Biomedical subjects

C Wagner

Publications and source records attributed to C Wagner.

At least 145 records · Page 8Linked to original sources

PubMed1998

Immune response against human primary malignant melanoma: a distinct cytokine mRNA profile associated with spontaneous regression.

Spontaneous regression of melanoma lesions is thought to be the result of an efficient immune response against melanoma cells in vivo. The outcome of immune responses is critically influenced by a complex network of interacting cytokines present in the local microenvironment. Analysis of cytokine gene transcription in melanoma lesions exhibiting or lacking a sufficient anti-tumor immune response thus may help to define cytokines or cytokine combinations critical to the development of this immune response. In the present study, we have investigated an extended panel of cytokine and cytokine receptor genes by reverse transcription-PCR and in situ hybridization in regressive and progressive primary human cutaneous melanoma samples. Whereas the presence of a lymphocyte infiltrate in tissue samples was associated with a TH1 cytokine mRNA profile (TNF-alpha, INF-gamma, IL12p35, IL12p40, IL2Rbeta, and IL2Rgamma), clinically and histologically regressive samples exhibited additionally increased transcript levels for GM-CSF, IL2, and IL15. mRNAs of TH2 cytokines IL4 and IL5 were detected only in a minor portion of progressive melanoma samples and regressive melanoma lesions. These results were further supported by comparison of progressive with regressive regions in three melanoma samples. Again, regressive regions contained higher transcript levels for GM-CSF, IL2, and IL15. In comparison to cutaneous metastatic melanoma lesions, regressive melanomas also overexpressed the same cytokine mRNA profile. These results provide evidence for an association of spontaneous regression with increased transcript levels for the cytokine combination GM-CSF, IL2, and IL15 in malignant melanoma. This cytokine combination could be relevant for experimental anti-tumor immune response studies and for immunotherapeutic and gene transfer studies in the treatment of melanoma patients.

Antibodies↗

PubMed1997

Transport of rat liver glycine N-methyltransferase into rat liver nuclei.

Rat liver cytosolic glycine N-methyltransferase (GNMT) catalyzes the S-adenosylmethionine-dependent methylation of glycine to sarcosine. It is comprised of four identical 292-amino acid residue subunits. Recently, evidence has been provided to show that GNMT is identical to the cytosolic receptor for benzo[a]pyrene, which induces cytochrome P450 1A1 gene expression. In the present study we show that chemical modification of purified rat liver GNMT with fluorescein isothiocyanate (FITC) resulted in dissociation of the tetrameric enzyme and was accompanied by loss of enzyme activity. Amino acid sequence analysis of the FITC-labeled peptides obtained by hydrolysis of the modified protein with Staphylococcus aureus V8 protease revealed that lysines 45, 89, 92, 96, 122, and 147 were modified. Lys-122 and Lys-147 were derivatized in tetrameric, dimeric, and monomeric forms of the enzyme. Lysines 45, 89, 92, and 96 were derivatized only in monomeric GNMT, suggesting that modification of these residues resulted in GNMT dissociation. The modified monomeric GNMT was quickly transported into isolated rat liver nuclei. This transport was specific for the GNMT monomer, since neither tetramer nor dimer was able to enter the nuclei. Bovine carbonic anhydrase, similar in size to the GNMT monomer, was labeled with FITC to a similar extent but was not transported into the nuclei. Disruption of the nuclei containing fluorescein-labeled GNMT and subsequent extraction of the nuclear lysate with both high and low salt buffers recovered FITC-GNMT only in the chromatin pellet. Our study supports the suggestion of an additional function for GNMT, probably connected with regulation of cytochrome P450 1A1 gene expression.

Amino Acid Sequence↗

PubMed1997

[Effect of diltiazem on concentration of cyclosporin metabolites in Sandimmune and Neoral treated kidney transplant patients].

