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PubMed2005

Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia.

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and is strongly associated with Epstein-Barr virus (EBV). We investigated the primary diagnostic value of circulating EBV DNA and anti-EBV immunoglobulin G (IgG) and IgA levels in Indonesian NPC patients (n = 149). By a 213-bp Epstein-Barr virus nuclear antigen 1 (EBNA1)-based real-time LightCycler PCR, 72.5% of patients were positive for EBV DNA in whole blood, with 29.5% having levels above a previously determined clinical cutoff value (COV) of 2,000 EBV DNA copies/ml, the upper level in healthy carriers. In a 99-bp LightCycler PCR, 85.9% of patients were positive and 60.4% had levels above the COV. This assay quantified a significantly higher EBV load than the 213-bp PCR assay (P < 0.0001), suggesting that circulating EBV DNA is fragmented. Using data from 11 different studies, we showed a significant inverse correlation between PCR amplicon size and the percentage of patients positive for circulating EBV DNA (Spearman's rho = -0.91; P < 0.0001). EBV DNA loads were unrelated to anti-EBV IgG or IgA levels, as measured by VCA-p18 and EBNA1-specific synthetic peptide-based enzyme-linked immunosorbent assays. The presence of circulating tumor cells was assessed by amplification of BamHI-A rightward frame 1 (BARF1) mRNA, a viral oncogene abundantly expressed in EBV-carrying carcinomas but virtually absent from EBV-associated lymphomas. Despite high EBV DNA loads and the presence of EBNA1 and human U1A small nuclear ribonucleoprotein mRNA, BARF1 mRNA was never detected in blood. We conclude that amplicon size significantly influences EBV DNA load measurement in NPC patients. The circulating EBV DNA load is independent of serological parameters and does not reflect intact tumor cells. The primary diagnostic value of the EBV DNA load for the detection of NPC is limited.

Antibodies, Viral↗

PubMed1985

Utilization of internal AUG codons for initiation of protein synthesis directed by mRNAs from normal and mutant genes encoding herpes simplex virus-specified thymidine kinase.

Previous studies (H.S. Marsden, L. Haarr, and C.M. Preston, J. Virol. 46:434-445, 1983) have shown that at least three polypeptides, with molecular weights of 43,000, 39,000, and 38,000, are encoded by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene. It has been suggested that the 39,000- and 38,000-molecular-weight polypeptides arise from preinitiation complexes bypassing the first and second AUG codons before commencement of translation since, according to previous work (M. Kozak, Nucleic Acids Res. 9:5233-5252, 1981), these codons are not of the most efficient structure for initiation. This possibility was investigated by using specific herpes simplex virus mutants with alterations in the TK gene. Mutant TK4 has an amber mutation between the first and second AUG codons, whereas mutant delta 1 has a deletion which removes the first AUG codon but leaves other AUG codons, as well as transcriptional promoter sequences, intact. Both mutants synthesized only the 39,000- and 38,000-molecular-weight polypeptides, and the amounts produced were normal in TK4-infected cells but increased in delta 1-infected cells. Furthermore, the levels of TK produced after infection with the mutant viruses correlated with the amounts of the 39,000- and 38,000-molecular-weight polypeptides synthesized. The 43,000-, 39,000-, and 38,000-molecular-weight polypeptides were shown to be related by their positive reaction with anti-TK serum in both immunoprecipitation and immunoblotting experiments. The production of the 39,000- and 38,000-molecular-weight polypeptides through bypassing of the first AUG codon was examined by hybrid arrest experiments with a DNA fragment complementary to only 50 bases at the 5' terminus of TK mRNA. This fragment arrested the synthesis of the 30,000- and 38,000-molecular-weight polypeptides when annealed to mRNA from wild-type HSV-1- or TK4-infected cells, showing that those polypeptides arise from an mRNA initiated upstream from the first AUG codon. mRNA from cells infected with mutant delta 1, which lacks DNA sequences upstream from the first AUG, was not affected by the 50-base-pair fragment. The data therefore confirm that three polypeptides encoded by the HSV-1 TK gene arise by differential use of in-phase AUG codons for the initiation of protein synthesis. This mechanism for the production of related but distinct polypeptides has not previously been demonstrated in a eucaryotic system, and the implications for the regulation of TK enzyme activities are discussed.

