight complexes. Because the neurofibrillary tangles in Alzheimer disease have similar biochemical characteristics, and because the microtubule-associated protein τ is the predominant component of these structures, the substrate properties of τ with respect to transglutaminase were investigated. Bovine τ and recombinant human τ isoforms rapidly form high molecular weight, cross-linked polymers on incubation with transglutaminase. Polyamine incorporation assays indicate that bovine τ is an excellent substrate of transglutaminase, with a Km of 10.4 ± 2.2 µM and a Vmax of 40.9 ± 4.5 nmol/mg of enzyme/min. Individual recombinant human τ isoforms are not equivalent with respect to transglutaminase, as the smallest isoform T3 (352 amino acids) is not as good a substrate as the larger isoforms T4 (383 amino acids) and T4L (441 amino acids). To determine which segments of the τ protein are susceptible to modification by transglutaminase, τ was labeled with [3H]putrescine by transglutaminase and proteolyzed with α-chymotrypsin, and the breakdown products were analyzed. These experiments demonstrate that the enzyme modifies τ at only one or a few discrete sites, primarily in the carboxyl half of the molecule. Thus, the reaction is specific for only a small number of the many glutamine residues in τ. Furthermore, a τ deletion construct (T264) containing a portion of the microtubule-binding domains, which is a substrate of transglutaminase, cannot be cross-linked by the enzyme. This provides evidence that the cross-linking reaction is specific, and requires that the substrates be appropriately associated for cross-linking to occur.

Notes: Abstract: The effect of energy failure on Cl−-dependent l-glutamate (l-Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose-deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl−-dependent l-[3H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased l-[3H]Glu uptake was inhibited by l-Glu, dl-2-amino-4-phosphonobutyrate, l-homocysteic acid, l-cystine, 4,4′-diisothiocyano-2,2′-disulfonic stilbene, and removal of Cl−. Uptakes of Na+-dependent l-[3H]-Glu, [3H]GABA, and [3H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the Vmax value without affecting the Km value. The increased l-[3H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose-containing solution. ATP content in brain slices decreased to 〈10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose-containing solution. Treatment with veratrine, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and NaCN in normoxic conditions increased l-[3H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl−-dependent l-Glu transport activity in synaptic membranes increases in ischemia- or hypoxia-induced brain energy failures.

Notes: Abstract: The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent Mr 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of Mr 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro “ischemia” was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N2 instead of O2 and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca2+ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca2+ was omitted from the incubation media. Reoxygenation of ischemic slices also resulted in the Ca2+-dependent, transient tyrosine phosphorylation of p42. The major PSD-associated, tyrosine-phosphorylated glycoprotein of molecular mass 180 kDa has recently been identified as the NR2B subunit of the NMDA receptor. The results suggest that changes in tyrosine phosphorylation after an ischemic insult may modulate the NMDA receptor or signal transduction pathways in the postsynaptic cell and are consistent with a role for tyrosine phosphorylation in the sequence of events leading to neuronal cell damage and death.

Notes: Abstract: Brefeldin A (BFA) has been used extensively to study the intracellular transport and processing of proteins and sphingolipids because of its dramatic alteration of the structural and functional organization of the Golgi. We have examined the effect of BFA on the synthesis of galactosylceramide sulfate (SGalCer) and its immediate precursor galactosylceramide (GalCer) in an immortalized Schwann cell line (S16) to determine the intracellular sites of synthesis of these two related glycolipids. During a 6-h labeling period, a dose-dependent inhibition of [35S]sulfate incorporation into SGalCer was observed with 95% inhibition occurring at 0.5 µg/ml BFA. Labeling of newly synthesized galactosphingolipids with [3H]-palmitic acid for 6 h in the presence of BFA resulted in increased incorporation of label into GalCer containing nonhydroxy fatty acids (NFA-GalCer) to 162% of control values, whereas labeling of GalCer containing 2-hydroxy fatty acids (HFA-GalCer) was reduced to 63% of control. After 24 h, these values were at 366 and 91%, respectively. These results indicate that at least some of the HFA-GalCer was initially synthesized at a location distal to the BFA block and separate from the site of NFA-GalCer synthesis. Examination of [3H]palmitic acid incorporation into free ceramides showed an increase of 133 and 161% for hydroxy and nonhydroxy fatty acid ceramides, respectively, in cells treated for 6 h with BFA in comparison with levels found in untreated control cells, indicating that BFA did not block fatty acid 2-hydroxylation or the formation of HFA ceramide. Incorporation of [3H]palmitic acid into glucosylceramide and GM3 was increased over control levels whereas labeling of GM2 was inhibited, consistent with what has been reported previously for the effect of BFA on these glycolipids in other cell types. These results suggest that there are at least two separate intracellular sites for the galactosylation of HFA and NFA ceramide, respectively, which can be distinguished by their sensitivity to BFA. Our results also indicate that the site of GalCer sulfation is not redistributed to the endoplasmic reticulum in the presence of BFA and therefore may be localized to the distal Golgi or trans-Golgi network.

Notes: Abstract: The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 µM fumonisin B1, a fungal inhibitor of mammalian ceramide synthase, inhibited incorporation of [3H]galactose/glucosamine and [14C]serine into complex sphingolipids of cultured cerebellar neurons. Under this condition, the expression of Purkinje cell-enriched sphingolipids, including GD1α, 9-O-acetylated LD1 and GD3, and sphingomyelin, was significantly decreased. After 2 weeks' exposure to fumonisin B1, dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag, but their branches began to degenerate. In some cells, formation of elongated dendrite trees was severely impaired. However, treatment with fumonisin B1 also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells, morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B1. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed, these effects of fumonisin B1 were reversed, but not completely, by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]caproyl]sphingosine (C6-NBD-ceramide), a synthetic derivative of ceramide. Thus, we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells.

Notes: Abstract: P0, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys153. To gain insights into the mechanism of P0 acylation, the in vitro palmitoylation of both P0 and a synthetic Cys153-containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P0 with [3H]palmitoyl-CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P0 was not affected by boiling the isolated P0 for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonezymatic nature of the reaction. After chemical deacylation, P0 was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [14C]palmitoyl-CoA, the synthetic peptide encompassing the natural P0 acylation site (I150RYCWLRR157) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37°C, peptide palmitoylation followed a second-order reaction (k2 = 246 ± 6 M−1 min−1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl-CoA. The activation energy calculated from the Arrhenius plot is ∼2 kcal/mol and much lower than that of enzyme-catalyzed transacylations. Finally, two other P0 peptides (V121PTRYG126 and K109TSQVTL115) as well as various unrelated thiol-containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.