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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neuroendocrinology 7 (1995), S. 0 
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The mechanisms involved in 2-deoxy-D-glucose (2-DG)-induced growth hormone (GH) suppression in the rat were examined. Conscious male rats were given 2-DG by intracerebroventricular (icv) injection and the pulsatile GH secretion was monitored for 6 h. The single icv injection of 2-DG (8 mg/rat) eliminated pulsatile GH secretion in conscious rats. Pretreatment with somatostatin (SS) antiserum completely restored the suppressed GH secretion in the 2-DG treated rats. Hypothalamic GH-releasing hormone (GRH) and SS mRNA levels were not altered by single and multiple icv injections of 2-DG. These findings suggest that 2-DG-induced GH suppression is primarily due to hypersecretion of SS without a significant change at the transcription level in the rat.
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  • 2
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The profiles of growth hormone (GH) secretion were examined by obtaining serial blood samples every 15 min for a 5 to 24 h observation period from freely-moving, conscious male rabbits chronically implanted with a right atrial cannula. The effects of restraint or surgical stress on GH secretion were also investigated in these animals. Four days after cannulation of the right atrium, plasma GH levels remained low without oscillation, during a 5 h observation period (1100 to 1600 h) with the mean (± SEM) value of 1.6±0.2 ng/ml. Individual rabbits exhibited a spontaneous, pulsatile GH secretion 7 days after the surgery. Mean 6 h GH levels were 5.6 ± 0.8 ng/ml at 7 days after the surgery, 6.3 ± 0.6 ng/ml at 14 days and 7.0 ± 1.2 ng/ml at 28 days. Therefore, the animals, 7 to 14 days after cannulation, were used to analyse the pulsatile pattern of GH secretion throughout 6 to 24 h. Two episodes of 45 min immobilization stress, separated by 75 min, caused a complete suppression of the spontaneous GH secretion (mean 6 h GH levels, 2.2 ± 0.1 ng/ml vs control, 5.0 ± 0.5 ng/ml, P〈0.01). No surges appeared after the first restraint stress. In 14 non-treated rabbits, plasma GH levels fluctuated in an episodic manner throughout the study with the peaks of 14.2 + 0.7 ng/ml, the nadirs of 2.6 ± 0.2 ng/ml and the peak to peak intervals of 2.20 ± 0.17 h. The iv administration of normal goat λ-globulin (NGG) affected neither GH secretory patterns nor baseline levels of plasma GH. In contrast, the iv administration of anti-sornatostatin goat λ-globulin (ASG) caused a significant increase in the amplitude of plasma GH peaks (38.8±1.9 vs NGG-treated, 13.7 ± 0.8 ng/ml, P〈0.001) as well as the trough level (13.5 ± 0.6 vs NGG, 2.9 ± 0.1 ng/ml, P〈0.001) during a 24 h observation period. Also, ASG treatment increased numbers of plasma GH peaks per day (18.8±2.7 vs NGG, 12.2 ± 0.8, P 〈 0.05) with concomitant shortening of the peak to peak interval (1.25 ± 0.10 vs NGG, 2.03±0.12h, P〈0.01).These findings suggest: 1) that GH is episodically secreted throughout the day in conscious male rabbits, 2) that surgical and restraint stresses suppress the spontaneous GH secretion, and 3) that endogenous somatostatin might rather play a tonic inhibitory role in GH release in conscious male rabbits, since ASG treatment resulted in sustained marked increases of plasma GH levels irrespective of the stage in GH pulsatile rhythm.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Journal of neuroendocrinology 9 (1997), S. 0 
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Basic fibroblast growth factor (FGF-2) is not only a potent mitogen for various cells but also a multifunctional factor with angiogenic and chemotactic activity, and the capacity to induce the synthesis of various proteinases and to modulate endocrine function. To clarify the role played by FGF-2 in the progression of pituitary tumor, we fused rat FGF-2 cDNA to the promoter SRα, consisting of the early promoter of SV40 and HTLV(I)-LTR, and we cotransfected GH3 cells with pSV2-neo by an electroporation method. After selection by G418, we obtained 7 neomycin-resistant clones. Southern blot analysis of genomic DNA revealed the presence of transfected rat FGF-2 cDNA in 4 of the 7 clones. To measure FGF-2 molecules, we established a new immuno-fluorometric assay system, using 3 monoclonal antibodies against different portions of human FGF-2. This assay had a minimum sensitivity of 10 pg/ml and cross-reacted neither with acidic fibroblast growth factor (FGF-1) nor insulin-like growth factor 1 (IGF-1), even at a concentration of 100 ng/ml. Although FGF-2 was undetectable in the culture medium of any of the clones, the cell homogenate contained a significant amount of FGF-2 (7.2 ng/mg protein) in 1 of the 4 FGF-2-transfected clones (GH3FGF(+)), whereas FGF-2 was not detected (〈5.2 pg/mg protein) in the cell homogenates of either the parent GH3 cells or the control cells transfected with pSV2-neo alone (GH3FGF(−)). GH3FGF(+) grew as adherent cells and formed epithelial sheets with a growth rate similar to that of control cells. The amount of prolactin(PRL) released by TRH was greater in GH3FGF(+) than that in GH3 or GH3FGF(−). On the other hand, the sensitivity to SRIF was increased in GH3FGF(+) compared with that in other clones. The findings of these in vitro studies indicate that FGF-2, if it is expressed in pituitary tumor cells, plays little if any role in cell growth but may modulate certain cell functions such as responsiveness to hormones.
