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Biomedical subjects

T Nowak

Publications and source records attributed to T Nowak.

At least 55 records · Page 3Linked to original sources
PubMed1993

Inactivation of hepatitis A virus by heat treatment in aqueous solution.

Hepatitis A virus infections have been reported recently among hemophilic patients in Italy and Germany, leading to speculation that infectious hepatitis A virus (HAV) might have been present in some factor VIII concentrates. In both cases, the implicated factor concentrates had been treated by a solvent/detergent method, which inactivates enveloped viruses but which would not be expected to inactivate HAV, a nonenveloped picornavirus. To determine whether HAV would be inactivated during pasteurization of factor VIII concentrate, an alternative method employed for virus inactivation, we determined the extent to which the infectivity of cell culture-adapted HAV, suspended either in cell culture medium or in a proprietary stabilizing buffer, was reduced by heat treatment at 60 degrees C for 10 hr. The titer of infectious HAV declined rapidly at 60 degrees C, but the stabilizer considerably delayed HAV inactivation. In cell culture medium, HAV was inactivated by > 3.6 log10 within 30 min, but 3.6 log10 inactivation of HAV was reached only after 6 hr in the presence of the stabilizer. Residual infectious HAV was present after even 10 hr of heat treatment in the stabilizer, indicating that < 5.2 log10 infectious HAV particles are inactivated under these conditions. In the presence of the stabilizer, HAV was significantly more stable than poliovirus type 1, which has been used to validate virus inactivation by pasteurization. We conclude that pasteurized factor VIII concentrate should pose little if any risk for transmission of HAV if pooled plasma used for its manufacture contained low levels of the virus.

Animals↗
PubMed1993

[Results of treating chronic myelogenous leukemia in children].

Retrospective analysis of the results of treatment chronic myeloid (CML) leukemia in 80 children was done. From among 72 children treated by conventional methods the probability of 10 years survival had 17%. Statistically significant better results was obtained in the group of children with adult than juvenile type of CML (23 and 8%). Allogenic bone marrow transplantation was performed in six children, two children were treated with interferon. New approaches of treatment may offer the better chance for constitution of normal bone marrow function.

Adolescent↗
PubMed1993

Theoretical and technical concerns in inactivation/elimination of viruses in plasma derivatives.

To know the virus eliminating/inactivating capacity of the manufacturing process of a plasma protein, it is essential to analyse it by adding virus to the source material or to different materials obtained at various stages of the manufacturing procedure and then to determine the elimination/inactivation of this virus. To carry out such experiments properly, three prerequisites have to be fulfilled: (i) the manufacturing procedure must be scaled down as exactly as possible; (ii) relevant test viruses have to be selected for the spiking experiments and (iii) the resulting samples must be assayed properly for infectious virus. The successful reduction of a manufacturing procedure to a more than 1000-fold smaller scale has to be validated to prove that it corresponds to the production scale. The most important viruses of risk in human plasma are hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). HIV is the only one of these viruses which can be tested in vitro. A decision has therefore to be made concerning which other viruses should be used. The selection of test viruses depends on (i) the relationship of these candidates to the viruses of risk; (ii) the possibility of growing them to high titres in vitro and (iii) the availability of accurate infectivity assays. The use of highly sensitive assays is necessary to be able to determine small amounts of residual viruses. Since such assays are based on a 7 to 28 day incubation of the virus samples on cell cultures, these samples must be sterile and non-cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Products↗
PubMed1993

Inactivation of HIV, HBV, HCV related viruses and other viruses in human plasma derivatives by pasteurisation.

Laboratory studies were made to verify the capacity and efficacy of the pasteurisation step (heat treatment at 60 degrees C for 10 hours in stabilized aqueous solution) in inactivating various pathogenic viruses (e.g. HIV, HBV, HCV related viruses, HAV, HSV, poliovirus). These experiments proved that pasteurisation effectively inactivates a broad spectrum of viruses with a range of different physicochemical properties. The results, summarized in inactivation kinetics, demonstrate that complete inactivation of high quantities of both enveloped (e.g. HIV, HCV related viruses) and non-enveloped (e.g. poliovirus) viruses takes place during pasteurisation. In clinical trials on the virus safety of plasma derivatives no case of AIDS, hepatitis B or C occurred. From these data we conclude that heat treatment at 60 degrees C in a stabilized aqueous solution confers a high margin of safety on human plasma protein preparations regarding the transmission of diseases such as AIDS or hepatitis.