BACKGROUND: Diltiazem reduces the cyclosporine dose required for blood levels in the therapeutic target range by 30 to 40%. The effect of diltiazem on the pharmacokinetic disposition of cyclosporine after oral Neoral application is unknown and it is unclear whether or not the diltiazem-cyclosporine interaction is affected by the galenic cyclosporine formulation. PATIENTS AND METHODS: Fifty-one stable renal allograft patients (19 females, 32 males) were enrolled in this prospective, randomized and double-blind study. The patients were assigned to 3 treatment groups: with diltiazem (I, n = 17), with nifedipine (II, n = 17) and without calcium channel blockers (III, n = 17). Nine patients in each group received Sandimmun and 8 patients Neoral. Blood concentrations of cyclosporine and its metabolites AM1 and AM9 were measured using HPLC for 12 weeks. The 3 treatment groups were not different in respect to age, gender distribution and serum creatinine concentration. Cyclosporine doses were adjusted on basis of the blood levels. RESULTS: The cyclosporine doses required to achieve target blood levels were significantly lower in group I compared with group II (-43%) and group III (-33%; p < 0.0001). Although the cyclosporine blood concentrations in all groups were in the therapeutic range, the blood levels in group I showed a much lower variability. The blood concentrations of the metabolite AM1 in group I were significantly higher than those in groups II and III after dose correction (p < 0.0001), those of AM9 were significantly lower in group I than in groups II and III (p < 0.0001). The average dose, and the blood concentration of cyclosporine was not different when patients receiving Neoral were compared with those receiving Sandimmun within the groups. In the patients in group I, the blood concentration of metabolite AM1 was significantly higher after Sandimmun application than after Neoral. No other differences in the metabolite concentrations were detected within the groups comparing patients taking Sandimmun or Neoral. The incidences of acute rejection were lower in group I (17.6%) than in the other groups (II: 52.9%; III: 41%). CONCLUSION: Diltiazem significantly reduced the necessary dose of cyclosporine. Compared with groups II and III, the blood concentrations were more stable in patients in group I. Diltiazem increased the blood concentration of AM1 in patients treated with Sandimmun to a larger extent than in patients taking Neoral. No additional pharmacokinetic differences of the 2 cyclosporine applications different with Sandimmun or Neoral were found.

Adult↗

PubMed1997

The acetyl-CoA synthetase gene ACS2 of the yeast Saccharomyces cerevisiae is coregulated with structural genes of fatty acid biosynthesis by the transcriptional activators Ino2p and Ino4p.

The yeast Saccharomyces cerevisiae contains two acetyl-CoA synthetase genes, ACS1 and ACS2. While ACS1 transcription is glucose repressible, ACS2 shows coregulation with structural genes of fatty acid biosynthesis. The ACS2 upstream region contains an ICRE (inositol/choline-responsive element) as an activating sequence and requires the regulatory genes INO2 and INO4 for maximal expression. We demonstrate in vitro binding of the heterodimeric activator protein Ino2p/Ino4p to the ACS2 promoter. In addition, the pleiotropic transcription factor Abf1p also binds to the ACS2 control region. The identification of ACS2 activating elements also found upstream of ACC1, FAS1 and FAS2 suggests a role of this acetyl-CoA synthetase isoenzyme for the generation of the acetyl-CoA pool required for fatty acid biosynthesis.

Acetate-CoA Ligase↗

PubMed1997

Pancreatic exocrine secretion is blocked by inhibitors of methylation.

A number of early experiments suggested a relationship between methyl group metabolism and the exocrine secretion of the pancreas. These included nutritional studies showing that ethionine, the ethyl analog of methionine which inhibits cellular methylation reactions, is a specific pancreatic toxin. Other studies indicated that protein carboxymethylation might be involved. We now show that in vivo ethionine inhibits amylase secretion from freshly isolated rat pancreatic acini, while in vitro ethionine inhibits amylase secretion from the AR42J pancreatic cell line. S-Adenosylhomocysteine (SAH) is a product inhibitor of all methyltransferase reactions involving S-adenosylmethionine (SAM), and treatments that elevate cellular levels of SAH such as inhibition of S-adenosylhomocysteine hydrolase and the in vitro addition of adenosine and homocysteine result in the inhibition of amylase secretion in both isolated pancreatic acini and AR42J cells. Measurement of SAM and SAH levels in AR42J cells shows that inhibition of secretion is more closely related to elevation of SAH levels than to a decrease in the SAM/SAH ratio. Small G-proteins are carboxymethylated on the C-terminal prenylated cysteine and inhibitors of membrane-associated prenylcysteine methyltransferase, N-acetylfarnesylcysteine, N-acetylgeranylgeranylcysteine, and farnesylthioacetic acid (FTA), block secretion in AR42J cells. N-Acetylgeranylcysteine is not an inhibitor of the methyltransferase and does not inhibit amylase secretion. FTA inhibits membrane-associated prenylcysteine methyltransferase from AR42J cells with a Ki in the 45-69 microm range. These results suggest that a methylation event is needed for pancreatic exocrine secretion which may be the reversible methylation of a G-protein involved in signal transduction or membrane trafficking.