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  • 4
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The synthetic hexapeptide GH-releasing peptide (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2; GHRP-6) and GH releasing hormone (GHRH) are both potent stimulators of GH release in rats. Using reverse hemolytic plaque assay (RHPA), we have compared the effects of human GHRH and GHRP-6 on GH release from the dispersed individual cells of rat anterior pituitary.In a single RHPA, we quantified the percentage of plaque forming cells (% PFC) and their mean plaque area (MPA) after 30 min-incubation, and calculated a total secretion index (TSI) by multiplying % PFC and MPA. 10 nM GHRH and 100 nM GHRP-6 each caused a significant increase in % PFC (%) (GHRH 39.15, GHRP-6 29.4, vs vehicle 24.3, P〈0.01), MPA (×10−2 μm2) (GHRH 124.04, GHRP-6 94.80, vs vehicle 44.57, P〈0.01) and TSI (×10−2) (GHRH 54.46, GHRP-6 32.87, vs vehicle 10.84, P〈0.01). Simultaneous addition of both secretagogues caused a further increase in GH release (%PFC 46.4, MPA 142.55, TSI 69.82, P〈0.01 vs vehicle), although the effect was additive but not synergistic. Somatostatin analog, SMS201–995 (SMS) partially suppressed all parameters in GH secretion after stimulation by GHRH and/or GHRP-6.A double RHPA was then performed to test whether all somatotrophs respond equally to GHRH and GHRP-6 or some cells formed plaques only by either GHRH or GHRP-6. There were somatotrophs responsive to only GHRH (23.3%vs control 6.2%, P〈0.01), those responsive to only GHRP-6 (11.9%vs control 6.1%, P〈0.01), and those responsive to both GHRH and GHRP-6 (7.8%vs control 0.2%, P〈0.01).These results confirmed the previous findings that GHRP-6 and GHRH directly but independently stimulate GH release from the pituitary cells, and further suggest the presence of at least three functionally distinct somatotroph subpopulations concerning the responsiveness to GHRP-6 and GHRH in rats.
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  • 5
    ISSN: 1432-2072
    Keywords: Lithium ; Prolactin ; Thyrotropin ; TRH ; Manic patients
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The plasma thyrotropin (TSH) and prolactin (PRL) responses to thyrotropin releasing hormone (TRH) were studied before and during lithium treatment for 3–4 weeks in 6 patients with manic states and 8 control subjects. The plasma TSH responses to TRH were not different between the two groups before lithium treatment. Lithium administration did not alter non-stimulated secretion of TSH in any groups, but resulted in exaggerated responses of plasma TSH to TRH in both groups. No difference between two groups was observed in plasma TSH responses to TRH. The basal plasma PRL concentration did not differ between the two groups and was not affected by lithium administration to either group. The plasma PRL responses to TRH in female subjects were greater than those in male subjects. In females, the plasma PRL responses to TRH in manic patients were significantly higher than those in control subjects before the treatment. Lithium administration caused enhanced responses to TRH in patients when compared to pretreatment levels, but not in control subjects. Although the small number of male subjects limits conclusions, pretreatment plasma PRL responses to TRH in male manic patients were apparently greater than those in control subjects. However, lithium administration appeared not to affect the responses of plasma PRL to TRH in manic patients when compared to pretreatment levels. The augmented responses of plasma PRL to TRH in patients with manic states suggest the existence of some abnormality in the hypothalamo-pituitary axis. Thus, the effect of the anti-manic agent on PRL secretion in manic patients may suggest the machanism by which the drug affects manic symptoms.