Cell Line↗

PubMed2001

Effects of mutations within the herpes simplex virus type 1 DNA encapsidation signal on packaging efficiency.

The cis-acting signals required for cleavage and encapsidation of the herpes simplex virus type 1 genome lie within the terminally redundant region or a sequence. The a sequence is flanked by short direct repeats (DR1) containing the site of cleavage, and quasi-unique regions, Uc and Ub, occupy positions adjacent to the genomic L and S termini, respectively, such that a novel fragment, Uc-DR1-Ub, is generated upon ligation of the genomic ends. The Uc-DR1-Ub fragment can function as a minimal packaging signal, and motifs have been identified within Uc and Ub that are conserved near the ends of other herpesvirus genomes (pac2 and pac1, respectively). We have introduced deletion and substitution mutations within the pac regions of the Uc-DR1-Ub fragment and assessed their effects on DNA packaging in an amplicon-based transient transfection assay. Within pac2, mutations affecting the T tract had the greatest inhibitory effect, but deletion of sequences on either side of this element also reduced packaging, suggesting that its position relative to other sequences within the Uc-DR1-Ub fragment is likely to be important. No single region essential for DNA packaging was detected within pac1. However, mutants lacking the G tracts on either side of the pac1 T-rich motif exhibited a reduced efficiency of serial propagation, and alteration of the sequences between DR1 and the pac1 T element also resulted in defective generation of Ub-containing terminal fragments. The data are consistent with a model in which initiation and termination of packaging are specified by sequences within Uc and Ub, respectively.

Animals↗

PubMed1990

Nuclease activity associated with mammalian mRNA in its native state: possible basis for selectivity in mRNA decay.

Polysome and messenger ribonucleoprotein (mRNP) preparations from various mammalian cells contain tightly bound nuclease activity that causes degradation of the mRNA in the preparations. This activity was found to cosediment with all polysome size classes as well as with free mRNPs and to remain associated with the mRNPs released from polysomes by treatment with EDTA. No association with ribosomal subunits was evident. The rates of mRNA degradation were not affected by serial dilution, an indication that enzyme and substrate are tightly associated. beta-Globin mRNA in purified reticulocyte polysomes was cleaved at AU sequences in the 3'-terminal region. Cleavages at the same sites occurred when deproteinized reticulocyte RNA was incubated with mouse sarcoma 180 (S-180) polysomes. The S-180 preparations caused additional cleavages, primarily at UG sequences. A P40 mRNA in S-180 polysomes was cleaved primarily in the 3' noncoding region, but the cleavages in a P21 mRNA were seen in the 5' noncoding region only. Actin mRNA was cleaved in an internal region, yielding large relatively stable 3'- and 5'-terminal fragments. These data suggest the occurrence of highly specific interactions between one or more mRNA-bound nucleases and individual mRNA species.

Animals↗

PubMed2004

Developmentally essential protein flightless I is a nuclear receptor coactivator with actin binding activity.

Hormone-activated nuclear receptors (NR) activate transcription by recruiting multiple coactivator complexes to the promoters of target genes. One important coactivator complex includes a p160 coactivator (e.g., GRIP1, SRC-1, or ACTR) that binds directly to activated NR, the histone acetyltransferase p300 or CBP, and the arginine-specific histone methyltransferase CARM1. We previously demonstrated that the coactivator function of CARM1 depends both on the methyltransferase activity and on additional unknown proteins that bind to CARM1. In this study a yeast two-hybrid screen for proteins that bind CARM1 identified the protein Flightless I (Fli-I), which has essential roles in Drosophila and mouse development. Fli-I bound to CARM1, GRIP1, and NRs and cooperated synergistically with CARM1 and GRIP1 to enhance NR function. Fli-I bound poorly to and did not cooperate with PRMT1, a CARM1-related protein arginine methyltransferase that also functions as an NR coactivator. The synergy between GRIP1, CARM1, and Fli-I required the methyltransferase activity of CARM1. The C-terminal AD1 (binding site for p300/CBP) and AD2 (binding site for CARM1) activation domains of GRIP1 contributed to the synergy but were less stringently required than the N-terminal region of GRIP1, which is the binding site for Fli-I. Endogenous Fli-I was recruited to the estrogen-regulated pS2 gene promoter of MCF-7 cells in response to the hormone, and reduction of endogenous Fli-I levels by small interfering RNA reduced hormone-stimulated gene expression by the endogenous estrogen receptor. A fragment of Fli-I that is related to the actin binding protein gelsolin enhanced estrogen receptor activity, and mutations that reduced actin binding also reduced the coactivator function of this Fli-I fragment. These data suggest that Fli-I may facilitate interaction of the p160 coactivator complex with other coactivators or coactivator complexes containing actin or actin-like proteins.

