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Biomedical subjects

R Krebs

Publications and source records attributed to R Krebs.

At least 19 recordsLinked to original sources
PubMed2006

The effect of platelet-derived growth factor ligands in rat cardiac allograft vasculopathy and fibrosis.

BACKGROUND: In chronic rejection, parenchymal fibrosis and cardiac allograft vasculopathy (CAV) characterized by neointimal growth are the leading causes of graft loss for heart transplant recipients. During alloimmune responses a variety of cytokines, adhesion proteins, and growth factors, such as platelet-derived growth factor (PDGF), are up-regulated. The PDGF family (AA, AB, BB, CC, DD), which acts mainly on connective tissue cells, is considered to be a potent mitogenic and chemotactic factor for fibroblasts and vascular smooth muscle cells. In this study, we evaluated the effects of PDGF ligands in chronic rejection. METHODS: Heterotopic heart transplantations were performed between fully major histocompatability complex-mismatched Dark Agouti to Wistar Furth rats receiving cyclosporine immunosuppression. Allograft coronary arteries were perfused with a recombinant adeno-associated virus (AAV) encoding enhanced green fluorescence protein (EGFP) as a control gene or PDGF-A, -B, -C, -D. Allografts were harvested at 100 days for morphometric analysis of CAV and fibrosis. RESULTS: AAV-mediated transgene expression was detected by EGFP immunoreactivity across the graft section (at 100 days) in AAV EGFP-perfused allografts. AAV PDGF-A, -C, and -D perfusion resulted in accelerated CAV and fibrosis. In contrast, AAV PDGF-B perfusion did not induce arteriosclerotic changes or fibrosis in cardiac allografts. CONCLUSIONS: AAV PDGF-A, -C, and -D overexpression accelerated the development of chronic rejection, whereas PDGF-B did not. Our results suggested that more targeted therapy with monoclonal antibodies blocking the active sites of PDGF-A, -C, and -D may produce beneficial effects on heart transplant survival.

Animals↗
PubMed2006

Effect of graft preservation and acute rejection on hypoxia-inducible factor-1 in rat cardiac allografts.

Hypoxia plays an integral part in cardiac transplantation as prolonged graft preservation is an individual risk factor for the development of cardiac allograft vasculopathy (CAV). In this study we characterized the role of hypoxia-inducible factor-1 (HIF-1) during prolonged graft preservation, ischemia-reperfusion (I/R), acute rejection, and chronic rejection. Heart transplantations were performed from Dark Agouti (DA) to Wister-Furth (allo) or DA to DA (syn) rats, without immunosuppression (acute rejection model, harvested at day 5) or with cyclosporine (chronic rejection model, harvested at day 60). To study the effect of preservation on HIF-1 regulation, normal DA hearts were subjected to different cold ischemia times with or without 45 minutes of additional warm ischemia. The role of I/R was studied by harvesting syngrafts at different time points after reperfusion. Real-time reverse-transcriptase polymerase chain reaction quantified total HIF-1 mRNA, while enzyme-linked immunosorbent assay and immunohistochemistry quantified and localized HIF-1 protein. Our results show that HIF-1 nuclear immunoreactivity is increased during graft preservation and I/R leads to loss of nuclear HIF-1 immunoreactivity. Acute rejection induced HIF-1 in mRNA level. Our findings thus indicated that HIF-1 is activated during transplantation and suggested that manipulation of the HIF-1 pathway might reveal new therapeutic options to manage CAV.

Animals↗
PubMed2006

Vascular endothelial growth factor plays a major role in development of experimental obliterative bronchiolitis.

