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Biomedical subjects

A Szabo

Publications and source records attributed to A Szabo.

At least 19 recordsLinked to original sources
PubMed1998

Electrostatic enhancement of diffusion-controlled protein-protein association: comparison of theory and experiment on barnase and barstar.

The electrostatic enhancement of the association rate of barnase and barstar is calculated using a transition-state theory like expression and atomic-detail modeling of the protein molecules. This expression predicts that the rate enhancement is simply the average Boltzmann factor in the region of configurational space where association occurs instantaneously in the diffusion-controlled limit. Based on experimental evidence, this "transition state" is defined by configurations in which, relative to the stereospecifically bound complex, the two proteins are shifted apart by approximately 8 A (so a layer of water can be accommodated in the interface) and the two binding surfaces are rotated away by 0 degrees to 3 degrees. The values of the average Boltzmann factor, calculated by solving the Poisson-Boltzmann equation, for the wild-type complex and 16 complexes with single mutations are found to correlate well with experimental results for the electrostatic rate enhancement. The predicted rate enhancement is found to be somewhat insensitive to the precise definition of the transition state, due to the long-range nature of electrostatic interactions. The experimental ionic strength dependence of the rate enhancement is also reasonably reproduced.

Bacterial Proteins↗
PubMed1998

Distinct prevalence of antibodies to the E2 protein of GB virus C/hepatitis G virus in different parts of the world.

Since the identification of the new human virus, GB virus C (GBV-C)/hepatitis G-virus (HGV), in 1995/1996, reverse transcription polymerase chain reaction remained the sole available diagnostic tool for GBV-C/HGV infection. Recently, a serologic test based on the detection of antibodies to the putative envelope protein 2 (anti-E2) has been introduced. We used this assay for a seroepidemiological survey including 3,314 healthy individuals from different parts of the world, 123 patients from Germany who were suspected to have an increased risk of acquiring GBV-C/HGV infection, 128 multiple organ donors, and 90 GBV-C/HGV RNA positive persons. In European countries, anti-E2 seropositivity ranged from 10.9% (Germany) to 15.3% (Austria). In South Africa (20.3%) and Brazil (19.5%), even higher anti-E2 prevalence rates were recorded. In Asian countries like Bhutan (3.9%), Malaysia (6.3%), and the Philippines (2.7%), anti-E2 positivity was significantly lower. GBV-C/HGV anti-E2 prevalence in potential "risk groups," i.e., patients on hemodialysis and renal transplant recipients, did not vary significantly from anti-E2 seroprevalence in German blood donors. Anti-E2 and GBV-C/HGV RNA were found to be mutually exclusive, confirming the notion that anti-E2 has to be considered as a marker of past infection.

Antibodies, Viral↗
PubMed1998

Hypertension accelerates the pace of chronic graft dysfunction in the rat.

In this study we compared the effects of hypertension on chronic rejection in a rat model of renal transplantation utilizing genetically normotensive (BBOK) and spontaneously hypertensive rats (SHR). SHR received either a BBOK (BBOK-->SHR) or an SHR (SHR-->SHR) kidney; normotensive isografts served as controls. Before transplantation, SHR recipients were treated with hydralazine (50 mg/kg per day). To prevent acute rejection, an anti-CD4 antibody (3 mg/kg per day for 3 weeks) in combination with cyclosporin A (3 mg/kg per day for 1 week) was given to all groups. Six weeks after transplantation, blood pressure was measured, and the kidneys removed for histological and immunohistological analysis. SHR-->SHR developed a significantly higher blood pressure than BBOK-->SHR. Blood pressure in BBOK-->BBOK was significantly lower than in the other two groups. The degree of glomerulosclerosis was similarly increased in allografted (BBOK-->SHR) and SHR-->SHR kidneys as compared with the BBOK-->BBOK kidneys (P < 0.05). Infiltration of ED-1+ monocyte/macrophages and OX19 pan-T-cells was most pronounced in allografts (BBOK-->SHR) and was also increased in SHR-->SHR as compared with BBOK-->BBOK. Our results indicate that hypertension accelerates the morphological and immunohistological changes characteristic of grafts undergoing chronic rejection. However, our findings support the hypothesis that alloantigen-dependent factors are of greater important.

Animals↗
PubMed1998

Hungarian population data for six STR loci.

Population genetic studies were carried out on a South Hungarian population (n = 111 individuals). The short tandem repeat (STR) systems Hum VWA, HumTH01, HumF13B, HumCD4, HumFES and HumFIBRA (FGA) were investigated by PCR amplification. All loci met Hardy-Weinberg expectations.

