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Biomedical subjects

G Kramer

Publications and source records attributed to G Kramer.

At least 55 records · Page 3Linked to original sources
PubMed1996

Tissue polypeptide-specific antigen in renal cell carcinoma.

OBJECTIVES: The usefulness of serum tissue polypeptide-specific antigen (TPS), a cytokeratin 18-associated marker, in renal cell carcinoma (RCC) was assessed in vitro and in vivo. METHODS: Indirect immunoperoxidase staining for TPS expression was performed on frozen sections of normal renal tissue and RCC specimens. By using a monoclonal TPS immunoradiometric assay, serum TPS concentrations were analyzed in 82 healthy controls, in 20 patients with locoregional RCC before and after surgery and in 18 patients with advanced disease following surgery receiving immunotherapy with interferon-gamma. RESULTS: Using immunohistochemistry, TPS was found to be expressed by both normal and cancerous renal epithelial cells. The mean TPS concentrations in 82 healthy controls was 56 +/- 49 U/1 with a 95% percentile of 78.5 U/1. Out of 20 patients with locoregional RCC, 8 presented with elevated values (mean 168 +/- 82 U/1) above the cut-off level (78.5 U/1, sensitivity 40%) which dropped to normal within 2 weeks after surgery. During a follow-up period of 1 year, none of the patients presented with tumor recurrence and TPS concentrations remained low (mean 52 +/- 36 U/1). In 18 patients receiving interferon-gamma therapy, serum TPS concentrations were monitored over a period of 12 months. In 5/18 patients, baseline levels were within the normal range (mean 37 +/- 21 U/1); interestingly, these at the same time were the only responders to immunotherapy (n = 2) or at least showed stable disease (n = 3). Response to therapy was reflected by low serum TPS levels (mean 28 +/- 23 U/1) over the entire observation period. Thirteen patients suffered progressive disease during therapy, all of them exhibiting significantly elevated (p < 0.005) pretherapeutic TPS concentrations (mean 186 +/- 124 U/1) that remained equally elevated throughout therapy (mean 192 +/- 102 U/1), reflecting tumor progression. CONCLUSIONS: TPS might have some clinical value as prognostic marker in RCC, possibly by reflecting the proliferative tendency of the tumor.

Adult↗
PubMed1995

Phenotypic patterns of mononuclear cells in dilated cardiomyopathy.

BACKGROUND: Immunological factors in the pathogenesis of idiopathic dilated cardiomyopathy (IDC) were suggested previously on the basis of the demonstration of mononuclear cell infiltrates and autoantibodies against the myocardium. The present study investigated whether tissue leukocyte subpopulations isolated from hearts with IDC (n = 6) differ in phenotype from those of tissues without IDC (n = 7). METHODS AND RESULTS: Leukocytes were quantified as reactive cells per square millimeter in perivascular, interstitial, and parenchymal tissue sections. Freshly isolated heart-tissue T cells and peripheral-blood T cells from the same patients were analyzed by triple staining and flow cytometry to identify T-cell subpopulations as well as their states of differentiation (expression of CD45RA and Leu-8 versus CD45RO) and activation (IL-2R, IL-7R, very late antigen-1, HLA-DR). All types of infiltrating cells (T cells, B cells, macrophages, granulocytes) are increased in hearts with IDC compared with normal hearts, but only CD8+ T cells and macrophages are increased relative to the other leukocyte subpopulations. CD45RO+/CD45RA-/Leu-8- cells constitute the majority of heart-tissue T cells in both normal hearts and hearts with IDC. Strikingly, hearts with IDC are infiltrated by eightfold greater numbers of perivascularly located IL-2R(+)- (26% of all T cells) and CD45RO(+)-activated memory T cells; moreover, in contrast to normal heart, approximately 40% of both CD4+ and CD8+ heart-tissue T cells express activation markers. CONCLUSIONS: Both normal hearts and hearts with IDC are populated by leukocytes. The quantitative increase in IDC, associated with a dramatically altered activation status of heart-tissue T cells, suggests a direct role of infiltrating leukocytes in the pathogenesis of IDC.

