Library

Notes: Summary Calcium-dependent exocytosis in ‘leaky’ bovine adrenal medullary cells has a requirement for Mg-ATP. One possibility is that exocytosis depends in some way on the operation of the ATP-dependent proton pump that serves to maintain the core of the secretory vesicles both acid and at a positive potential with respect to the cytosol. This possibility has been tested in ‘leaky’ cells by monitoring exocytosis under conditions where the secretory vesicle pH and potential gradients are measuredin situ. The results show rather clearly that exocytosis can persist, with unchanged Ca-activation kinetics, in the virtual absence both of a difference in pH between the cytosol and secretory vesicle core and also of a difference in potential across the vesicle membrane. The results do not, however, exclude a small modulating effect of vesicle pH or potential on exocytosis and shed no light on whether or not the plasma membrane potential, which is maintained close to zero in these experiments, influences exocytosis.

Notes: Summary The calcium sensitivity of exocytosis from electroper-meabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electropermeabilized cells are exposed to Ca2+ levels much in excess of 50 μm. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca2+ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.

Notes: Summary By subjecting isolated adrenal medullary cells to intense electric fields of brief duration it is possible to gain access to the cell interior without impairing the ability of the cell to undergo exocytosis. After a single exposure to a field of 2 kV/cm, τ=200 μsec, adrenal medullary cells behave as if their plasma membrane contains two pores of effective radius 2 nm. At 37°C these ‘equivalent pores’ remain patent for up to 1 hr. The formation and stability of these ‘pores’ is not affected by the Ca content of the bathing solution. The ‘pores’ permit externally applied catecholamine and Ca-EGTA to equilibrate rapidly with the cell water. Cells rendered ‘leaky’ in K glutamate medium containing 5mm Mg-ATP and EGTA to give an ionized Ca close to 10−8 m release less than 1% of their total catecholamine. These same cells can release up to 30% of their catecholamine when exposed to 10−5 m Ca. This Ca-dependent release is unaffected by Ca-channel blockers such as D600. Catecholamine release in response to a calcium challenge only seems to occur during the first few minutes whilst the Ca concentration is changing, and the extent of release depends on the final Ca concentration achieved. Half-maximal release occurs at about 1 μm Ca, and this value is independent of the EGTA concentration used to buffer the ionized Ca. The relation between ionized Ca and catecholamine release is best fitted by a requirement for 2 Ca ions. Calcium-evoked release of catecholamine is associated with the release of dopamine-β-hydroxylase (DβH) but not lactate dehydrogenase. The ratio DβH/catecholamine released is the same as that in stimulated intact cells and perfused glands. The time course of appearance in the external medium of DβH and catecholamine is identical. Transmission electron microscopy of ‘leaky’ cells exposed to 10−8 m Ca reveals no marked differences from unstimulated intact cells. The cytoplasm of ‘leaky’ cells exposed to 10−5 m Ca contains large membrane-bounded vacuoles. When secretion is caused to take place in the presence of horseradish peroxidase, this marker is found within the vacuoles. Ca-dependent release of both catecholamine and DβH requires Mg-ATP. Cells equilibrated with Ca in the absence of Mg-ATP can be triggered to undergo exocytosis by the addition of Mg-ATP. In the absence of Mg, ATP alone is ineffective. Of a variety of other nucleotides tested, none is as effective as ATP. Mg-ATP affects the extent of exocytosis and not its apparent affinity for Ca. Replacement of glutamate as the major anion by chloride results in a marked reduction in Ca-dependent release of both catecholamine and DβH. Chloride causes a small increase in Ca-independent release of catecholamine, a large reduction in the extent of exocytosis, and a decrease in the apparent affinity of exocytosis for Ca. Of a variety of anions examined, their order of effectiveness at supporting Ca-dependent exocytosis is glutamate−〉acetate−〉Cl−〉Br−〉SCN−. Exocytosis is not obviously affected by replacing K by Na or sucrose or by altering the pH over the range pH 6.6 to 7.8. Raising the free Mg concentration reduces the extent of Ca-dependent exocytosis and also its apparent affinity for calcium. Calcium-dependent exocytosis in ‘leaky’ cells is largely unaffected by (i) a variety of agonists and antagonists of the nicotinic receptor; (ii) agents that disrupt microtubules and microfilaments; (iii) phalloidin; (iv) vanadate; (v) inhibitors of anion permeability; (vi) protease inhibitors; and (vii) agents that dissipate the vesicle pH gradient and potential. It is partially inhibited by (i) certain antipsychotic drugs; (ii) a rise in osmotic pressure, (iii) lowering the temperature below 20°C, and (iv) N-ethyl maleimide.