Alleles↗
PubMed1998

Hepatitis G virus infection in children on dialysis and after renal transplantation.

The aim of the present study was to use a combination of the reverse transcription-polymerase chain reaction and a new diagnostic test for antibodies against the viral envelope protein E2 to assess the prevalence of hepatitis G virus (GBV-C/HGV) infection in sera of Hungarian children on hemodialysis (HD) or peritoneal dialysis (CAPD), as well as in sera of renal transplant patients (RTx). The GBV-C/HGV RNA prevalence was significantly higher in the whole group of children with renal failure (18.5%) than in the control group (children with urinary tract infection, 2.5%). The difference between the GBV-C/HGV RNA prevalence in the RTx group (33.3%) and in the control group (2.5%) was significant (P = 0.007). Anti-E2, which is considered an indicator of a past GBV-C/ HGV infection, was detected in 10% (1/10) of HD patients, in 33.3% (4/12) of RTx patients, but in none of the children on CAPD. These differences were not significant. Children receiving a renal graft are at an increased risk of developing GBV-C/HGV infection, which may be attributed to the immunosuppressive drugs necessary to maintain the grafts.

Adolescent↗
PubMed1998

Tacrolimus rescue therapy in patients with rejection and long-term dialysis after kidney transplantation.

Therapy with tacrolimus has been proven effective in patients with steroid-resistant rejection episodes. It was the objective of the present study to evaluate the efficacy of tacrolimus in patients with long-lasting primary or secondary non-function (defined as being on dialysis for at least 28 days) converted for biopsy proven acute rejection and to determine parameters of prognostic value. Eight patients with biopsy proven acute interstitial rejection unsuccessfully treated with steroids were converted from a cyclosporine based immunosuppressive regimen to tacrolimus. Three patients were additionally pretreated with OKT3 without success. The tacrolimus whole blood through level was 7.5 +/- 2.6 ng/ml at one week and 8.1 +/- 3.4 ng/ml at one month after conversion. The follow-up period after conversion to tacrolimus ranged from 12-18 months. In four patients hemodialysis was stopped after 78 +/- 49 days (range 36-145 days). One year thereafter, creatinine was 3.9 +/- 2.7 mg/dl. In conclusion, our observations, although obtained in a small number of patients, offer some hope in this selected group and may encourage further studies in patients with long-term dialysis after renal transplantation.

Adult↗
PubMed1997

Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66.

A gene encoding the extracellular domain of the human erythropoietin receptor (EPO-R) was constructed using oligonucleotides, with a view to maintaining preferred codon usage for the Streptomycetes. The gene was subcloned into a multicopy Streptomyces-Escherichia coli shuttle vector, pCAN46 (derived from pIJ680), containing a strong constitutive promoter from the S. fradiae aph gene, a signal peptide coding region derived from the protease B gene of S. griseus, and a transcription terminator sequence also derived from the S. fradiae aph gene. Extracellular expression of authentic EPO-R by S. lividans was demonstrated using SDS-PAGE and Western blot analysis, followed by direct amino terminal sequencing of the purified product. Specific binding of S. lividans-expressed EPO-R to recombinant human glycosylated EPO was demonstrated using BIAcore (surface plasmon resonance) analysis and native gel shift assays.

Base Sequence↗
PubMed1997

The effect of vocalization on the heart rate response to mental arithmetic.

The heart rate (HR) responses to verbal and nonverbal mental arithmetic tasks were studied in twenty undergraduate males. The two problem-sets were presented in counterbalanced order to the subjects. Greater HR responses to verbal, in contrast to nonverbal, arithmetic were only observed when the verbal arithmetic task was presented first. The peak (maximal) HR response was also the highest when the verbal arithmetic was presented first to the subjects. It is concluded that physiological or mechanical components of vocalization do not affect the HR response to mental arithmetic. However, certain psychological components, including test-anxiety or motivation, may increase the HR when subjects have to provide verbal answers during a first exposure to a challenge. This effect dissipates during the second exposure to the challenge due to habituation.

Adult↗
PubMed1997

GBV-C/HGV infection in renal dialysis and transplant patients.