Biological Products↗
PubMed1992

Influence of pH on the Mn2+ activation of and binding to yeast enolase: a functional study.

The influence of pH on the activation of yeast enolase by Mn2+ was measured by steady-state kinetics. The pH influence on the binding of Mn2+ to apoenolase and the enolase-substrate complex was measured by EPR spectroscopy. At pH values above 6.6, activation by Mn2+ is fit by Michaelis-Menten kinetics, but at higher concentrations of Mn2+, inhibition is observed. Under conditions analogous to the kinetic studies, the enzyme binds two Mn2+ per dimer with a Kd in the micromolar range. In the presence of the substrate 2-phosphoglycerate, three thermodynamically distinct cation binding sites per monomer are detected and the binding constants are determined by a fit to the data. As the pH decreases, the reaction velocity decreases and the cation inhibition becomes minimal. Under these conditions, only two Mn2+ binding sites per monomer are observed; the third site must be the inhibitory site. The velocity and kinetic constants are minimally affected by buffer except at pH 5.8 with PIPES. Under these conditions, the velocity is only about 40% that observed with other buffers and only a single binding site for Mn2+ per monomer is detected in the presence or absence of substrate. A direct role in the catalytic mechanism by the second cation is called to question. The binding constant for Mn2+ at site I is independent of pH over the range from 7.5 to 5.2, and the binding at site II increases only slightly over this same pH range. These results indicate that the cation sites at positions I and II contain ligands that are pH independent over this range.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

Metal ion specificity at the catalytic site of yeast enolase.

A new, more gentle enzyme purification for yeast enolase was developed. A series of kinetic experiments was performed with yeast enolase where the concentration of Mg(II) is kept constant and at the Km' level; the addition of Mn(II), Zn(II), or Cu(II) gives a hyperbolic decrease in the enzyme activity. The final velocity of these mixed-metal systems is the same as the velocity obtained only with Mn(II), Zn(II), or Cu(II), respectively. The concentration of the second metal that gives half-maximal effect in the presence of Mg(II) is approximately the same as the apparent Km (Km') value measured for that cation alone. Direct binding of Mn(II) to apoenolase in the absence and presence of Mg(II) shows that Mn(II) and Mg(II) compete for the same metal site on enolase. In the presence of D-2-phosphoglycerate (PGA) and Mg(II), only a single cation site per monomer is occupied by Mn(II). Water proton relaxation rate (PRR) studies of enzyme-ligand complexes containing Mn(II) and Mn(II) in the presence of Mg(II) are consistent with Mn(II) binding at site I under both conditions. PRR titrations of ligands such as the substrate PGA or the inhibitors orthophosphate or fluoride to the enolase-Mn(II)-Mg(II) complex are similar to those obtained for the enolase-Mn(II) complex, also indicating that Mn(II) is at site I in the presence of Mg(II). High-resolution 1H and 31P NMR was used to determine the paramagnetic effect of enolase-bound Mn(II) on the relaxation rates of the nuclei of the competitive inhibitor phosphoglycolate. The distances between the bound Mn(II) and the nuclei were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

Virus safety of human immunoglobulins: efficient inactivation of hepatitis C and other human pathogenic viruses by the manufacturing procedure.