Obliterative bronchiolitis (OB) is the major limitation for long-term survival of lung allograft recipients. The exact molecular and cellular mechanisms contributing to obliterative lesion formation are unknown. Pathological characteristics of OB are epithelial damage, peribronchial inflammation, and increasing obliteration of bronchioli. Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that exerts proinflammatory effects by increasing endothelial permeability and inducing expression of endothelial adhesion molecules. We investigated the role of VEGF in the development of OB in rat tracheal allografts and the role of VEGF receptors (VEGFR)-1 and -2 in the development of OB in mouse tracheal allografts. In nontreated allografts, with increasing loss of epithelium and airway occlusion, VEGF messenger RNA (mRNA) and protein expression vanished in the epithelium and increased in smooth muscle cells and mononuclear inflammatory cells compared with syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF164 gene led to a decrease in epithelial necrosis but increased luminal occlusion by >50% compared with AdLacZ-treated rat tracheal allografts. When compared with the control immunoglobulin (Ig)G group, simultaneous treatment with antibodies against VEGFR-1 and -2 significantly lowered the degree of luminal occlusion of mouse tracheal allografts.

Adenoviridae↗
PubMed2001

Halide dependence of the halorhodopsin photocycle as measured by time-resolved infrared spectra.

Time-resolved Fourier transform infrared (FTIR) difference spectra of the halorhodopsin (hR) photocycle have been collected from 3 micros to 100 ms in saturating concentrations of KCl or KBr. Kinetic analysis of these data revealed two decay processes, with time constants of tau(1) approximately 150 micros and tau(2) approximately 16 ms in the presence of either halide, with tau(2) describing the return to the starting (hR) state. Comparison to previous low-temperature FTIR spectra of hR intermediates confirms that characteristic hK and hL spectral features are both present before the tau(1) decay, in a state previously defined as hK(L) (Dioumaev, A., and M. Braiman. 1997. Photochem. Photobiol. 66:755-763). However, the relative sizes of these features depend on which halide is present. In Br-, the hL features are clearly more dominant than in Cl-. Therefore, the state present before tau(1) is probably best described as an hK(L)/hL(1) equilibrium, instead of a single hK(L) state. Different halides affect the relative amounts of hK(L) and hL(1) present, i.e., Cl- produces a much more significant back-reaction from hL(1) to hK(L) than does Br-. The halide dependence of this back-reaction could therefore explain the halide selectivity of the halorhodopsin anion pump.

Bacteriorhodopsins↗
PubMed2001

Activin A and activin receptors in thyroid cancer.

Proliferation is controlled by a network of mitogenic and growth inhibitory factors. Transforming growth factor-beta1 (TGF-beta1) and activin A are the most important growth inhibitors of benign follicular epithelial cells of the human thyroid. The effects of these substances on malignant primary thyrocytes are not known. We have examined the growth regulatory effects of activin A and TGF-beta1 in primary cultures derived from four papillary cancers, two follicular thyroid cancers, and three benign thyroid tissues. Malignant cells demonstrated resistance to activin and TGF-beta1 or reversal to a weak but significant mitogenic effect (p < 0.001). We also evaluated the activin receptor transcription pattern. Isoforms alk4-1, 4-2, and 4-3 were found in benign (n = 12) and malignant (n = 22) tissues. Two subtypes of type I and type II activin receptors were demonstrated. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) demonstrated a significant threefold downregulation of alk4-1 receptors in papillary (n = 25) and follicular (n = 18) thyroid cancers as compared to normal thyroids (n = 12) (p < 0.001). To our knowledge these are the first data to demonstrate reversal of activin and TGF-beta1 effects in thyroid malignancy and to demonstrate changes of the type Ib activin receptor expression in thyroid malignancy.

Activin Receptors↗
PubMed2000

Activin A and activin receptors in the human thyroid: a link to the female predominance of goiter?

