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C Bergmann

Publications and source records attributed to C Bergmann.

At least 37 records · Page 2Linked to original sources
PubMed2000

Nitric oxide synthase inhibition promotes carcinogen-induced preneoplastic changes in the colon of rats.

l-Arginine is metabolized either to polyamines through arginase and ornithine decarboxylase (ODC) activities or to citrulline and nitric oxide (NO, nitrogen monoxide) through the NO synthase (NOS) pathway. Polyamine levels and ODC activity are high in tumor cells. The aim of this study was to test whether N(G)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NOS, modulates colon carcinogenesis. Adult male Wistar rats were treated with azoxymethane (AOM, 15 mg/kg ip), a chemical carcinogen, once a week for 2 weeks. One week after the second injection the rats were randomly divided into two groups. One group (n = 8) received l-NAME (10 mg/kg body wt/day) in drinking water. The control group (n = 8) received tap water. After 5 weeks, the rats receiving l-NAME showed enhanced mean basal arterial blood pressure, decreased heart rate, and a significant decrease of the cGMP content in the colonic mucosa. In both groups, AOM induced the formation of colonic aberrant crypt foci (ACF). In l-NAME-treated rats, the number of ACF was higher than in controls by 47%. ODC activity was enhanced by 11-fold. S-Adenosyl-methionine-decarboxylase activity and putrescine concentration were significantly increased in the colonic mucosa of l-NAME-treated rats. The data suggest that l-NAME promotes carcinogen-induced preneoplastic changes in the colon by inhibiting NOS activity and by stimulating polyamine biosynthesis.

Adenosylmethionine Decarboxylase↗
PubMed2000

Charcot-Marie-Tooth neuropathy type 2 and P0 point mutations: two novel amino acid substitutions (Asp61Gly; Tyr119Cys) and a possible "hotspot" on Thr124Met.

Mutations in the gene for the major protein component of peripheral nerve myelin, myelin protein zero (MPZ, P0), cause hereditary disorders of Schwann cell myelin such as Charcot-Marie-Tooth neuropathy type 1B (CMT1B), Dejerine-Sottas syndrome (DSS), and congenital hypomyelinating neuropathy (CHN). More recently, P0 mutations were identified in the axonal type of CMT neuropathy, CMT2, which is different from the demyelinating variants with respect to electroneurography and nerve pathology. We screened 49 patients with a clinical and histopathological diagnosis of CMT2 for mutations in the P0 gene. Three heterozygous single nucleotide changes were detected: two novel missense mutations, Asp61Gly and Tyr119Cys, and the known Thr124Met substitution, that has already been reported in several CMT patients from different European countries. Haplotype analysis for the P0 locus proved that our patients with the 124Met allele were not related to a cohort of patients with the same mutation, all of Belgian descent and all found to share a common ancestor. Our data suggest that P0 mutations account for a detectable proportion of CMT2 cases with virtually every patient harbouring a different mutation but recurrence of the Thr124Met amino acid substitution. The high frequency of this peculiar genotype in the European CMT population is presumably not only due to a founder effect but Thr124Met might constitute a mutation hotspot in the P0 gene as well.

Adult↗
PubMed1999

Selection of CTL escape mutants in mice infected with a neurotropic coronavirus: quantitative estimate of TCR diversity in the infected central nervous system.

Variant viruses mutated in the immunodominant cytotoxic T cell epitope surface (S) glycoprotein S-510-518 are selected in mice chronically infected with mouse hepatitis virus, strain JHM. We determined whether this selection occurred in the presence of an oligoclonal or polyclonal T cell response using soluble MHC/peptide tetramers in direct ex vivo analyses of CNS-derived lymphocytes. A total of 42% (range, 29-60%) of CD8 T cells in the CNS of mice with acute encephalitis recognized epitope S-510-518. A total of 34% (range, 18-62%) of cells from mice with hind limb paralysis (and chronic demyelination) were also epitope specific, even though only virus expressing mutated epitope is detected in these animals. Sequence analysis of the beta-chain CDR3 of 487 tetramer S-510-518-positive cDNA clones from nine mice showed that a majority of clonotypes were identified in more than one mouse. From these analyses, we estimated that 300-500 different CD8 T cell clonotypes responsive to epitope S-510-518 were present in each acutely infected brain, while 100-900 were present in the CNS of each mouse with chronic disease. In conclusion, a polyclonal CD8 T cell response to an epitope does not preclude the selection of T cell escape mutants, and epitope-specific T cells are still present at high levels even after RNA-encoding wild-type sequence is no longer detectable.