Acetylcysteine↗

PubMed1997

The homodimeric form of glycine N-methyltransferase acts as a polycyclic aromatic hydrocarbon-binding receptor.

In the rat, cytochrome P4501A1 gene expression is thought to be regulated by several trans-acting factors including the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein, which was recently identified as glycine N-methyltransferase [Raha, A., Wagner, C., MacDonald, R. G., and Bresnick, E. (1994) J. Biol. Chem. 268, 5750-5756]. Glycine N-methyltransferase (GNMT) is one of those unique proteins which exhibit diversity in function. Different subunit configurations are involved in its enzymatic role as a methyltransferase and as PAH-binding receptor. Here we report a systematic study of the oligomeric state of GNMT in the presence of benzo[a]pyrene (B[a]P) in vivo and in vitro. We have used chemical cross-linking and denaturing polyacrylamide gel electrophoresis to show that the B[a]P-binding unit of GNMT is a homodimer. We recently reported that phosphorylation is involved in the interaction of B[a]P with the 4S PAH-binding protein [Bhat, R., Weaver, J. A., Wagner, C., Bodwell, J. E., and Bresnick, E. (1996) J. Biol. Chem. 271, 32551-32556]. In the present study, this observation has been amplified by using bacterially expressed GNMT, which was post-translationally modified by a reticulocyte lysate and ATP-generating system. This modification was also accompanied by the formation of homodimers in the presence of B[a]P. These results indicate that post-translational modification is involved in determining the final configuration, i.e., dimeric form, of GNMT which then acts as a PAH-binding receptor.

Animals↗

PubMed1997

Transport of S-adenosylmethionine in isolated rat liver mitochondria.

Mitochondria do not have the enzyme, methionine adenosyltransferase (ATP: L-methionine S-adenosyltransferase, EC 2.5.1.6), necessary for the biosynthesis of S-adenosylmethionine. Nevertheless, about 30% of total hepatic S-adenosylmethionine resides in the mitochondria and radiolabeled S-adenosylmethionine may be isolated from the mitochondria after administration of radiolabeled methionine. This leads to the hypothesis that a carrier-mediated system is responsible for S-adenosylmethionine transport from the cytosol into the mitochondria. We have characterized such a system in isolated rat liver mitochondria. Uptake of S-adenosylmethionine consisted of two components. One component was incorporation of the methyl group into phospholipids as shown by thin-layer chromatography. The second component represented uptake into the mitochondria since addition of excess unlabeled S-adenosylmethionine resulted in efflux of labeled substrate. This countertransport is characteristic of a carrier-mediated transport system. Uptake (corrected for incorporation into phospholipids) was saturable with an apparent Km = 8.9 microM and Vmax = 54.3 pmol x mg protein(-1) x min(-1). Uptake was not inhibited by methionine, adenosine, 5'-methylthioadenosine, carnitine, choline, betaine, quinine, or hemicholinium-3. Uptake was inhibited by sinefungin and by S-adenosylhomocysteine (Ki = 53.4 microM). Uptake of S-adenosylmethionine was not dependent on the electrical potential across the mitochondrial membrane. These results indicate that S-adenosylmethionine is taken up into mitochondria via a specific, carrier-mediated system.

Animals↗

PubMed1997

Direct measurement of nucleation and growth rates in lysozyme folding.