Activin A belongs to the TGF-beta family of growth factors involved in control of tissue formation by growth inhibition and to a family of hormones involved in human reproduction. Serum levels of dimeric activin A display a sexual dimorphism with significantly higher circulating hormone concentrations in men than in women after menopause. Since goiter is far more frequent in women than in men, we investigated the role of this sex-related growth factor in the human thyroid. Primary cultures were obtained from 3 patients with goiter, and 4 with recurrent goiter. Activin A significantly inhibited the proliferation of human thyroid follicular epithelial cells in vitro in concentrations between 0.5 and 50 ng/ml and was almost as potent as TGFbeta-1. Analysis in native tissues from 12 normal thyroids, 16 goiters, and 5 Graves thyroids demonstrated mRNA expression of the activin subunit betaA, TGFbeta-receptor type I and II, activin receptors type I receptors alk2 and alk4, and activin type II receptors actRII and actRIIb. Isoform analysis of the major functional type I activin receptor alk4 revealed the full-length transcript SKR2-1 and transcripts for the partially truncated proteins SKR2-2 and SKR2-3 in normal thyroids and goiters. Semi-quantitative RT-PCR revealed a significant 2.5-fold decrease of mRNA for alk4-1 type I activin receptors in goiter as compared to normal thyroid (p < 0.05), whereas expression of the type II receptor actRII was unchanged. These data identify a novel growth inhibitory pathway by activin in the human thyroid. Down-regulation of activin receptor type lb in goiter hints towards end-organ changes which could contribute to deficient growth inhibition. These data provide a mechanistic model how the sexually dimorphic hormone activin A may protect males from goitrogenesis.

Activin Receptors↗
PubMed1999

Refined model for primer/template binding by HIV-1 reverse transcriptase: pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation events distinguish between different binding modes depending on the nature of the nucleic acid substrate.

The kinetic mechanism of nucleic acid substrate binding and nucleotide incorporation by human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was analysed using synthetic DNA/DNA and DNA/RNA primer/templates (p/t) without predicted secondary structures in the single-stranded region. Determination of the pre-steady-state kinetics of p/t binding by a combination of stopped-flow and quench flow methods indicate a branched binding mechanism for the HIV-1 RT/nucleic acid interaction. Analysis of p/t-RT association by stopped-flow measurements suggest a three-step binding mode with an initial second-order step followed by two isomerisation steps with rates of about 6 s(-1)and 0.5 s(-1), respectively. Determination of the rate-limiting step of the association process via single turnover, single nucleotide incorporation analysis by quench flow measurements revealed two binding events (the initial second-order step cannot be detected with this experimental set-up) with rates of 4 - 7 s(-1)and 0.4 - 0. 7 s(-1), respectively, indicating that both binding events exist in parallel. Thorough pre-steady-state analysis of single turnover, single nucleotide incorporation kinetics showed that dNTP incorporation occurs with a biphasic exponential burst followed by a linear phase. The exponential burst consists of a fast phase with rates of 20 - 60 s(-1) and a slow phase with rates of 0.5 - 2 s(-1), respectively. The relative distribution of these two burst amplitudes differs significantly depending upon which substrate is used. The DNA/RNA-RT complex shows primarily fast incorporation (>80 %) whereas less than 45 % of the DNA/DNA-RT complex incorporate dNTP rapidly. The same relative distribution of amplitudes concerning the two substrates is also found for the association process of RT and p/t. Analysis of dNTP incorporation of the preformed RT-p/t complex in the presence of a nucleic acid competitor shows no effect on the biphasic burst amplitude, however the linear phase disappears. Here, a refined model of the mechanism of RT-p/t binding is presented which is based on the suggestion that two different RT-p/t complexes are formed, i.e. a productive enzyme/substrate complex which is capable of nucleotide incorporation and a non-productive complex which has to undergo an isomerisation before dNTP incorporation can occur. In addition, binding of RT to its substrate can lead to a dead end complex that is not capable of dNTP incorporation.

Binding, Competitive↗
PubMed1998

Pre-steady-state kinetic characterization of RNA-primed initiation of transcription by HIV-1 reverse transcriptase and analysis of the transition to a processive DNA-primed polymerization mode.