Notes: Abstract Cytokines and their related enzyme pathways may play a part in the development of insulin-dependent diabetes mellitus (IDDM). We have therefore studied the activity of the enzyme 2′–5′ oligoadenylate synthetase (which is induced by both interferon and the tumour necrosis factors) in circulating mononuclear cells from 40 subjects with IDDM and 32 healthy control subjects. There was no difference in mean basal enzyme activity between the two groups. A polymorphism of the 2′–5′ oligoadenylate synthetase gene, not previously described, was found using the restriction enzymeBam HI. There was no association of 2′–5′ oligoadenylate synthetase genotypes with IDDM, but there was a significant correlation between basal 2′–5′ oligoadenylate synthetase activity and 2′–5′ oligoadenylate synthetase genotypes. Significantly higher mean basal levels of 2′–5′ oligoadenylate synthetase activity were associated with HLA-DQA 4.6 phenotype (determined using the restriction enzymeTaq 1 and a DQA probe) and HLA-DR3 (determined serologically), whereas significantly lower mean levels of enzyme activity were associated with HLA-DQA 5.5 and HLA-DR7, in both IDDM and control subjects. An analysis of variance confirmed that these associations were independent 2′–5′ oligoadenylate synthetase genotype. Likewise, a significantly higher mean level of enzyme activity was associated with the heterozygous 1/3 insulin-related genotype in the IDDM subjects only. This study therefore suggests that the possession of certainHLA haplotypes might be associated with differing levels of basal 2′–5′ oligoadenylate synthetase activity.

Notes: Abstract Heavy rainfall and dense vegetation on tropical volcanoes produce abundant carbonized wood in pyroclastic deposits, in addition to easy contamination of this wood by root systems and soluble humic material. Because the physical nature of the charcoal varies, some samples are more prone to contamination. Two independent studies of the same volcano, Mt Liamuiga on St Kitts in the Lesser Antilles, sometimes using samples from the same carbonized tree, yielded a systematic difference in radiocarbon ages. An exchange of samples and a re-investigation of three physically distinct types of charcoal yielded the following results. Rare, hard, dense charcoal, lacking contamination, which had yielded a spurious age of 2860 years bp, was redated at 1845±58 years bp. Common soft, friable charcoal with good cellular structure proved to be susceptible to contamination. A field decontamination technique utilized by one group seems significant as it yields older ages than when only routine laboratory pre-treatment was used, indicating that the latter technique only partly removes the dried and hard residue produced by the decomposition of modern plant rootlets. A previous date of 24870 years bp obtained from powdery charcoal in a horizon beneath the Mansion ‘Series’ contradicted ages older than 41000 years bp from common friable charcoal in the lower Mansion ‘Series’. The soft powdery charcoal was re-investigated using a sample collected a few centimeters from the original, although field decontamination of this sample was not possible, more extensive laboratory treatment yielded an age of ca. 43000 years bp, again proving that routine laboratory pretreatments are inadequate. A revised geochronology for the Mansion ‘Series’ is described and a cautionary discussion is presented for the benefit of investigators using radiocarbon ages to date volcanic deposits.