Acute Disease↗
PubMed1999

X-linked dominant Charcot-Marie-Tooth neuropathy: clinical, electrophysiological, and morphological phenotype in four families with different connexin32 mutations(1).

The sensorimotor neuropathy of the Charcot-Marie-Tooth type (CMT) is the most common hereditary disorder of the peripheral nervous system. The X-linked dominant form of CMT (CMTX) is associated with mutations in the gene for the gap junction protein connexin32. We examined four CMTX pedigrees two of which had potentially novel mutations in the only coding exon of connexin32. One previously unreported missense mutation, Ala39Val, was found in a family displaying a CMT phenotype with additional upper limb postural tremor reminiscent of a Roussy-Lévy syndrome. A novel single base insertion, 679insT, is among the first mutations found in the fourth transmembrane domain of connexin32. Frameshift and premature stop of translation are supposed to result in a non-functional carboxy-terminus. Two further families had the known missense mutations Arg15Trp and Arg22Gln. Several female carriers were found normal on clinical presentation, however, the genotype was paralleled by decreased nerve conduction velocities (NCV) and slowed central conduction of brain stem auditory evoked responses (BAER). Median motor NCVs showed mild (in women) to intermediate (in males) reduction, indicating a peripheral neuropathy with a predominating axonal component. Nerve biopsy findings were consistent with the electrophysiological data showing a marked loss of large myelinated fibres and clusters of regenerating axons. Electron microscopy revealed various alterations of the axoglial attachment zone. This suggests defective axon-Schwann cell interactions which may induce the axonopathy in CMTX.

Adolescent↗
PubMed1999

Characterization of the glycosylation of recombinant endopolygalacturonase I from Aspergillus niger.

The carbohydrate chains of recombinant endopolygalacturonase I (EPG I) from Aspergillus niger were characterized using a combination of mass spectrometric techniques. High performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry was used to separate the components of EPG I liberated by trypsin digestion. In-source collision-induced dissociation (CID) was utilized to fragment the digestion products entering the mass spectrometer, and the generation of carbohydrate fragment ions allowed for the identification of glycopeptides. The masses of the resulting glycans were calculated and entered into a carbohydrate database to search for possible structures. The primary sequences of the carbohydrate chains were confirmed by digesting aliquots of the intact glycopeptide with endo- and exoglycosidases and then analyzing the digestion products using matrix-assisted laser desorption/ionization mass spectrometry. These experiments demonstrated that one of the two N-linked sites of EPG I was occupied by a series of high-mannose structures, the second N-linked site was not occupied, and no O-linked sites were detected.

Amino Acid Sequence↗
PubMed1999

Characterization of the N-linked glycosylation site of recombinant pectate lyase.

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.

Aspergillus nidulans↗
PubMed1999

Promotion of intestinal carcinogenesis by dietary methionine.