Single-turnover and equilibrium measurements were carried out to determine the basis of the apparently slow, nonprocessive polymerization reaction catalyzed by HIV-1 reverse transcriptase (RT) during transcription initiation, when both the primer and template are composed of RNA. Comparison of the binding and kinetic parameters of a 20-mer, all-RNA primer/35-mer template substrate to one identical in sequence but composed of a 20-mer, all-DNA primer/35-mer RNA template reveals striking differences. Equilibrium titrations yielded a dissociation constant (Kd) >200 nM for the RNA/RNA-RT complex which is at least 200-fold higher than that of the DNA/RNA-substrate (Kd approximately 1 nM). The affinity of the RT-RNA/RNA complex for dTTP was found to be at least 500 times lower (Kd approximately 3.4 mM) than that of the RT-DNA/RNA complex (Kd approximately 6.6 microM). The single-turnover dNTP incorporation time course using the RNA-primer substrate, the DNA-primer substrate, or a series of RNA-primer substrates preextended with one to eight deoxynucleotides showed that dNTP incorporation occurs with a biphasic exponential burst of +1 extension product, followed by a linear phase. At least three different RT-bound forms of the p/ts exist: a fast, kinetically competent form (single-turnover rate approximately 10-50 s-1); a slow form (rate approximately 0.3-1 s-1); and a form that is dead-end (no turnover). The studies further revealed that a switch to a fast, kinetically competent p/t occurs after six dNTPs are incorporated into the RNA primer, with the switch being defined as the transition from a minority to a majority of the p/t bound in the optimal manner.

Base Composition↗
PubMed1998

Analysis of the polymerization kinetics of homodimeric EIAV p51/51 reverse transcriptase implies the formation of a polymerase active site identical to heterodimeric EIAV p66/51 reverse transcriptase.

Homodimeric EIAV p51/51 and heterodimeric EIAV p66/51 reverse transcriptase were purified in order to compare the different modes of DNA synthesis supported by the enzymes. Analysis of the dimerization behavior of the EIAV enzymes indicates that the dimer stability of EIAV reverse transcriptase enzymes is higher than that of their HIV-1 reverse transcriptase counterparts. EIAV p51/51 polymerizes DNA distributively whereas DNA synthesis by EIAV p66/51 is processive. Steady-state and pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation were performed with both enzymes to determine the reasons for the different polymerization behavior. Equilibrium fluorescence titrations demonstrated that the Kd values of EIAV p51/51 for binding of DNA/DNA and DNA/RNA substrates are increased 10-fold and 28-fold, respectively, as compared to EIAV p66/51. Stopped-flow measurements with DNA/DNA show that the increase in the Kd is in part due to a 17. 4-fold higher dissociation rate constant (k-1) for EIAV p51/51. Additionally, with EIAV p51/51, kdiss is increased 7-fold for DNA/DNA and 14-fold for DNA/RNA primer/template substrates, respectively. The lack of the RNase H domain in EIAV p51/51 leads to differences in the pre-steady-state kinetics of nucleotide incorporation on DNA/DNA and DNA/RNA templates. The burst of both enzymes is composed of two phases for both substrates, and the values for the corresponding pre-steady-state burst rates, kpol1 and kpol2, are similar for both enzymes, implying the formation of identical polymerase active sites. However, the amplitudes of the two phases differ with DNA/DNA templates, indicating a different distribution between two states varying greatly in their kinetic competence.

Binding Sites↗
PubMed1997

Single-step kinetics of HIV-1 reverse transcriptase mutants responsible for virus resistance to nucleoside inhibitors zidovudine and 3-TC.

Two mutants of HIV-1 reverse transcriptase (RT) associated with high-level resistance of the virus to AZT (RT-AZT: D67N, K70R, T215Y, K219Q, and M41L) or 3-TC (RT-3TC: M184V) were expressed in Escherichia coli and purified. None of these mutants showed significant changes in the affinity and kinetics of binding to a DNA/DNA primer/template. RT-AZT was investigated in detail with respect to its kinetics of incorporation of nucleotides. No change in the relative rates of TMP and AZTMP incorporation could be detected for RT-AZT with respect to wild type RT. These results imply that there is no increased discrimination against AZTTP in the mutant. This was found for DNA/DNA and DNA/RNA primer/template. Additionally, rapid kinetics of incorporation of 3'-amino-3'-deoxythymidine 5'-monophosphate (a possible metabolite of AZT) were investigated and compared with TMP incorporation, but no difference in its relative rates of incorporation between wild type RT and RT-AZT was detected. In contrast, the already very slow rate of incorporation of 3-TCMP seen with wild type enzyme was drastically reduced (by a factor of 23 and 36 with DNA/DNA primer/template and DNA/RNA primer/template, respectively) for RT-3TC, showing a clear correlation between in vitro and in vivo effects. The affinity of 3-TCTP to the RT-3TC-primer/template complex was not affected by the mutation M184V. A 1.6-fold cross-resistance to ddATP, the converted form of the prodrug ddI, could also be shown for RT-3TC, but no cross-resistance to ddCTP was detected. Additionally, rapid kinetics of AZTMP incorporation by RT-3TC were investigated. There was an indication of a slightly higher rate of incorporation of AZTMP by RT-3TC than wild type RT.