A kinetic folding intermediate of hen lysozyme is shown to form in a nucleation/growth type of mechanism. Under native solvent conditions, a nucleated state is formed slowly during refolding (tau = 14 +/- 1 ms at 0 M GdmCl) and is rapidly converted to the folding intermediate (tau = 300 +/- 150 micros at 0 M GdmCl). Under these conditions the nucleated state represents a high-energy state compared to the folding intermediate (delta deltaG0 = 13.7 +/- 3 kJ/mol). At elevated concentrations of GdmCl, the nucleated state becomes more stable than the intermediate and it consequently becomes transiently populated during unfolding of the intermediate state. This allowed us to measure the rate constant of the growth step using stopped-flow double-jump experiments. At high concentrations of GdmCl (>5 M), the growth step becomes rate-limiting in unfolding, leading to the frequently observed rollover in the GdmCl dependence of the logarithm of the apparent rate constant of the unfolding reaction.

Animals↗

PubMed1997

Expression, purification, and properties of the aldehyde dehydrogenase homologous carboxyl-terminal domain of rat 10-formyltetrahydrofolate dehydrogenase.

The liver cytosolic enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH) (EC 1.5.1.6) catalyzes two reactions: the NADP+-dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP+-independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the enzyme (residues 420-902) is about 48% identical to a family of NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3), and FDH possesses aldehyde dehydrogenase activity. We expressed the COOH-terminal domain (residues 420-902) of FDH in insect cells using a baculovirus expression system. The recombinant protein was released from insect cells to the culture medium and was purified from the medium by a two-step procedure: precipitation with 35% saturated ammonium sulfate followed by chromatography on hydroxyapatite. The purified COOH-terminal domain displayed aldehyde dehydrogenase activity similar to that of native FDH but had neither dehydrogenase nor hydrolase activity toward folate substrates. Aldehyde dehydrogenase activity of the COOH-terminal domain and FDH was independent of the presence of 2-mercaptoethanol while 10-FDDF dehydrogenase activity of FDH occurred only in the presence of 2-mercaptoethanol. The COOH-terminal domain existed as a tetramer showing that the sites for oligomerization of subunits in native FDH resides in this domain. Using titration of tryptophan fluorescence, it was found that the COOH-terminal domain bound NADP+ to the same extent as FDH (Kd 0.2 and 0.3 microM, respectively) but did not bind folate. Both FDH and its COOH-terminal domain also bound NAD+ (Kd 11 and 16 microM, respectively) as measured by fluorescence titration. Both proteins were able to catalyze the aldehyde dehydrogenase reaction utilizing NADP+ or NAD+, but the Km for NAD+ was three orders higher than that for NADP+ (2 mM and 1.5-2.0 microM, respectively). The concentration of NAD+ required for the reaction was high compared with the physiological level of NAD+, suggesting that the reaction does not occur in vivo. NAD+ at physiological concentrations stimulated the aldehyde dehydrogenase reaction performed by FDH or its COOH-terminal domain using NADP+.

Aldehyde Dehydrogenase↗

PubMed1997

Domain structure of rat 10-formyltetrahydrofolate dehydrogenase. Resolution of the amino-terminal domain as 10-formyltetrahydrofolate hydrolase.

We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.

Amidohydrolases↗

PubMed1997

Regulation of angiotensin II receptor AT1 subtypes in renal afferent arterioles during chronic changes in sodium diet.