Actins↗

PubMed2004

Monocrotaline pyrrole-induced endothelial cell megalocytosis involves a Golgi blockade mechanism.

Pyrrolizidine alkaloids initiate disease in the lung (pulmonary hypertension), liver (veno-occlusive disease and cirrhosis), and kidneys (afferent arteriolar block and mesangiolysis) by inducing a megalocytotic phenotype in target endothelial and parenchymal cells. A "hit-and-run" type of exposure to the bioactive pyrrolizidine results, within 2-3 days, in enlarged cells with large nuclei and enlarged Golgi and endoplasmic reticulum, while the cells remain in G2/M block. In the present study, we recapitulated monocrotaline pyrrole (MCTP)-induced megalocytosis in cultures of bovine pulmonary arterial endothelial cells (PAEC), human Hep3B hepatocytes, human type II-like alveolar epithelial cells (A549), and human pulmonary arterial smooth muscle cells (PASMC) and investigated the subcellular mechanism involved. There was an inverse relationship between reduction in caveolin (Cav)-1 levels and stimulation of promitogenic STAT3 and ERK1/2 cell signaling. In megalocytotic PAEC, the Golgi scaffolding protein GM130 was shifted from membranes with heavy density to those with a lighter density. This lighter Golgi fraction was enriched for hypo-oligomeric Cav-1, indicating dysfunctional trafficking of cargo. Immunofluorescence imaging studies confirmed the trapping of Cav-1 in a GM130-positive Golgi compartment. There was an increase in Ser25 phosphorylation of GM130 (typically a prelude to Golgi fragmentation and mitosis) and increased association between pGM130, cdc2 kinase, and Cav-1. Nevertheless, megalocytotic MCTP-treated cells showed reduced entry into mitosis upon stimulation with 2-methoxyestradiol (2-ME), reduced 2-ME-induced Golgi fragmentation, and a slowing of Golgi reassembly after nocodazole-induced fragmentation. These data suggest that a disruption of the trafficking and mitosis sensor functions of the Golgi may represent the subcellular mechanism leading to MCTP-induced megalocytosis ("the Golgi blockade hypothesis").

Animals↗

PubMed2004

Models of chromosome aberration induction: an example based on radiation track structure.

A few examples of models of chromosome aberration induction are summarised and discussed on the basis of the three main theories of aberration formation, that is "breakage-and-reunion", "exchange" and "one-hit". A model and code developed at the Universities of Milan and Pavia is then presented in detail. The model provides dose-response curves for different aberration types (dicentrics, translocations, rings, complex exchanges and deletions) induced in human lymphocytes by gamma rays, protons and alpha particles of different energies, both as monochromatic fields and as mixed fields. The main assumptions are that only clustered - and thus severe - DNA breaks ("Complex Lesions", CL) can participate in the production of aberrations, and that only break free ends in neighbouring chromosome territories can interact and form exchanges. The yields of CLs induced by the various radiation types of interest are taken from a previous modelling work. These lesions are distributed within a sphere representing the cell nucleus according to the radiation track structure, e.g. randomly for gamma rays and along straight lines for light ions. Interphase chromosome territories are explicitly simulated and configurations are obtained in which each chromosome occupies an intranuclear domain with volume proportional to its DNA content. In order to allow direct comparisons with experimental data, small fragments can be neglected since usually they cannot be detected in experiments. The presence of a background level of aberrations is also taken into account. The results of the simulations are in good agreement with experimental dose-response curves available in the literature, that provides a validation of the model both in terms of the adopted assumptions and in terms of the simulation techniques. To address the question of "true" incompleteness, simulations were also run in which all fragments were assumed to be visible.

Animals↗

PubMed2004

Renin-angiotensin system gene polymorphisms: its impact on IgAN and its progression to end-stage renal failure among Chinese in Singapore.