Anti-HIV Agents↗
PubMed1997

Kinetic analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip region of p66. Implications for DNA synthesis and dimerization.

The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the beta12-beta13 hairpin (residues 227-235). It has been proposed to play a role in aligning the 3'-OH end of the primer in a position for nucleophilic attack on an incoming dNTP. To analyze the importance of the primer grip for RT function, mutant RTs were used that contain single alanine substitutions of residues Trp229, Met230, Gly231, and Tyr232 in the p66 subunit of the heterodimeric p66/51 enzyme. Steady-state and pre-steady-state kinetic analyses of the enzymes were performed. All mutant enzymes revealed reduced polymerase activity. Mutation of Y232A showed the smallest effect on polymerase function. Equilibrium fluorescence titrations demonstrated that the affinity of the mutants for tRNA was only slightly affected. However, the affinity for primer-template DNA was reduced 27-fold for mutant p66(W229A)/51 and 23-fold for mutant p66(G231A)/51, and the maximal pre-steady-state rate of nucleotide incorporation, kpol, was reduced 27-fold for p66(W229A)/51 and 70-fold for p66(G231A)/51, respectively. Mutant p66(M230A)/51 revealed no reduced affinity for primer-template but showed a 71-fold reduced affinity for dTTP. Additionally, the mutations Trp229 and Gly231 affected the stability of the RT heterodimer.

Chromatography, High Pressure Liquid↗
PubMed1997

A dose-dependent inhibitory effect of cyclosporine A on obliterative bronchiolitis of rat tracheal allografts.

Inbred DA and WF rats were used as donors and recipients of heterotopic tracheal allografts. To investigate cellular and molecular mechanisms as well as inhibitory effects of differently acting immunosuppressive drugs on the development of obliterative bronchiolitis (OB), the recipient rats received either cyclosporine A (CsA; 1, 2, or 5 mg/kg/d), 15-deoxyspergualin (DSG; 1 or 2 mg/kg/d), or mycophenolate mofetil (MMF; 20 or 40 mg/kg/d), or were left without immunosuppression. The grafts were removed at various time points for histology and immunohistochemistry. In tracheal allografts removed from nonimmunosuppressed animals, respiratory epithelium was replaced by cuboidal or squamous cell epithelium, with markedly enhanced expression of epithelial major histocompatibility class (MHC) Class II antigens at 3 d after transplantation. This was associated with a pronounced inflammatory episode and proliferation of T cells and macrophages, with the prominence of lymphoid activation markers, MHC Class II antigens and interleukin-2R (IL-2R). Later, total epithelial necrosis developed and intense proliferation of granulation tissue occluded the airway lumen producing a condition resembling OB in humans. In syngeneic tracheal grafts, no such changes could be observed. CsA decreased the development of OB in a dose-dependent fashion, in association with downregulation of epithelial MHC Class II antigen expression, IL-2R expression, and the infiltration of T cells. The new immunosuppressive drugs DSG (suppression of the monocyte/macrophage action and lymphocyte proliferation) and MMF (blocking of the de novo pathway of purine synthesis), in the dose range tested, showed no significant effect on the development of OB. These results suggest that an acute alloimmune response to airway targets, perhaps to epithelium, is the primary cause of OB, since CsA, with a nearly exclusive effect on the transcription of immune cytokines, entirely inhibited the generation of OB, and that preventive intervention for OB must occur early in the T-cell activation pathway.

Animals↗
PubMed1996

Cytomegalovirus infection accelerates obliterative bronchiolitis of rat tracheal allografts.