Studies determined the effects of chronic changes in sodium diet on the expression, regulation, and function of different angiotensin II (ANG II) receptor subtypes in renal resistance vessels. Rats were fed low- or high-sodium diets for 3 wk before study. Receptor function was assessed in vivo by measuring transient renal blood flow responses to bolus injections of ANG II (2 ng) into the renal artery. ANG II produced less pronounced renal vasoconstriction in rats fed a low- compared with high-sodium diet (16% vs. 56% decrease in renal blood flow, P < 0.001). After acute blockade of ANG II formation by iv enalaprilat injection in sodium-restricted animals, ANG II produced a 40% decrease in renal blood flow, a level between untreated dietary groups and less than high salt diet. Intrarenal administration of angiotensin II receptor type 1 (AT1) receptor antagonists losartan or EXP-3174 simultaneously with ANG II caused dose-dependent inhibition of ANG II responses. Based on maximum vasoconstriction normalized to 100% ANG II effect in each group, AT1 receptor antagonists produced the same degree of blockade in all groups, with an apparent maximum of 80-90%. In contrast, similar doses of the angiotensin II receptor type 2 (AT2) receptor ligand CGP-42112 had only a weak inhibitory effect. In vitro equilibrium-saturation 125I-ANG II binding studies on freshly isolated afferent arterioles indicated that ANG II receptor density was lower in the low- vs. high-sodium animals (157 vs. 298 fmol/mg, P < 0.04); affinity was similar (0.65 nM). Losartan and EXP-3174 displaced up to 80-90% of the ANG II binding; fractional displacement was similar in both diet groups. In contrast, the AT2 receptor analogues PD-123319 and CGP-42112 at concentrations < 10(-6) M had no effect on ANG II binding. RT-PCR assays revealed the expression of both angiotensin II receptor type 1A (AT(1A)) and angiotensin II receptor type 1B (AT(1B)) subtypes in freshly isolated afferent arterioles, while there was very little AT2 receptor expression. Total AT1 receptor mRNA expression was suppressed by low sodium intake to 66% of control levels, whereas it was increased to 132% of control by high-sodium diet, as indicated by ribonuclease protection assay. Receptor regulation was associated with parallel changes in AT(1A) and AT(1B) expression; the AT(1A)/AT(1B) ratio was stable at 3.7. We conclude that AT1 receptors are the predominant ANG II receptor type in renal resistance vessels of 7-wk-old rats. Chronic changes in sodium intake caused parallel regulation of expression and amount of receptor protein of the two AT1 receptor genes that modulate receptor function and altered reactivity of renal vessels to ANG II.

Actins↗

PubMed1997

Analysis of Pmel17/gp100 expression in primary human tissue specimens: implications for melanoma immuno- and gene-therapy.

Pmel17/gp100-encoded tumor-associated antigens are recognized by cytotoxic T lymphocytes in melanoma patients and may represent attractive target antigens for immuno- and gene-therapeutic strategies. An important prerequisite for identification and monitoring of melanoma patients that could potentially benefit from Pmel17/gp100-based immuno- and gene-therapies is the detailed knowledge of Pmel17/gp100 expression in vivo. Immunophenotyping is considerably hampered by the different immunoreactivities of Pmel17/gp100-reactive antibodies. Therefore, we analyzed an extended series of different primary normal and malignant human tumor specimens for Pmel17/gp100 expression at the mRNA level. Transcripts were detectable in all malignant melanoma tissue specimens representing all stages of tumor progression, with significant levels even in early and amelanotic melanoma lesions. In contrast, normal melanocytes exhibited significantly less Pmel17/gp100 mRNA in vivo, as determined by comparative in situ hybridization. Tissue specimens from the retina and substantia nigra also contained Pmel17/gp100 mRNA, whereas other normal and malignant human tissues were negative. As determined by comparative in situ hybridisation and HMB-45 immunostaining, even tumor tissue lacking Pmel17/gp100 immunoreactivity contained Pmel17/gp100 transcripts. Our results indicate a melanocytic-cell-lineage-restricted expression of Pmel17/gp100 with significant transcript levels in all stages of melanoma progression, including early and amelanotic melanoma lesions, and a significantly differential expression between melanoma cells and normal melanocytes in vivo. Owing to its higher sensitivity, phenotyping of individual tumor specimens by mRNA expression analysis seems to be more valuable than phenotyping by immunostaining.

Antibodies, Neoplasm↗

PubMed1997

Quality systems in Dutch health care institutions.

The implementation of quality systems in Dutch health care was supervised by a national committee during 1990-1995. To monitor the progress of implementation a large survey was conducted in the beginning of 1995. The survey enclosed all subsectors in health care. A postal questionnaire--derived from the European Quality Award--was sent to 1594 health care institutions; the response was 74%. The results showed that in 13% of the institutions a coherent quality system had been implemented. These institutions reported, among other effects, an increase in staff effort and job satisfaction despite the increased workload; 59% of the institutions had implemented parts of a quality system. It appeared that management pay more attention to human resource management compared to documentation of the quality system. The medical staff pay relatively more attention to protocol development than to quality-assurance procedures. Patients were hardly involved in these quality activities. The research has shown that it is possible to monitor the progress of implementation of quality systems on a national level in all subsectors of health care. The results play an important role in the discussions and policy on quality assurance in health care.

Data Collection↗

PubMed1997

Role of sympathetic nerves for the stimulation of the renin system by angiotensin II receptor blockade.