BACKGROUND: Gene polymorphisms in angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II type 1 receptor (ATR) had been associated with IgA nephropathy (IgAN) and its progression. Several studies on Caucasian and Japanese had reported contradicting results. We determined these polymorphisms in 118 Chinese patients with IgAN and 94 healthy Chinese to assess their clinical impact. METHODS: Genotyping was performed with DNA from peripheral leukocytes, PCR amplification of the polymorphic sequence, restriction enzymes digestion, separation and identification of DNA fragments. Clinical data at renal biopsy and final status on renal function were determined from patients' records. RESULTS: Among controls, genotype distributions were in Hardy-Weinberg equilibrium. Comparing all IgAN patients with controls, AGT and ATR genotype distributions were similar whereas there was significant increase in the ACE DD genotype (p < 0.05). Comparing patients with end-stage renal failure (IgAN-ESRF) and without (IgAN-nonESRF), there was no difference in any of the three gene polymorphisms. But in contrast, there were significant differences in higher male prevalence (p < 0.05), increased serum creatinine at presentation (p < 0.05), more sclerosis (p < 0.01) and higher tubulointerstitial lesion score (p < 0.001) in the IgAN-ESRF group. CONCLUSION: Among the ACE, AGT and ATR gene polymorphisms, only the DD genotype may predispose the individual to IgAN in our Chinese population. In contrast to clinical and histological risk factors, these genetic variations showed no impact on disease progression to ESRF. It is unlikely that genotyping more patients will prove these genes useful. Nevertheless, preclinically determined genetic markers are very useful as risk factors for disease occurrence and as prognostic indices for disease progression. Therefore, continuing efforts should be made to look at other genes to find those with significance.

Adult↗

PubMed1997

Fas is expressed in human atherosclerotic intima and promotes apoptosis of cytokine-primed human vascular smooth muscle cells.

The membrane protein Fas/Apo-1/CD95 signals programmed cell death or apoptosis in activated T lymphocytes. Vascular smooth muscle cells (SMCs) bear markers of programmed cell death or apoptosis in advanced atherosclerotic plaques that contain immune cells e.g., macrophages and T lymphocytes. This study tested the hypothesis that the Fas death-signaling pathway contributes to apoptosis of SMCs exposed to proinflammatory cytokines produced by these immune cells during atherogenesis. All atherosclerotic plaques examined (n = 14) contained immunoreactive Fas. The majority of the Fas+ SMCs localized in the intima of the plaques, whereas the medial SMCs expressed Fas antigen less prominently. Double staining for DNA fragments (TUNEL) and Fas or cell identification markers colocalized Fas with TUNEL+ SMCs in the areas that contained CD3+ T cells and CD68+ macrophages, suggesting a role for Fas in the induction of SMC apoptosis by activated T cells during atherogenesis. In culture, stimulation with interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta increased expression of Fas in SMCs. Incubation with an activating anti-Fas antibody triggered apoptosis of the cytokine-primed but not the untreated SMCs, as demonstrated by TUNEL and electrophoresis of oligonucleosomal DNA fragments. These data suggest that activation of the Fas death-signaling pathway contributes to the induction of SMC apoptosis during atherogenesis and furnish a mechanism whereby immune cells and their cytokines promote this cell death process related to vascular remodeling and plaque rupture.

Apoptosis↗

PubMed1999

Apoptosis during regression of cardiac hypertrophy in spontaneously hypertensive rats. Temporal regulation and spatial heterogeneity.