The metabolism of the polyamines spermidine and spermine is known to be enhanced in rapidly proliferating cells. Methionine is a precursor of the aminopropyl moieties of these amines. Therefore, it was of interest to study the effects of a methionine supplemented diet on polyamine metabolism and preneoplastic changes occurring in the intestinal tract of rats treated with the chemical carcinogen azoxymethane (AOM). Adult Wistar rats received 15 mg AOM/kg body wt (i.p.) once each week for 2 weeks. Thereafter, the rats were randomly divided into two groups and received controlled isoenergetic diets containing the same amount of folate, choline and vitamin B12 during 12 weeks: one group was kept on a standard diet; the other was fed the same diet, except that 1% L-methionine was added at the expense of carbohydrates. After 12 weeks, the administration of the methionine-supplemented diet stimulated the turnover rate of ileal epithelial cells, indicating enhanced crypt cell proliferation. Furthermore, in this group, a 2-fold increase in the number of aberrant hyperproliferative crypts and the appearance of tumors was observed in the colon. These effects were accompanied by the increased formation of spermidine and spermine due to the enhancement of S-adenosylmethionine decarboxylase activity and by the upregulation of Cdx-1, a homeobox gene with oncogenic potentials. The experimental data do not support the view of a chemopreventive effect of dietary methionine supplementation on intestinal carcinogenesis in rats, even at an early phase of preneoplastic development, but rather suggest that methionine promotes intestinal carcinogenesis.

Animals↗
PubMed1998

Concomitant changes in polyamine pools and DNA methylation during growth inhibition of human colonic cancer cells.

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, of S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine-DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.

Adenosylmethionine Decarboxylase↗
PubMed1998

Variability of persisting MHV RNA sequences constituting immune and replication-relevant domains.

Survivors of acute infection with the neurotropic JHM strain of mouse hepatitis virus develop a persistent infection of the central nervous system associated with chronic ongoing demyelination. Persistence is characterized by viral RNA in the absence of infectious virus. To associate persistence with possible immune evasion and/or replication defects, viral RNA from brains of acutely and persistently infected mice was examined for mutations by reverse transcriptase-PCR. Sequences analyzed included the encapsidation sequence (ECS), the transmembrane domains of the matrix (M) protein, and a cytotoxic T cell (CTL) epitope within the nucleocapsid (N) protein. The ECS, present only on genomic RNA, revealed minimal variability and was detected out to 120 days postinfection, suggesting low levels of replication. The M gene sequence also remained stable during persistence despite random mutations during the acute phase. Although the N gene sequence exhibited the greatest diversity, mutations were random and not selected for during persistence. A single exception was detected comprising a prominent Pro to Ser substitution in a region of N not associated with any known regulatory or immune function. Of the N gene mutations found within the CTL epitope in responder mice (H-2d), one resulted in reduced CTL recognition with no evidence of antagonist activity. However, this mutation was also detected in nonresponder mice (H-2b), suggesting that escape variants arising from CTL pressure play no role in establishing persistence in immunocompetent hosts infected as adults.

Animals↗
PubMed1998

X-linked dominant Charcot-Marie-Tooth disease: nerve biopsies allow morphological evaluation and detection of connexin32 mutations (Arg15Trp, Arg22Gln).

X-linked Charcot-Marie-Tooth neuropathy (CMTX) is caused by mutations in the connexin32 gene on Xq13. Because of overlapping morphological and clinical data, CMTX patients often meet the criteria of autosomal-dominant CMT2, the neuronal type of CMT. Hence, it might be useful to analyse the connexin32 gene in suspected CMT2 patients when there is no male-to-male transmission. We selected a cohort of 30 patients who were considered having CMT2 on the basis of previous clinical and histopathological evaluation. DNA was extracted from paraffin-embedded sural nerve biopsy samples and screened for connexin32 mutations to verify the possible diagnosis of CMTX. In 2 patients mutations were found corresponding to amino acid substitutions of arginine for tryptophan in codon 15 and arginine for glutamine in codon 22 of connexin32. This study illustrates that archival material allows genetic classification of suspected CMT cases. Furthermore, there is additional proof that connexin32 mutations represent the underlying genetic defect in some cases of predominantly neuronal CMT.

Adolescent↗
PubMed1997

Identification of the glycosylation site and glycan structures of recombinant endopolygalacturonase II by mass spectrometry.