Cardiomyopathy, Dilated↗
PubMed1995

Folding of an enzyme into an active conformation while bound as peptidyl-tRNA to the ribosome.

Rhodanese bound to bacterial ribosomes as peptidyl-tRNA can be folded into an enzymatically active conformation by generating C-terminal extensions of the wild-type enzyme. Rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system from plasmids containing the coding sequences for the wild-type enzyme or its C-terminally extended mutants. Two proteins with extensions of 23 amino acids or longer were enzymatically active while bound to the ribosomes whereas wild-type protein and a 13-amino acid extension were not. All forms of the enzyme were active after termination and release of the full-length protein from the ribosomes. All five of the bacterial chaperones were required to substantially increase the specific enzymatic activity of the extended rhodanese while the nascent protein was bound to ribosomes. The results provide direct support for the hypothesis that proteins acquire tertiary structure as they are formed in ribosomes.

Cell-Free System↗
PubMed1995

Elongation and folding of nascent ricin chains as peptidyl-tRNA on ribosomes: the effect of amino acid deletions on these processes.

Ricin A-chain was used as a test protein to study the effects of deletion of codons on the ribosomal synthesis, release and chaperone-mediated folding of the proteins. Synthesis of wild-type ricin and five mutant proteins was carried out in an Escherichia coli cell-free coupled transcription/translation system from otherwise identical non-linearized plasmids. The deletions involved small numbers of contiguous amino acid residues at different points from the N terminus to the C terminus of the wild-type protein. Deletion of the N-terminal 20 amino acid residues caused a 45% reduction in total protein synthesis whereas deletion of the next three amino acid residues caused a 1.5-fold increase in synthesis compared with wild-type with an accumulation of full-length polypeptides as peptidyl-tRNA in the ribosomal P site. Intermediate levels of synthesis and release were seen with the other three mutants. Enzymatic activity was detected only with wild-type protein and a mutant lacking the C-terminal five amino acid residues. These were the only ricin species in which chaperone-dependent reactions could be detected by fluorescence from coumarin incorporated with methionine at the N terminus of the proteins. By using sparsomycin to block termination of full-length peptidyl-tRNA, it was demonstrated that the chaperone-mediated reactions detected by fluorescence occur on the ribosomes and involve folding of the nascent protein as peptidyl-tRNA. The results presented provide a direct demonstration of two points of fundamental importance: folding of nascent proteins involving chaperone-mediated reactions can occur on ribosomes and is directly related to the conformation of the native enzyme. Deletion of amino acid residues at different points from the N terminus to the C terminus affects the reactions of elongation, chaperone-mediated folding and release of full-length protein.

Cell-Free System↗
PubMed1995

The importance of the N-terminal segment for DnaJ-mediated folding of rhodanese while bound to ribosomes as peptidyl-tRNA.

Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound rhodanese into the native conformation. The effect of DnaJ is monitored by fluorescence from coumarin cotranslationally incorporated at the N terminus of nascent rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-type protein interferes with the synthesis of wild-type rhodanese but has much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length rhodanese polypeptides that accumulate during in vitro synthesis. The results lead to the hypothesis that the N-terminal segment of rhodanese is required for its chaperone-dependent folding on the ribosome.

Escherichia coli Proteins↗
PubMed1995

Inhibition of the release factor-dependent termination reaction on ribosomes by DnaJ and the N-terminal peptide of rhodanese.