We previously reported that increased apoptosis participates in the regression of aortic hypertrophy in spontaneously hypertensive rats. To further document the potential role of apoptosis in cardiovascular therapy, we examined apoptosis during regression of hypertrophy in the heart of spontaneously hypertensive rats receiving the antihypertensive drug enalapril (30 mg. kg(-1). d(-1)), losartan (30 mg. kg(-1). d(-1)), nifedipine (35 mg. kg(-1). d(-1)), hydralazine (40 mg. kg(-1). d(-1)), propranolol (50 mg. kg(-1). d(-1)), or hydrochlorothiazide (75 mg. kg(-1). d(-1)) for 1 to 4 weeks, starting at 10 to 11 weeks of age. Systolic blood pressure and heart rate were measured by the tail-cuff method. Markers of apoptosis included oligonucleosomal DNA fragmentation in extracted cardiac DNA or in situ in ventricular cross sections labeled with terminal deoxynucleotidyl transferase. Cardiac DNA synthesis was evaluated by [(3)H]-thymidine incorporation in vivo. All drugs reduced cardiac workload, defined as the product of blood pressure and heart rate, by >20% at 4 weeks. However, only nifedipine, enalapril, losartan, and propranolol reduced cardiac mass (>19%) within 4 weeks. Regression of cardiac hypertrophy was accompanied by a 50% to 300% increase in DNA fragmentation and a >20% reduction in DNA synthesis, resulting in a >20% reduction in cardiac DNA content after 4 weeks. Apoptosis induction occurred early and was transient within 4 weeks of nifedipine, enalapril, or losartan administration. With all regression-inducing drugs, the increase in DNA fragmentation occurred mainly in the subepicardium. Thus, transient induction of apoptosis in the subepicardium appears to be a characteristic feature of the early response to drug-induced regression of cardiac hypertrophy in spontaneously hypertensive rats.

Adrenergic beta-Antagonists↗

PubMed2001

Phenotypic alteration of vascular smooth muscle cells precedes elastolysis in a mouse model of Marfan syndrome.

Marfan syndrome is associated with early death due to aortic aneurysm. The condition is caused by mutations in the gene (FBN1) encoding fibrillin-1, a major constituent of extracellular microfibrils. Prior observations suggested that a deficiency of microfibrils causes failure of elastic fiber assembly during late fetal development. Mice homozygous for a targeted hypomorphic allele (mgR) of Fbn1 revealed a predictable sequence of abnormalities in the vessel wall including elastic fiber calcification, excessive deposition of matrix elements, elastolysis, and intimal hyperplasia. Here we describe previously unrecognized concordant findings in elastic vessels from patients with Marfan syndrome. Furthermore, ultrastructural analysis of mgR mice revealed cellular events that initiate destructive changes. The first detectable abnormality was an unusually smooth surface of elastic laminae, manifesting the loss of cell attachments that are normally mediated by fibrillin-1. Adjacent cells adopted alteration in their expression profile accompanied by morphological changes but retained expression of vascular smooth muscle cell markers. The abnormal synthetic repertoire of these morphologically abnormal smooth muscle cells in early vascular lesions included elastin, among other matrix elements, and matrix metalloproteinase 9, a known mediator of elastolysis. Ultimately, cell processes associated with zones of elastic fiber thinning and fragmentation. These data suggest that the loss of cell attachments signals a nonproductive program to synthesize and remodel an elastic matrix. This refined understanding of the pathogenesis of vascular disease in Marfan syndrome will facilitate the development of therapeutic strategies.

Actins↗

PubMed2001

Cancer gene therapy using a survivin mutant adenovirus.

We have constructed a replication-deficient adenovirus encoding a nonphosphorylatable Thr(34)-->Ala mutant of the apoptosis inhibitor survivin (pAd-T34A) to target tumor cell viability in vitro and in vivo. Infection with pAd-T34A caused spontaneous apoptosis in cell lines of breast, cervical, prostate, lung, and colorectal cancer. In contrast, pAd-T34A did not affect cell viability of proliferating normal human cells, including fibroblasts, endothelium, or smooth muscle cells. Infection of tumor cells with pAd-T34A resulted in cytochrome c release from mitochondria, cleavage of approximately 46-kDa upstream caspase-9, processing of caspase-3 to the active subunits of approximately 17 and 19 kDa, and increased caspase-3 catalytic activity. When compared with chemotherapeutic regimens, pAd-T34A was as effective as taxol and considerably more effective than adriamycin in induction of tumor cell apoptosis and enhanced taxol-induced cell death. In three xenograft breast cancer models in immunodeficient mice, pAd-T34A suppressed de novo tumor formation, inhibited by approximately 40% the growth of established tumors, and reduced intraperitoneal tumor dissemination. Tumors injected with pAd-T34A exhibited loss of proliferating cells and massive apoptosis by in situ internucleosomal DNA fragmentation. These data suggest that adenoviral targeting of the survivin pathway may provide a novel approach for selective cancer gene therapy.

Adenoviruses, Human↗

PubMed2001

Cell surface tissue transglutaminase is involved in adhesion and migration of monocytic cells on fibronectin.