BACKGROUND: Recently a new human virus (GBV-C/HGV) was identified. With the use of the polymerase chain reaction (PCR) the possibility of a high prevalence of the GBV-C/HGV infection in haemodialysis patients was demonstrated. The aim of the present study was to use a combination of the PCR and a new diagnostic test for antibodies to the viral envelope protein E2 to assess the prevalence of the GBV-C/HGV infection in German patients with renal failure. METHODS: RT-PCR and ELISA were used to detect GBV-C/HGV RNA and antiviral antibodies (anti-E2) in the sera of 31 patients on a maintenance haemodialysis (HD)), 25 patients on a perioneal dyalisis (CAPD), and 92 renal transplant patients (RT). RESULTS: A statistical trend was noted for a higher rate of the GBV-C/HGV RNA in the whole group of patients with renal failure (11.5%) than in the control group of organ donors (5.5%); The difference between GBV-C/HGV RNA prevalence in RT patients (15.2%) and in organ donors (5.5%) was found to be significant. Anti-E2, which are considered as an indicator of a past GBV-C/HGV infection, were detected in 12.9% of HD patients, in 20.0% of CAPD patients, in 10.9% of RT patients, in 11.1% of organ donors, and in 10.9% of blood donors. These differences were not significant. Time on haemodialysis was significantly longer in GBV-C/HGV infected patients compared to uninfected patients and all patients with the GBV-C/HGV RNA have a history of blood transfusions. CONCLUSIONS: Patients with renal failure treated with dialysis or subjected to renal transplantation are at increased risk of aquiring the GBV-C/HGV infection. Higher rates of the GBV-C/HGV RNA and a similar prevalence of anti-E2 in patients with renal failure as compared to donors suggest that the rate of GBV-C/HGV infection resolution in immunosuppressed patients with renal failure might be lower than in immunocompetent patients.

Adult↗
PubMed1996

On the decomposition of free energies.

The decomposition of free energies and entropies into components has recently been discussed within the framework of the free energy perturbation (FEP) and thermodynamic integration (TI) methods. In FEP, the cumulant expansion of the excess free energy contains coupling terms in second and higher orders. It is shown here that this expansion can be expressed in terms of temperature derivatives of the mean energy, suggesting a natural decomposition of the free energy into components corresponding to each term in the Hamiltonian. This result is derived in such a way that it establishes the equivalence to a particular form of component analysis based on TI in which all terms in the interaction energy are turned on simultaneously using 1/kT as the coupling parameter.

Computer Simulation↗
PubMed1996

Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding.

How fast can a protein fold? The rate of polypeptide collapse to a compact state sets an upper limit to the rate of folding. Collapse may in turn be limited by the rate of intrachain diffusion. To address this question, we have determined the rate at which two regions of an unfolded protein are brought into contact by diffusion. Our nanosecond-resolved spectroscopy shows that under strongly denaturing conditions, regions of unfolded cytochrome separated by approximately 50 residues diffuse together in 35-40 microseconds. This result leads to an estimate of approximately (1 microsecond)-1 as the upper limit for the rate of protein folding.

Cytochrome c Group↗
PubMed1996

A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates.

The Escherichia coli heat-shock protein DnaJ cooperates with the Hsp70 homolog DnaK in protein folding in vitro and in vivo. Little is known about the structural features of DnaJ that mediate its interaction with DnaK and unfolded polypeptide. DnaJ contains at least four blocks of sequence representing potential functional domains which have been conserved throughout evolution. In order to understand the role of each of these regions, we have analyzed DnaJ fragments in reactions corresponding to known functions of the intact protein. Both the N-terminal 70 amino acid 'J-domain' and a 35 amino acid glycine-phenylalanine region following it are required for interactions with DnaK. However, only complete DnaJ can cooperate with DnaK and a third protein, GrpE, in refolding denatured firefly luciferase. As demonstrated by atomic absorption and extended X-ray absorption fine structure spectroscopy (EXAFS), the 90 amino acid cysteine-rich region of DnaJ contains two Zn atoms tetrahedrally coordinated to four cysteine residues, resembling their arrangement in the C4 Zn binding domains of certain DNA binding proteins. Interestingly, binding experiments and cross-linking studies indicate that this Zn finger-like domain is required for the DnaJ molecular chaperone to specifically recognize and bind to proteins in their denatured state.

Absorptiometry, Photon↗
PubMed1996

Theory and simulation of the time-dependent rate coefficients of diffusion-influenced reactions.