A peptide consisting of the 17 N-terminal amino acids of native bovine rhodanese in combination with the chaperone DnaJ specifically inhibits release factor- and stop codon-dependent hydrolysis of N-formylmethionine from N(formyl)-methionyl-tRNA bound with AUG to salt-washed ribosomes. Neither the peptide nor DnaJ by itself causes this inhibition. The N-terminal peptide and DnaJ both singularly and combined do not affect the peptidyltransferase reaction per se. The total amount of rhodanese synthesized in the cell-free coupled transcription-translation system is reduced by the peptide, with concomitant accumulation of full-length enzymatically inactive rhodanese polypeptides on ribosomes. In combination with DnaJ, the N-terminal polypeptide inhibits the termination and release of full-length rhodanese peptides that have accumulated on Escherichia coli ribosomes during the course of uninhibited coupled transcription-translation in the cell-free system. This inhibition appears to involve release factor 2-mediated termination at the UGA termination codon in the coding sequence for rhodanese. It is suggested that the N-terminal peptide inhibits the binding of the release factor to ribosomes. These data appear to provide the first report of differential inhibition of the termination reaction on ribosomes without inhibition of the peptidyltransferase reaction and peptide elongation.

Amino Acid Sequence↗
PubMed1995

Low plasma homovanillic acid levels in recently abstinent alcoholic men.

OBJECTIVE: To explore dopaminergic mechanisms in alcohol dependence, the authors measured plasma homovanillic acid (HVA) in recently detoxified alcohol-dependent men. METHOD: Plasma HVA was measured in 83 male patients with a diagnosis of alcohol dependence who had maintained documented abstinence for at least 3 weeks and in 69 healthy male comparison subjects. RESULTS: The alcoholic patients as a group had significantly lower levels of plasma HVA than the comparison subjects. This difference was not influenced by any other measured covariate, including a family history of alcohol dependence. CONCLUSIONS: These results imply that factors such as alcohol dependence should be taken into account in future studies of plasma HVA.

Alcohol Drinking↗
PubMed1995

High expression of a CD38-like molecule in normal prostatic epithelium and its differential loss in benign and malignant disease.

PURPOSE: We characterize an undescribed antigen on prostatic epithelial cells, which is recognized by anti-CD38. MATERIALS AND METHODS: Normal (3 cases), benign hyperplastic (10) and carcinomatous (10) prostatic tissues were analyzed using immunohistochemistry, immuno-electron microscopy and the Western blot test. RESULTS: Comparison of prostatic and lymphatic CD38 antigen revealed identical bands at 45 kD. Electron microscopy demonstrated anti-CD38 reactivity on the cytoplasmic membrane and within the secretory vacuoles. Double labeling with anti-cytokeratin types 5/15 and 8/18 confirmed that basal and secretory normal prostatic epithelial cells express CD38. By contrast, a complete loss was found in malignant (52.8%), tumor-surrounding nonmalignant (16.3%) and benign prostatic hyperplasia derived glands (3.7%). CONCLUSIONS: CD38 is a novel prostatic antigen. Its role in intracellular calcium mobilization may contribute to smooth muscle cell contraction and/or sperm motility.

ADP-ribosyl Cyclase↗
PubMed1995

Effects of electroconvulsive therapy on plasma GABA.

There are no published data on the effects of seizures on indices of gamma-aminobutyric acid (GABA) function in human subjects. In study 1, the effects of electroconvulsive therapy (ECT) on free plasma GABA were studied in 39 inpatients with major depressive disorder. Acutely after ECT, free plasma GABA was significantly reduced for up to 1 h after seizure termination, and this finding replicated strongly in a subgroup of six patients who received a second course of ECT. In a second study at a different site that compared sham ECT and real ECT in seven patients, some doubt was raised about the replicability of the acute effect of ECT on GABA levels. Nonetheless, the strength of the findings in the larger study 1 sample suggests that, unlike virtually all other biochemical indices, free plasma GABA may be reduced acutely after ECT. This acute decrease could reflect decreased levels of GABA in brain extracellular space or decreased brain turnover. In study 1, compared with ECT nonresponders, ECT responders had higher GABA levels at both baseline and after a course of ECT. Because plasma GABA levels are known to be low in a subset of patients with major depression, the higher GABA levels observed in clinical responders before and after the ECT course indirectly suggest that patients least abnormal in GABA levels may show superior clinical response. This also suggests that low plasma GABA is not a state marker for depression.