OBJECTIVE: To assess the relevance of sympathetic nerves for the stimulation of renin secretion and renin gene expression during effective angiotensin II type 1 receptor blockade in vivo. METHODS: Male Sprague-Dawley rats were treated with the angiotensin II type 1-receptor blocker losartan (40 mg/kg) for 3 days. To examine the role of renal sympathetic nerves in the stimulation of the renin system by losartan, left kidneys were denervated 4 days prior to the treatment with losartan. Also, to examine the role of circulating catecholamines in the stimulation of the renin system by losartan, the animals were administered a combination treatment of losartan with the beta1-adrenoreceptor blocker metoprolol (50 mg/kg per day) for 3 days. RESULTS: Losartan treatment increased plasma renin activity about sevenfold and renal renin messenger RNA (mRNA) levels about fivefold and decreased systolic blood pressure from 118 to 95 mmHg. Administration of losartan elevated renin mRNA both in the innervated and in the denervated kidneys to the same level as it did in kidneys of normal animals. Losartan treatment increased plasma renin activity and renal renin mRNA levels in the beta1-blocker-treated rats to the same extent as it did in animals administered losartan only. CONCLUSION: These findings suggest that, under sub-chronic treatment with hypotensive doses of angiotensin II receptor blockers, sympathetic outflow plays no important mediator role in the characteristic stimulation of renin secretion and renin gene expression, suggesting that it is mainly a direct disinhibition of angiotensin II's action on the level of juxtaglomerular cells that accounts for the effect.

Adrenergic beta-Antagonists↗

PubMed1997

Recombinant human growth hormone-binding protein fails to enhance the in vivo bioactivity of human growth hormone in normal rats.

GH circulates in the plasma partially bound with a GH-binding protein (GHBP), but the physiological significance of the GHBP and how it affects GH bioactivity in vivo is still unknown. In the present study, we took advantage of the known biological action of exogenous human (h) GH to inhibit endogenous rat (r) pulsatile GH release and examined the effect of combining hGH with recombinant hGHBP on this response in normal rats. Spontaneous 7-h plasma rGH and hGH profiles were obtained from four groups of free-moving adult male rats s.c. administered either: 1) 200 microg hGH alone; 2) a mixture of 200 microg hGH and 200 microg hGHBP preincubated for 30 min before injection; 3) 200 microg hGHBP alone; or 4) Tris buffer (vehicle) alone. Rats administered the vehicle or hGHBP separately exhibited the typical pulsatile pattern of rGH secretion. Injection of hGH alone resulted in a marked (P < 0.01) suppression of spontaneous rGH pulses for approximately 3.5 h after the injection compared with vehicle-injected controls; during the subsequent 3.5- to 7-h period, recovery of spontaneous rGH peaks was evident. Plasma levels of hGH in these animals reached a peak within 1 h after hGH injection and declined to near undetectable levels by the end of the sampling period. In contrast, the disappearance rate of hGH was markedly slower in rats administered the hGH + hGHBP complex; plasma hGH concentrations at 7 h after injection were 14-fold higher than those in animals administered hGH alone, and hGH was still readily detectable up to 24 h after injection. However, despite the markedly higher levels of hGH persisting throughout the sampling period in complex-injected rats, both the time course of hGH-induced inhibition of rGH and the recovery of spontaneous rGH pulses were similar to those of animals administered hGH alone. Moreover, there were no significant modifications of plasma insulin-like growth factor-1 levels for up to 24 h after injection of the hGH + hGHBP complex. Computer simulations revealed that most of the total hGH observed during the 3.5- to 7-h period was circulating in the bound form. These results demonstrate that, despite hGHBP's ability to markedly prolong the bioavailability of hGH, precomplexing hGH with hGHBP failed to enhance hGH's in vivo bioactivity in the inhibition of endogenous pulsatile rGH release. Our findings do not provide support for the concept that the GHBP enhances the bioactivity of GH in vivo, at least over the time course examined here.

Animals↗

PubMed1997

Covalent binding of acetaminophen to N-10-formyltetrahydrofolate dehydrogenase in mice.