A cascade of inflammation and injury of the airway wall followed by a fibroproliferative process that results in airway obstruction has been suggested as the explanation of the process of obliterative bronchiolitis (OB) in lung allograft recipients. To determine the impact of rat cytomegalovirus (RCMV) infection on the development of OB, heterotopic rat tracheal allografts were transplanted from DA donors to WF recipients immunosuppressed with 2 mg/kg per day cyclosporine A. Chronic RCMV infection was similarly established 8 weeks before transplantation in donors alone (D+/R-), recipients alone (D-/R+), and both donors and recipients (D+/R+). The control rats were left non-infected, but were similarly immunosuppressed. The results of this study demonstrate that both acute and chronic recipient RCMV infection, but not donor infection, amplify the development of experimental OB in the rat and suggest that RCMV infection-associated immune response, rather than the viral load in the graft, is essential for the development of the accelerated form of OB.

Animals↗
PubMed1994

The role of an agency medical director.

No two agencies provide care in the exact same way. Thus, it is difficult to come up with a definitive description of what a medical director should do. But one medical director undertook a description; through intense examination of his personal perspective and his professional experience, he presents a total view of the role of a home care agency medical director.

Community Health Nursing↗
PubMed1993

Cell vitality in cartilage tissue culture following excimer laser radiation: an in vitro examination.

Excimer laser is used for cartilage debridement, although the resulting cell damage is yet unclear. For examination of cartilage survival after treatment, we used short-term tissue cultures of human joint cartilage. Specimens were treated with a XeCl-Excimer laser using different laser parameters, pulse energies, and repetition rates. Following treatment, discs were cultured for 8 days prior to examination. In contrast to the 20 microns damage zone as instant visible effect in histomorphologic examinations, we found a 0.3 mm zone in which approximately 50% of cartilage cells had morphological signs of damage on light microscopic examinations. Autoradiography revealed that cartilage cells in an 0.5-0.7 mm area surrounding the laser craters had no collagen synthesis. This examination indicates that cell damage of excimer laser is higher than expected from prior studies.

Adult↗
PubMed1993

Intensive dynamic back exercises with or without hyperextension in chronic back pain after surgery for lumbar disc protrusion. A clinical trial.

Sixty-two patients with chronic low back pain occurring 14-60 months after undergoing discectomy for the first time were randomized to two physical treatment groups: 24 sessions of intensive dynamic back exercises with hyperextension or 24 sessions of intensive dynamic back exercises without hyperextension. At the conclusion of therapy and at one-year follow-up, no difference was seen between the randomized groups, with regard to the combined assessments of pain, disability and objective measurements. A difference for back exercises without hyperextension to be superior to the other treatment regimen was statistically significant at the three-month follow-up. In the patient's qualitative assessment of treatment outcome there were seen no significant differences between back exercises with or without hyperextension. There was a similar and significant improvement of the isometric endurance of back muscles in both groups, but the flexibility of the spine was significantly improved only in the group using hyperextension exercises. The overall response rate of an earlier published investigation was reproduced. It is concluded that chronic back patients after first time discectomy may benefit from an intensive rehabilitation protocol including intensive exercises. The added use of hyperextension exercises does not confer any independent benefit. Furthermore, the training had to continue for more than 2-3 months before a statistical significant decrease in back pain was reported in the patient pain diary.

Adult↗
PubMed1989

Trauma malpractice claims related to trauma level designation.

The purpose of the present study was to review a one-state experience (Arizona) with trauma malpractice claims as a function of hospital trauma level designation. A total of 191 cases covering 7 years was reviewed and analyzed. Three categories of treatment facilities were compared: American College of Surgeons-categorized Level I hospitals, all other hospitals, and outpatient treatment facilities. Only 22 percent of the malpractice claims were found in Level I facilities, whereas 58 percent were found in other hospitals and 20 percent came from outpatient facilities. Not surprisingly, the indemnity awards were more than two times higher in other hospitals than in Level I hospitals. However, the legal dollars spent in defense per claim were two times higher in Level I than either outpatient hospitals or outpatient treatment facilities, suggesting that Level I malpractice claims are more defensible. We have identified six areas of high potential liability and have made some suggestions for the reduction of risk management in those areas.

Arizona↗