Adult↗
PubMed1994

Chaperone-dependent folding and activation of ribosome-bound nascent rhodanese. Analysis by fluorescence.

Fluorescently labeled rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system. A derivative of coumarin was co-translationally incorporated at the N terminus of the polypeptide. Molecules released from the ribosomes during the incubation are enzymatically active; however, continued incubation results in accumulation of enzymatically inactive full-length rhodanese polypeptides on the ribosomes. These can be activated and released in the presence of the added chaperones, DnaJ, DnaK, GrpE, GroEL, GroES and ATP. Fluorescence parameters (quantum yield, anisotropy and the emission maximum) of ribosome-bound coumarin-labeled rhodanese are affected differentially by addition of the chaperones individually or sequentially. Rhodanese released from the ribosomes in the presence of all chaperones (enzymatically active) differs in fluorescence properties from rhodanese released by GroES or DnaK only or by puromycin (enzymatically inactive) indicating a difference in conformation. Using sparsomycin, an inhibitor of the peptidyl transferase reaction, full-length rhodanese can be trapped on the ribosomes. A ribosome-bound intermediate formed by DnaJ or DnaJ plus DnaK was demonstrated by the effect of these chaperones on fluorescence spectra resulting from binding of anticoumarin antibodies to the N terminus of newly synthesized rhodanese. The results support the hypothesis that folding of nascent proteins can take place on the ribosome.

Chaperonins↗
PubMed1994

Activation and release of enzymatically inactive, full-length rhodanese that is bound to ribosomes as peptidyl-tRNA.

Synthesis of rhodanese in a cell-free coupled transcription/translation system derived from Escherichia coli leads to an accumulation of full-length rhodanese protein on the ribosomes as well as to enzymatically active protein that is released from the ribosomes into the supernatant fraction. The ribosome-bound protein is enzymatically inactive but can be activated and released from the ribosomes without additional protein synthesis by subsequent incubation in the presence of the added chaperones DnaJ, DnaK, GrpE, GroEL, and GroES plus ATP. Efficient activation requires that all of the chaperones are present together during incubation which yields fully active rhodanese. Incubation in the presence of DnaJ only inhibits release whereas incubation with only GroES or DnaK promotes the release of enzymatically inactive protein. Incubation of the ribosome with puromycin leads to the release of enzymatically inactive protein whereas release and activation in the presence of all of the chaperones is blocked by sparsomycin. The effect of these antibiotics provides very strong evidence that enzymatically inactive, full-length rhodanese is bound to the ribosomes as peptidyl-tRNA and that the peptidyl transferase reaction is required for its release. Considered together, the data indicate that chaperone-mediated late stages of rhodanese folding into the enzymatically active, native conformation are intimately associated with the process of termination and release that occurs as part of the reaction cycle of protein synthesis.

Animals↗
PubMed1994

Learned helplessness sensitizes hippocampal norepinephrine to mild restress.

A proportion of rats exposed to inescapable tailshock stress displayed a performance deficit, termed learned helplessness, in a subsequent shuttlebox avoidance task. The technique of in vivo microdialysis was used to determine hippocampal norepinephrine levels in learned helpless, nonhelpless and nonprestressed control rats. Similar basal norepinephrine levels were detected in samples between rat groups. Following an exposure to a milder form of inescapable shock, an increase in norepinephrine output was detected in learned helpless rats, which was significantly greater than nonhelpless, nonprestressed, or control animals. Thus, inescapable stress appears to sensitize the hippocampus to increase norepinephrine release in response to a subsequent smaller stressor. This hypersensitivity might underlie the avoidance impairment of learned helplessness. Therefore, the possibility exists that similar neurochemical changes may also be responsible for some of the symptoms of human posttraumatic stress disorder (PTSD), such as the poor coping associated with seemingly mild stress.