A series of mass spectrometric experiments was performed to characterize the carbohydrate chains attached to endopolygalacturonase II (EPG-II) overexpressed in Aspergillus niger. First, an aliquot of trypsin-digested EPG-II was analyzed by capillary high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass spectrometry (HPLC/MS). The ESI mass spectrometer was operated in the tandem mass spectrometric (MS/MS) mode and utilized precursor ion scanning of the HexNAc+ oxonium ion at m/z 204 to selectively detect glycopeptides eluting from the HPLC. Aliquots of the fraction identified by HPLC/MS/MS to contain a glycopeptide were then sequentially digested with Aspergillus saitoi alpha-1-2-mannosidase and peptide N:glycosidase F (N-glycanase), followed by matrix-assisted laser-desorption/ionization mass spectrometric analysis to provide structural information from the carbohydrate chain. The masses of the carbohydrate chains in the glycopeptides were thus determined and subsequently used to search the Complex Carbohydrate Structure Database for the probable structures of the glycans. This analysis determined that recombinant EPG-II has a single N-glycosylation site, the carbohydrate chain at this site is heterogeneous, and the glycan structure is of the high-mannose type. No sites of O-glycosylation were detected in EPG-II.

Amino Acid Sequence↗
PubMed1995

Inadequate response to H2-receptor antagonists. Absence of parietal cell cAMP-stimulating autoantibodies.

In peptic ulcer patients with adequate (AR; N = 16) and inadequate response (IR; N = 20) to H2-receptor antagonists, the presence of parietal cell cAMP-stimulating autoantibodies was studied. Serum Ig fractions from these patients and 10 control subjects were examined to test whether they could stimulate cAMP production in a gastric cell line model. The human cell line HGT-1 was found to be a sensitive in vitro model for the cAMP stimulation assay as histamine (10 microM) increased by 11-fold the production of cAMP. Neither IgG (4 mg/ml) nor IgG-free Ig fractions (1 mg/ml) isolated from the blood of AR or IR affected cAMP production in the HGT-1 cells. The results obtained with the cAMP stimulation assay were confirmed by indirect immunofluorescence measurements based on frozen sections of rat stomach and kidney. No specific staining of rat parietal cells could be observed with patient sera. In addition, human gastric biopsies of the oxyntic mucosa from the same patients were studied for immunoreactive cell populations to assess organ-specific autoimmune processes. Biopsy specimens from AR and IR showed increased lymphocytic infiltrates, usually associated with gastritis. However, no significant differences in location of various cell populations between AR and IR could be observed. Our findings do not support a recent hypothesis that poor response to treatment with H2-receptor antagonists is due to the presence of autoantibodies.

Animals↗
PubMed1995

The backbone of the pectic polysaccharide rhamnogalacturonan I is cleaved by an endohydrolase and an endolyase.

Rhamnogalacturonan I (RG-I), a major pectic component of the primary walls of plant cells, is believed to play an important role in determining both the structure and functions of the walls. A more detailed structural description of RG-I is likely to lead to a greater understanding of the biological roles of this polysaccharide. Two enzymes secreted by Aspergillus aculeatus that have been cloned and expressed in a fungal system (Kofod et al., J. Biol. Chem., 269, 29182-29189, 1994) cleave the RG-I backbone in an endo fashion and should assist in the further structural characterization of this polysaccharide. We found that both of the available preparations of the cloned enzymes were contaminated with exoglycanases, reducing their utility in structurally characterizing RG-I. We purified the enzymes to apparent homogeneity by ion-exchange chromatography and then used the purified enzymes to generate backbone oligosaccharide fragments from partially debranched sycamore RG-I. The backbone oligosaccharides, which were separated from larger pieces of partially debranched RG-I by gel-permeation chromatography, have been structurally characterized by 1H-NMR spectroscopy, electrospray MS, GC-MS, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and UV spectroscopy. The results of these analyses establish that rhamnogalacturonase A (RGase A) is an endohydrolase that cleaves the -4)-alpha-D-GalpA-(1-2)-alpha-L-Rhap glycosidic linkage. However, the purported rhamnogalacturonase B (RGase B) is, in fact, an endolyase that cleaves the -2)-alpha-L-Rhap-(1-4)-alpha-D-GalpA glycosidic linkage, thereby generating oligosaccharides terminating at the non-reducing end with a hex-4-enopyranosyluronic acid residue.