A general formalism is developed for calculating the time-dependent rate coefficient k(t) of an irreversible diffusion-influenced reaction. This formalism allows one to treat most factors that affect k(t), including rotational Brownian motion and conformational gating of reactant molecules and orientation constraint for product formation. At long times k(t) is shown to have the asymptotic expansion k(infinity)[1 + k(infinity) (pie Dt)-1/2 /4 pie D + ...], where D is the relative translational diffusion constant. An approximate analytical method for calculating k(t) is presented. This is based on the approximation that the probability density of the reactant pair in the reactive region keeps the equilibrium distribution but with a decreasing amplitude. The rate coefficient then is determined by the Green function in the absence of chemical reaction. Within the framework of this approximation, two general relations are obtained. The first relation allows the rate coefficient for an arbitrary amplitude of the reactivity to be found if the rate coefficient for one amplitude of the reactivity is known. The second relation allows the rate coefficient in the presence of conformational gating to be found from that in the absence of conformational gating. The ratio k(t)/k(0) is shown to be the survival probability of the reactant pair at time t starting from an initial distribution that is localized in the reactive region. This relation forms the basis of the calculation of k(t) through Brownian dynamics simulations. Two simulation procedures involving the propagation of nonreactive trajectories initiated only from the reactive region are described and illustrated on a model system. Both analytical and simulation results demonstrate the accuracy of the equilibrium-distribution approximation method.

Algorithms↗
PubMed1995

The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system.

The Gram-positive soil bacterium Bacillus subtilis, generally regarded as an aerobe, grows under strict anaerobic conditions using nitrate as an electron acceptor and should be designated as a facultative anaerobe. Growth experiments demonstrated a lag phase of 24 to 36 hours after the shift from aerobic, to the onset of anaerobic respiratory growth. Anaerobically adapted cells grew without further lag phase after their transfer to fresh anaerobic growth medium. The cells change their morphology from rods to longer filament-like structures when moved from aerobic to anaerobic respiratory growth conditions. Surprisingly, anaerobically grown B. subtilis lost the capacity for sporulation. An investigation of the molecular basis of the switch between aerobic and anaerobic growth was initiated by the cloning of the genes encoding the respiratory nitrate reductase from B. subtilis. Oligonucleotides deduced from conserved amino acid sequence regions of eubacterial respiratory nitrate reductases and related enzymes were used for the isolation of the genes. Four open reading frames with significant homology to the E. coli respiratory nitrate reductase operons (narGHIJ, narZYWV) were isolated and termed narGHJI. A chromosomal knock-out mutation of the B. subtilis nar operon totally abolished nitrate respiration.

Amino Acid Sequence↗
PubMed1995

Spectral density function mapping using 15N relaxation data exclusively.

A method is presented for the determination of values of the spectral density function, J(omega), describing the dynamics of amide bond vectors from 15N relaxation parameters alone. Assuming that the spectral density is given by the sum of Lorentzian functions, the approach allows values of J(omega) to be obtained at omega = 0, omega N and 0.870 omega H, where omega N and omega H are Larmor frequencies of nitrogen and proton nuclei, respectively, from measurements of 15N T1, T2 and 1H-15N steady-state NOE values at a single spectrometer frequency. Alternatively, when measurements are performed at two different spectrometer frequencies of i and j MHz, J(omega) can be mapped at omega = 0, omega iN, omega jN, 0.870 omega iH and 0.870 omega iH, where omega iN, for example, is the 15N Larmor frequency for a spectrometer operating at 1 MHz. Additionally, measurements made at two different spectrometer frequencies enable contributions to transverse relaxation from motions on millisecond-microsecond time scales to be evaluated and permit assessment of whether a description of the internal dynamics is consistent with a correlation function consisting of a sum of exponentials. No assumptions about the specific form of the spectral density function describing the dynamics of the 15N-NH bond vector are necessary, provided that dJ(omega)/d omega is relatively constant between omega = omega H + omega N to omega = omega H - omega N. Simulations demonstrate that the method is accurate for a wide range of protein motions and correlation times, and experimental data establish the validity of the methodology. Results are presented for a folded and an unfolded form of the N-terminal SH3 domain of the protein drk.

Animals↗
PubMed1995

Surface plasmon resonance and its use in biomolecular interaction analysis (BIA).

Since the advent of surface plasmon resonance (SPR)-based interaction analysis techniques in 1990 the field has grown rapidly. So far, more than 220 publications and hundreds of laboratories have reported useful applications for this label-free real-time binding approach. Milestones passed during the past year include the direct detection of low molecular mass (200 Da) binding events and applications in several new fields as disparate as chaperonins, cellular adhesion, molecular biology, transcription and small-molecule screening.

Adsorption↗