Animals↗
PubMed1994

Development of a chaperone-deficient system by fractionation of a prokaryotic coupled transcription/translation system.

A coupled transcription/translation system from Escherichia coli has been developed that is very active for protein synthesis but deficient in chaperone proteins. The chaperones GroEL and DnaK distribute during the first ultracentrifugation of the E. coli extract partially with the ribosomes and partially in a liquid, viscous fraction above the ribosomes. Gel filtration chromatography of this latter fraction separates GroEL and DnaK as high-molecular-weight components from the peak of activity of the factors required for protein synthesis. Thus, a chaperone-deficient transcription/translation system can be reconstituted with salt-washed ribosomes. This chaperone-deficient system was used to study synthesis and folding of bacterial dihydrofolate reductase and of rhodanese, a eukaryotic mitochondrial enzyme. Both enzymes were synthesized from nonlinearized plasmids that had the respective coding sequence under the SP6 promoter. Both enzymes were synthesized in active form and with high specific activity in the chaperone-deficient system. A high proportion, about 20% of newly synthesized dihydrofolate reductase and about 50% of rhodanese, stayed with the ribosomes after coupled transcription/translation. No enzymatic activity was detected in this fraction. Addition of the chaperones GroEL/ES and DnaK resulted in a shift of rhodanese molecules from the ribosomes into the supernatant fraction. Nearly all molecules in the supernatant were enzymatically active.

Bacterial Proteins↗
PubMed1994

In vivo release of catecholamines from xenogeneic chromaffin cell grafts with antidepressive activity.

Previous studies in our laboratory have demonstrated that allografts of adrenal medullary tissue and xenografts of isolated bovine chromaffin cells to the rat frontal cortex can increase antidepressive activity in two separate animal models. Biochemical and pharmacological evidence suggest that the most likely mechanism of these antidepressive effects is via local release of catecholamines into the surrounding cortical parenchyma. The aim of the present study was to directly characterize the antidepressive mechanism of chromaffin cell xenografts by utilizing in vivo microdialysis to measure extracellular catecholamine levels from bovine chromaffin cell and control implanted rat frontal cortex. Following transplantation, only bovine chromaffin cell grafted rats displayed significant increases in antidepressive activity, as assessed by the forced swimming test, compared to rats with grafts of bovine adrenal medullary fibroblasts or nontransplanted rats. In vivo microdialysis results revealed remarkably elevated levels of epinephrine (EPI) and norepinephrine (NE), but not dopamine, in dialysates from bovine chromaffin cell-transplanted frontal cortex. The most likely source of these enhanced EPI and NE levels is the grafted xenogeneic chromaffin cells. The results of this study directly demonstrate that xenografts of bovine chromaffin cells to the rat frontal cortex provide a releasable pool of catecholamines for antidepressive activity.

Animals↗
PubMed1994

Does learned helplessness induction by haloperidol involve serotonin mediation?

Learned helplessness (LH) is a behavioral depression following inescapable stress. Helpless behavior was induced in naive rats by the dopamine D2 receptor blocker haloperidol (HDL) in a dose-dependent manner, with the greatest effects seen at 20 mg/kg (IP). Rats were tested 24 h after injection. Haloperidol (IP) increased release of serotonin (5-HT) in medial prefrontal cortex (MPC) as measured by in vivo microdialysis. Perfusion of HDL through the probe in MPC caused increased cortical 5-HT release, as did perfusion of both dopamine and the dopamine agonist apomorphine. Our previous work found that increased 5-HT release in MPC correlates with the development of LH. The present work suggests that increased DA release in MPC, known to occur with both inescapable stress and with HDL, may play a necessary but not sufficient role in the development of LH. Also, this suggests that increased DA activity in MPC leads to increased 5-HT release in MPC and to subsequent behavioral depression.

Animals↗