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  • 6
    ISSN: 1432-0843
    Keywords: Key words Pirarubicin ; HL60 cell lines ; Transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  We studied the transport mechanism of pirarubicin (THP) in HL60 and its THP-resistant (HL60/THP) cells, which showed no expression of mdr1 mRNA on Northern blot analysis. Under physiological conditions, the uptake of THP by both types of cell was time- and temperature-dependent. The amount of drug transport in the resistant cells was significantly less than that in the parent cells within 3 min of incubation. THP uptake was significantly higher in the presence than in the absence of 4 mM 2,4-dinitrophenol (DNP) in glucose-free Hanks’ balanced salt solution in both HL60 and HL60/THP cells and the increases were approximately equal. In the presence of DNP, the uptake of THP by both types of cell was concentration-dependent, and there were no significant differences in the apparent kinetic constants (Michaelis constant (K m), maximum velocity (V max) and V max/K m) for THP uptake between HL60 and HL60/THP cells. Additionally, THP transport was competitively inhibited by its analogue doxorubicin. The efflux of THP from HL60/THP cells was significantly greater than that from HL60 cells, and the release from both types of cell was completely inhibited by decreasing the incubation temperature to 0°C and by treatment with DNP in glucose-free medium. In contrast, the P-glycoprotein inhibitors verapamil and cyclosporin A did not inhibit THP efflux. However, genistein, which is a specific inhibitor of multidrug resistance-associated protein (MRP), increased the THP remaining in the resistant cells, and the value was approximately equal to that of the control group in the sensitive cells. These results suggest that THP is taken up into HL60 and HL60/THP cells via a common carrier by facilitated diffusion, and then pumped out in an energy-dependent manner. Furthermore, the accelerated efflux of THP by a specific mechanism, probably involving MRP, other than the expression of P-glycoprotein, resulted in decreased drug accumulation in the resistant cells, and was responsible, at least in part, for the development of resistance in HL60/THP cells.
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  • 7
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The expression and localization of parathyroid hormone-related protein (PTHrP), a major factor responsible for humoral hypercalcemia of malignancy (HHM), was investigated in 14 cases of surgically resected normal parathyroid glands. For light microscopic immunohistochemistry, formalin-fixed and paraffin-embedded specimens were stained with avidin-biotin-peroxidase complex (ABC) using the anti-PTHrP monoclonal antibody (MoAb), 4B3. Four percent paraformaldehyde (PFA)-fixed and OCT compound-embedded specimens were used for pre-embedded immunoelectron microscopy. For in situ hybridization, 4% PFA-fixed, frozen sections were studied using a bromodeoxyuridine (BrdU)-labeled PTHrP cDNA probe. Immunohistochemically, 12 of the 14 cases were positive for PTHrP, which was observed mainly in the oxyphil and transitional oxyphil cells. The chief and clear cells, on the other hand, were faintly positive. Electron microscopically, secretory granules positive for PTHrP were observed in cells containing abundant mitochondria. Consistent with the PTHrP immunoreactivity, transcripts of PTHrP were observed also in the oxyphilic cells by in situ hybridization. Thus the production and secretion of PTHrP was shown by the oxyphil cell lineage in the normal parathyroid glands.
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  • 8
    ISSN: 1435-5604
    Keywords: parathyroid hormone ; parathyroid hormone fragments ; alkaline phosphatase ; osteoblasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effect of a series of truncated carboxyl-terminal parathyroid hormone (PTH) fragments on alkaline phosphatase (ALP) activity was further examined in dexamethasone-treated rat osteoblastic osteosarcoma cells, ROS 17/2.8. As we previously reported, dexamethasone-induced ALP activity was inhibited not only by hPTH(1-84) and aminoterminal PTH fragment hPTH(1-34), but also by carboxyl-terminal PTH fragment hPTH(69-84). The longer carboxyl-terminal PTH fragment hPTH(53-84) stimulated ALP activity, and the shorter carboxyl-terminal PTH fragment hPTH(71-84) did not affect ALP activity. The longest newly synthesized carboxyl-terminal PTH fragment hPTH(35-84), which is complementary to amino-terminal PTH fragment hPTH(1-34), stimulated ALP activity as potently as hPTH(53-84), but not more potently than hPTH(53-84). Another newly synthesized carboxyl-terminal PTH fragment hPTH(64-84), which has an intermediate peptide length between hPTH (53-84) and hPTH(69-84), inhibited ALP activity as potently as hPTH(69-84). These results suggest that the 35-52 amino acid portion of the PTH molecule might not be crucial for the stimulatory effect of carboxyl-terminal PTH fragments on ALP activity, and that the 53–63 portion, but not the 64–68 portion, of the PTH molecule might be essential for the stimulatory effect of carboxyl-terminal PTH fragments on ALP activity. Furthermore, the importance of the 69th and 70th amino acid of the PTH molecule for the inhibitory effect of carboxyl-terminal PTH fragments on ALP activity was confirmed.