Notes: Effects of folate deficiency on the rate of apoptosis in human cytotrophoblastic cells has been investigated. Apoptosis was determined in cytotrophoblastic cells after culture in 1. control medium, 2. folate-free medium and 3. folate-free medium plus 10% fetal calf serum. Apoptosis rates in cells cultured in mediums 2 and 3 were significantly higher than those cultured in the control medium (P 〈 0.02 and P 〈 0.03, respectively). In conclusion, human cytotrophoblastic cells show a significantly increased rate of apoptosis in vitro after culture in a folate-free medium. Possible explanations for the association between folate deficiency and pregnancy complications are suggested.

Notes: Objective To compare the angiotensinogen genotypes in normotensive and pre-eclamptic pregnancies in maternal and fetal samples.Design Prospective observational study.Setting University Hospital, Queen's Medical Centre, Nottingham.Population Forty-three women with pre-eclampsia and 84 normotensive pregnant women. Fetal samples were available for genotyping from 96% of the pregnancies.Methods Maternal and fetal DNA was genotyped at angiotensinogen codon 235 and at a dinucleotide repeat polymorphism in the 3′ flanking region of the gene. Angiotensinogen and renin concentrations were measured in maternal plasma by radioimmunoassay.Results In contrast to earlier studies, no association was demonstrated between the angiotensinogen 235 Thr variant and pre-eclampsia. Normotensive pregnant women homozygous for this variant had significantly lower plasma angiotensinogen concentrations (median 2.2 ng AI/mL; IQR 1.8–3.0) than women homozygous for the 235 Met allele (3.6 ng AI/mL; IQR 2.5–4.1; P= 0.04). In pre-eclamptic pregnancies, 79% (11/14) of mothers heterozygous for the dinucleotide repeat allele designated A9 transmitted this allele to the fetus, more frequently than would be expected by chance (P= 0.02). The A9 allele was associated with low plasma angiotensinogen concentrations (P= 0.001) and high renin concentrations (P= 0.02) in normotensive women.Conclusions There is no evidence that the angiotensinogen 235 Thr allele is associated with pre-eclampsia in the Nottingham population. The angiotensinogen 235 Thr allele is associated with low plasma angiotensinogen concentrations in normotensive pregnant women, in contrast to the high levels associated with this variant in non-pregnant women, suggesting that regulation of angiotensinogen expression in normal pregnancy may differ significantly from that in the non-pregnant state. There is preliminary evidence that maternal-fetal transmission of an angiotensinogen allele associated with low plasma angiotensinogen concentrations is associated with pre-eclampsia. Impaired generation of angiotensin II at the maternal-fetal interface may be a factor in the pathogenesis of pre-eclampsia.

Notes: Objective To investigate the function of neutrophils in normal pregnancy and pre-eclampsia.Design Baseline levels and activated responses of peripheral blood neutrophils were measured in response to the physiological agonists, n-formyl-met-leu-phe (fMLP) and zymosan activated serum.Sample Neutrophils of 16 pre-eclamptic, 17 normal pregnant (third trimester) and 15 nonpregnant age-matched control women were calculated.Setting Antenatal Clinic, City Hospital, Nottingham.Methods Neutrophil superoxide anion production was determined by lucigenin-enhanced chemilumi-nescence; the release of secondary granule lactoferrin by ELISA; and the expression of cell surface adhesion molecules CD1 1b, CD 18 and L-selectin (CD62L) by flow cytometric analysis.Results Superoxide anion generation was reduced in the pregnant group compared with nonpregnant controls [fMLP by 51 % (P= 0.03) and zymosan activated serum by 56% (P= 〉 0.01)] but pre-eclamptic measurements did not show a similar reduction. There were no differences between the three study groups in the plasma levels of lactofenin, or in the stimulated expression and release of CD1 Ib and CD18, or lactoferrin and L-selectin. The base-line levels for the production of superoxide anions; the expression of CD11b or CD 18 or L-selectin; and the release of lactofemn showed no significant differences.Conclusions Circulating neutrophils in pregnancy and pre-eclampsia are neither activated nor primed in vivo, however the release of reactive oxygen species is diminished in normal pregnancy. In comparison, an elevation of reactive oxygen generation in pre-eclampsia may highlight a role for neutrophils in the oxidative stress and pathophysiology of this disease.