The analgesic acetaminophen is frequently used as a model chemical to study hepatotoxicity; however, the critical mechanisms by which it produces toxicity within the cell are unknown. It has been postulated that covalent binding of a toxic metabolite to crucial proteins may inhibit vital cellular functions and may be responsible for, or contribute to, the hepatotoxicity. To further understand the importance of covalent binding in the toxicity, a major cytosolic acetaminophen-protein adduct of 100 kDa has been purified by a combination of anion exchange chromatography and preparative electrophoresis. N-Terminal and internal amino acid sequences of peptides from the purified 100-kDa acetaminophen-protein adduct were found to be homologous with the deduced amino amino acid sequence from the cDNA of N-10-formyltetrahydrofolate dehydrogenase. Antiserum specific for N-10-formyltetrahydrofolate dehydrogenase and acetaminophen react in a Western blot with the purified 100-kDa acetaminophen-protein adduct. Administration of a toxic dose of acetaminophen (400 mg/kg) to mice resulted in a 25% decrease in cytosolic N-10-formyltetrahydrofolate dehydrogenase activity at 2 hr. The covalent binding of acetaminophen to proteins such as N-10-formyltetrahydrofolate dehydrogenase and the subsequent decreases in their enzyme activity may play a role in acetaminophen hepatotoxicity.

Acetaminophen↗

PubMed1997

ProC Global: the first functional screening assay for the complete protein C pathway.

In clinical practice, venous thromboembolic complications are much more frequent than bleeding disorders. In fact, disturbances within the protein C pathway due to coagulation factor V (FV) Leiden mutation and deficiency of protein C or protein S are the most frequent abnormalities in hereditary thrombophilia. Furthermore, acquired dysfunctions of the protein C system may predispose the single individual to an increased thrombotic risk. A routine-suited screening assay that would allow the monitoring of the proper interplay of factors in the protein C pathway could add an important factor to the basic coagulation profile. This consists of the prothrombin time and of the activated partial thromboplastin time, which currently allow only a screening for increased risk for bleeding but not for venous thromboembolism. A new functional screening test for the protein C system such as the presented ProC Global should therefore facilitate detection of FV Leiden as well as deficiency of protein C and protein S. The results of the present evaluation indicate that ProC Global is highly sensitive to activated protein C resistance/FV Leiden (100%) and protein C deficiency (90%) and sensitive to protein S deficiency (63%). Furthermore, the assay gives a quantitative measure of the net potential of the protein C pathway in relation to the intrinsic procoagulant system. The use of this assay for a prospective assessment of thromboembolic risk is the subject of current studies.

Blood Coagulation Disorders↗

PubMed1996

ATP depletion affects the phosphorylation state, ligand binding, and nuclear transport of the 4 S polycyclic aromatic hydrocarbon-binding protein in rat hepatoma cells.

In the rat, cytochrome P4501A1 gene expression is thought to be regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein. Phosphorylation and dephosphorylation have been suggested to influence the function of many cytosolic receptors and transcription factors. The ATP level within H4IIE rat hepatoma cells could be depleted by treatment with sodium azide or 2,4-dinitrophenol; restoration of the original ATP levels occurred with addition of glucose to the cell culture. ATP depletion reduced the phosphate content of the 4 S protein by approximately 25-30%, which lowered the binding of benzo[a]pyrene (B[a]P) to the 4 S protein by >60%. This effect could not be reversed by the addition of ATP to the binding reaction mixtures. Alkaline phosphatase treatment of the purified 4 S protein in a cell-free system also reduced the B[a]P binding to the protein. Cells treated with a protein phosphatase inhibitor, okadaic acid, and a protein kinase inhibitor, staurosporin, affected the B[a]P binding of the 4 S protein positively and negatively, respectively. These data suggested that phosphorylation is involved in the interaction of the 4 S protein with the PAH. The nuclear translocation of the predominantly cytosolic binding protein has been investigated after ligand binding. Western blots with the immunopurified 4 S PAH-binding protein from cytosolic and nuclear lysates showed significant differences in the distribution of the 4 S receptor between cytosolic and nuclear compartments in control and ATP-depleted cells. Ligand binding stimulated the movement of the receptor into the nucleus, which was completely blocked by reducing the intracellular ATP concentration. These findings provide new information on the role of ATP and phosphorylation on the interaction of B[a]P with 4 S PAH-binding protein and its nuclear translocation.

2,4-Dinitrophenol↗