Expression of tissue transglutaminase (transglutaminase II, tTG) was shown to increase drastically during monocyte differentiation into macrophages; however, its role in monocytic cells remains largely unknown. This study describes a novel function of cell surface tTG as an adhesion and migration receptor for fibronectin (Fn). Two structurally related transglutaminases, tTG and the A subunit of factor XIII (FXIIIA), are expressed on the surface of monocytic cells, whereas only surface tTG is associated with multiple integrins of the beta1 and beta3 subfamilies. Both surface levels of tTG and the amounts of integrin-bound tTG are sharply up-regulated during the conversion of monocytes into macrophages. In contrast, a reduction in biosynthesis and surface expression of FXIIIA accompanies monocyte differentiation. Cell surface tTG is colocalized with beta1- and beta3-integrins in podosomelike adhesive structures of macrophages adherent on Fn. Down-regulation of surface tTG by expression of antisense tTG construct or its inhibition by function-blocking antibodies significantly decreases adhesion and spreading of monocytic cells on Fn and, in particular, on the gelatin-binding fragment of Fn consisting of modules I6II1,2I7-9. Likewise, interfering with the adhesive function of surface tTG markedly reduces migration of myeloid cells on Fn and its gelatin-binding fragment. These data demonstrate that cell surface tTG serves as an integrin-associated adhesion receptor that might be involved in extravasation and migration of monocytic cells into tissues containing Fn matrices during inflammation.

Antigens, CD↗

PubMed2005

Herpes simplex virus interferes with amyloid precursor protein processing.

BACKGROUND: The early events underlying Alzheimer's disease (AD) remain uncertain, although environmental factors may be involved. Work in this laboratory has shown that the combination of herpes simplex virus type 1 (HSV1) in brain and carriage of the APOE-epsilon4 allele of the APOE gene strongly increases the risk of developing AD. The development of AD is thought to involve abnormal aggregation or deposition of a 39-43 amino acid protein--beta amyloid (Abeta)--within the brain. This is cleaved from the much larger transmembranal protein 'amyloid precursor protein' (APP). Any agent able to interfere directly with Abeta or APP metabolism may therefore have the capacity to contribute towards AD. One recent report showed that certain HSV1 glycoprotein peptides may aggregate like Abeta; a second study described a role for APP in transport of virus in squid axons. However to date the effects of acute herpesvirus infection on metabolism of APP in human neuronal-type cells have not been investigated. In order to find if HSV1 directly affects APP and its degradation, we have examined this protein from human neuroblastoma cells (normal and transfected with APP 695) infected with the virus, using Western blotting. RESULTS: We have found that acute HSV1 (and also HSV2) infection rapidly reduces full length APP levels--as might be expected--yet surprisingly markedly increases levels of a novel C-terminal fragment of APP of about 55 kDa. This band was not increased in cells treated with the protein synthesis inhibitor cycloheximide CONCLUSION: Herpes virus infection leads to rapid loss of full length APP from cells, yet also causes increased levels of a novel 55 kDa C-terminal APP fragment. These data suggest that infection can directly alter the processing of a transmembranal protein intimately linked to the aetiology of AD.

Alzheimer Disease↗

PubMed1983

Role of calcium in the stimulus-secretion coupling of antral gastrin release.

The role of calcium in the stimulus-secretion coupling of antral gastrin release was examined in isolated sheets of canine antral mucosa. Mucosa was obtained from 137 dogs and mounted in Ussing chambers to separate the luminal from nutrient surfaces. The influence of verapamil, LaCl3, A23187, and EGTA on the release of gastrin by luminal calcium and ethyl alcohol was examined and the release of immunoreactive gastrin (IG) was measured in luminal perfusates. Release of IG by luminal calcium was dose-related, unsaturable, and impaired by verapamil and by LaCl3. Release of IG by alcohol was prevented by leaching the mucosa of calcium and restored by repletion of the calcium. IG release induced by alcohol was prevented by topical, but not by nutrient, application of LaCl3. Molecular sieve and affinity chromatography of the endogenous IG released in the absence of luminal calcium, and of the exogenous gastrin added, indicated that antral gastrin was released as heptadecapeptide gastrin, and that which was released in the presence of CaCl2 degraded rapidly into a C-terminal fragment. The data indicate that calcium may participate in the stimulus-secretion coupling of canine antral gastrin release in vitro.