Carbohydrate Sequence↗
PubMed1994

Location of antibody epitopes within the mouse hepatitis virus nucleocapsid protein.

Thirteen monoclonal antibodies (Mab) specific for the nucleocapsid (N) protein of mouse hepatitis virus were mapped using a panel of carboxy-terminal N protein truncations expressed by recombinant vaccinia viruses. All of the Mab recognized both native protein and full-length N protein expressed in this vector by both Western blot and enzyme-linked immunoabsorbent assays (ELISA), indicating that they recognized linear epitopes. The results obtained by both Western blot and ELISA for binding to the truncated N proteins coincide for seven of the Mab tested. The linear epitopes recognized localize to four domains dispersed between amino acids 171 and 196, 231 and 277, and 374 and 455. The epitopes for six Mab were localized to domains comprising 29 amino acids or less as determined by ELISA. Seven Mab showed different reactivity patterns in Western blot versus ELISA, suggesting binding may be influenced by local conformation. Therefore, the fine specificity of these Mab could not be determined with certainty. These data represent the first determination of antibody binding domains within the mouse hepatitis virus N protein which forms the viral helical nucleocapsids and appears to perform a number of regulatory functions during virus replication.

Animals↗
PubMed1994

Ions, parathyroids, and genetic hypertension.

Several reports suggested an involvement of parathyroid function in blood pressure regulation in animals and humans: hyperparathyroid subjects frequently display an elevated systolic blood pressure and young mild hypertensive patients show enhanced serum PTH levels. Moreover, removal of parathyroid glands (PTX) in young rats attenuates and delays the development of mineralocorticoid and genetic hypertension. In addition, in vivo cardiovascular reactivity to norepinephrine in PTX rats from both spontaneously the hypertensive rat (SHR) and Lyon hypertensive rat (LH) strains is decreased, as is calcium content in aortic and heart fragments. Moreover, parathyroid grafts from SHR, stroke-prone SHR (SHR-SP), LH, or Milan hypertensive rats (MHS) into previously parathyroidectomized normotensive recipient rats have been shown to induce an increase in blood pressure. Recently, in essential hypertensive patients and in SHR, a circulating hypertensive factor has also been described. Produced by the PTX in SHR, this factor is inversely related to the amount of dietary calcium. It appears, therefore, that the PTX plays a major role in experimental and probably also in human hypertension.

Animals↗
PubMed1993

An endogenously synthesized decamer peptide efficiently primes cytotoxic T cells specific for the HIV-1 envelope glycoprotein.

The immunodominant H-2Dd-restricted cytotoxic T lymphocyte (CTL) response to the HIV-1 gp160 envelope glycoprotein maps to a single determinant in the V3 loop, designated p18. Using a series of peptides synthesized on pins we have determined that the minimal core sequence of this determinant required for CTL recognition comprises 8 amino acids (residues 320-327). However, 9mer and 10mer peptides containing this core sequence were more effective than the 8mer peptide at sensitizing Dd-expressing target cells. To analyze the antigenicity of endogenously synthesized p18, minigenes encoding a 10-amino acid determinant (residues 318-327) and a 67-amino acid peptide (residues 281-348; containing the V3 loop) were expressed using vaccinia virus (Vac) recombinants. Both peptides were as effective as wild-type gp160 in their ability to sensitize target cells for lysis by gp160-specific CTL. Immunization of BALB/c mice with Vac recombinants encoding both gp160 peptides elicited gp160-specific CTL. These data demonstrate that both the V3 loop itself and a 10-residue epitope are sufficient to prime CTL in vivo and strongly support the potential use of minigene-encoded CTL epitopes for recombinant vaccines designed to induce protective T cell-mediated immunity against HIV-1.

AIDS Vaccines↗