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  • 9
    ISSN: 1435-5604
    Keywords: osteoclast ; Pi transport ; integrin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The characteristics of the transport system for inorganic phosphate (Pi) have been investigated in chicken osteoclasts. Pi transport in these cells was mainly Nadependent and also energy-dependent, suggesting the existence of Na-Pi cotransport system in these cells. While the time course of Pi transport in chicken osteoclasts was comparable to that in chicken mononuclear cells, the precursors of the osteoclasts, Pi transport was stimulated in a time- and dose-dependent fashion, when the osteoclasts were exposed to bone particles. The stimulation was not due tode novo synthesis of Pi carrier, since the stimulatory effect was very rapid with the maximal effect observed at 1 h and could not be blocked by 50 µM cycloheximide. On the other hand, inhibition of osteoclast attachment to bone particles by adding an excess of Arg-Gly-Asp-Ser peptide blocked the stimulation. In summary, stimulation of Pi transport in chicken osteoclasts was induced by bone particles. Attachment of the osteoclasts to bone particles with integrin receptors appeared to be crucial to this stimulation.
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  • 10
    ISSN: 1435-5604
    Keywords: parathyroid hormone ; parathyroid hormone-related protein ; c-fos ; bone
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Although there is a recent evidence that PTH induces c-fos gene expression in osteoblasts, the physiological role of this expression remains unknown. We, therefore, employed c-fos antisense oligodeoxynucleotide (as-ODN) and sought to clarify the role of c-fos gene in the regulation of osteoblast proliferation and differentiation as well as osteoclast differentiation and bone-resorbing activity in the presence of osteoblasts by PTH and PTH-related protein (PTHrP). We employed osteoclastlike cell formation from mouse bone cells for the evaluation of osseoclast differentiation and the pit formation assay on the dentin slice in mouse bone cells for the evaluation of bone-resorbing activity by mature osteoclasts. Northern blot analysis revealed that both human (h)PTH-(1-34) and hPTHrP-(1-34) (10−8M) induced a transient c-fos gene expression to a similar degree in osteoblastic UMR-106 cells. Sp-cAMPS (10−4M), an activator of cAMP-dependent protein kinase (PKA), as well as dbcAMP induced a weak c-fos gene expression and Rp-cAMPS (10−4M), an inhibitor of PKA, almost completely antagonized these expressions. However, Rp-cAMPS only slightly blocked c-fos gene expression by PTH and PTHrP. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC) (10−7 to 10−6M), but not 4 alpha-phorbol 12, 13-didecanoate, incapable of activating PKC induced an intense expression of c-fos gene. Calcium ionophores (A23187 and ionomycin, 10−7 to 10−6M) did not induce the expression of c-fos gene. An inhibitor of PKC (H-7, 50µM) almost completely blocked the c-fos gene expression by PTH and PTHrP as well as PMA. Pretreatment with 1µM as-ODN significantly antagonized the inhibition of [3H] thymidine incorporation into UMR-106 cells and the stimulation of osteoclast-like cell formation by PTH and PTHrP, compared to pretreatment with the control oligodeoxynucleotide consisting of same nucleotides as as-ODN but with a random sequence. On the other hand, as-ODN did not affect an increase in alkaline phosphatase activity of UMR-106 cells and pit formation by PTH and PTHrP. In all experiments so far, the effects of PTHrP were virtually the same as those of PTH. The present study indicates, first, the direct involvement of PKC as well as PKA in PTH- and PTHrP-induced c-fos gene expression and, second, the participation of its expression in the regulation of osteoblast proliferation and osteoclast differentiation by PTH as well as PTHrP.
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