Animals↗

PubMed1999

Leydig cell apoptosis after the administration of ethane dimethanesulfonate to the adult male rat is a Fas-mediated process.

Leydig cells undergo apoptosis in response to the cytotoxin ethane dimethanesulfonate (EDS), with numbers declining at 12-18 h and maximal apoptosis at 24 h postinjection. The Bcl-2 family members, Bcl-2, Bcl-xl, and Bax, appear not to be involved in this process. To further investigate this phenomena, a single dose of EDS was administered to adult rats to induce the killing of Leydig cells. The interstitial cells were examined up to 3 days after EDS administration by Western blot analysis for the Bcl-2 family members (Bak and Bcl-w). Western blotting showed that Bak expression in the interstitial cell preparations was unchanged after EDS, and immunohistochemistry showed that it was not up-regulated in Leydig cells in response to EDS. Bcl-w expression in the Leydig cells and interstitial cell preparations was unchanged until 48 h when it became undetectable, suggesting that Leydig cell-associated Bcl-w is not involved in initiating apoptosis. We also investigated the role of the Fas system in Leydig cell apoptosis. Both Fas receptor and Fas ligand protein levels increased after EDS, peaking at 12-18 h and declining thereafter. Fas receptor and ligand were shown by immunohistochemistry to be present in Leydig cells, and after EDS all Leydig cells became strongly positive for both proteins. The intensity of staining increased in the early stages of apoptosis and decreased as the nuclear morphology became more fragmented. These data suggest that Bcl-2 family members are not involved in Leydig cell apoptosis after EDS administration. However, up-regulation of the Fas system does occur, implicating activation of Fas receptor in the induction of Leydig cell apoptosis.

Animals↗

PubMed1997

Suppression of growth hormone (GH) hypersecretion due to ectopic GH-releasing hormone (GHRH) by a selective GHRH antagonist.

We have recently demonstrated that a competitive antagonist of GHRH, (N-Ac-Tyr1,D-Arg2)GHRH-(1-29)NH2 (GHRH-Ant), eliminates nearly all nocturnal GH pulsatility in normal subjects, supporting the hypothesis that GH pulsatility is driven by GHRH. In this study, we compared the effects of every 12 h i.v. boluses of either GHRH-Ant or saline on 24-h GH profiles in a patient with acromegaly due to a metastatic GHRH-secreting carcinoid tumor. Bolus doses of GHRH-Ant (400 micrograms/kg, i.v.) acutely suppressed GH concentration to 30-40% of the pretreatment baseline, and this effect lasted 3-4 h. Administration of GHRH (0.33 microgram/kg, i.v.) bolus resulted in a small rise in GH, and this effect was blocked by GHRH-Ant (400 micrograms/kg). During saline treatment, the secretory patterns of both GH and ectopic GHRH were pulsatile; however, there was no correlation between changes in plasma GHRH and GH concentrations. This lack of correlation was probably due to the majority of circulating GHRH immunoreactivity consisting of nonbiologically active GHRH fragments. These data support the hypothesis that GH hypersecretion in the ectopic GHRH syndrome requires GHRH receptor occupancy and validates the use of GHRH-Ant to probe the potential involvement of endogenous GHRH in patients with acromegaly due to pituitary somatotropinoma.

Acromegaly↗

PubMed2005

Shotgun sequencing small oligonucleotides by nozzle-skimmer dissociation and electrospray ionization mass spectrometry.

Nozzle-skimmer dissociation in combination with de novo sequencing was investigated as an approach for increasing the throughput of oligonucleotide analysis attainable by electrospray ionization mass spectrometry. An experimental method allowing for the sequential generation of precursor and fragment ion data during direct infusion of sample was developed. These data can then be used with readily available de novo sequencing software to characterize small oligonucleotides. When this approach was applied to mixtures of oligonucleotides, it was found that de novo sequencing becomes limited due to spectral congestion and overlapping oligonucleotide m/z dissociation product values. Self-packed C(18) microspray emitters were investigated as a means of reducing spectral complexity. It was found that such emitters allow for the analysis of oligonucleotide mixtures with minimal component overlap, and these emitters provide additional benefits of pre- concentrating and desalting the sample. These developments can provide a route for the more rapid characterization of ribonucleic acid endonuclease digestion mixtures.

Base Sequence↗