Human immunoglobulins are plasma derivatives with a low risk of transmitting viral infections. To the present, no proven case of human immunoglobulins transmitting human immunodeficiency viruses has been reported. However, there have been a few reports on the transmission of hepatitis C virus by these plasma proteins. To improve further the safety of both 5s iv human immunoglobulins and 7s im immunoglobulins, we introduced a 10-hour heat treatment of the aqueous solutions at 60 degrees C (i.e., pasteurization) into the manufacturing procedure. This treatment was not added to the manufacturing procedure of 7s iv immunoglobulin that already contained the S-sulfonation as a virus inactivating method. We now report on experimental data that show that the whole manufacturing procedures of the above immunoglobulins inactivate efficiently hepatitis C virus and that the specific virus inactivation methods alone, namely, pasteurization or S-sulfonation, also inactivate completely viruses of the flavivirus family, to which the hepatitis C virus belongs. The inactivation of the Flaviviridae bovine viral diarrhea virus, tick-borne encephalitis virus, and yellow fever virus by pasteurization or S-sulfonation was at least 10(5). The clearance of HCV achieved by the entire manufacturing process of each of these immunoglobulins was also at least 10(5). The experiments therefore show that pasteurization or S-sulfonation provides a high margin of safety to human immunoglobulins regarding the transmission of hepatitis C virus.

Animals↗
PubMed1992

25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase.

25Mg NMR spectroscopy was used to study the interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase (PK). Titration of Mg2+ with enolase allows for the calculation of 1/T2 for Mg2+ bound at site I of 1510 s-1 and a quadrupolar coupling constant chi = 0.30 MHz. Titration of Mg2+ with enolase in the presence of 2-phosphoglycerate (PGA) and Zn2+, where Zn2+ binds specifically at site I, gives a 1/T2 for Mg2+ bound at site II of 4000 s-1 (chi = 0.49 MHz). The Mg2+ at site II appears to be more anisotropic than Mg2+ at site I. The titration of site I of the enolase-Mg-PGA-Mg complex with Zn2+ or Mn2+ shows a simple displacement of the Mg2+. No paramagnetic effects by Mn2+ on 25Mg relaxation were observed. Temperature studies of the 25Mg resonance show that fast exchange of the Mg2+ occurs under these conditions. From the lack of a paramagnetic effect, the distance between the cations at sites I and II must be more than 6-9 A. This distance limits the location, hence the function, of the cation at site II for catalytic activity. Titration of Mg2+ with PK gives a 1/T2 for bound Mg2+ of 2200 s-1 (chi = 0.24 MHz). A titration of Mg2+ with PK in the presence of the inhibitor oxalate gives a 1/T2 of 400 s-1. The temperature dependence of 25Mg relaxation in the PK-Mg-oxalate complex is consistent with slow exchange (Ea = 6.1 +/- 1.6 kcal/mol). The enzyme-bound cation is more tightly sequestered by the addition of a ligand that binds directly to the cation. An investigation of the 25Mg relaxation in the PK-Mn-oxalate-Mg-ATP complex, where the Mg2+ is bound to the nucleotide and the Mn2+ was enzyme bound, was not successful due to precipitation of PK under experimental conditions and the short T2 relaxation for 25Mg in this complex. The applications of 25Mg NMR have been useful in partially describing the properties of the bound Mg2+ in these two metal-requiring enzymes.

Adenosine Triphosphate↗
PubMed1992

Follow-up of hepatitis C virus infection in chimpanzees: determination of viraemia and specific humoral immune response.

Chimpanzees were inoculated intravenously with the H strain of hepatitis C virus (HCV), and analysed for viraemia using the polymerase chain reaction and for a humoral immune response using first and second generation anti-HCV ELISAs and an immunoblot assay (4-RIBA). In all seven chimpanzees studied, viraemia occurred several weeks before a significant increase in serum alanine transferase (ALT) activity, whereas the first circulating anti-HCV antibodies became detectable at the time of significant increase in ALT levels, provided the second generation ELISA or 4-RIBA was used. On the basis of the duration of viraemia the chimpanzees studied could be assigned to two different groups: those in which viraemia disappeared in conjunction with or shortly after seroconversion, and those remaining viraemic for many weeks after the appearance of antibodies. The clearance of HCV from the circulation did not correlate with the antibody pattern determined using 4-RIBA, i.e. the HCV-specific assays currently available do not enable us to predict whether an infected chimpanzee will develop persistent viraemia. Only two of the seven chimpanzees analysed developed anti-core protein (c-22) antibodies, which appeared at the same time as the first ALT peak, whereas all animals developed antibodies to the non-structural protein, c-33, and these antibodies persisted.

Alanine Transaminase↗
PubMed1991

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase.

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

Animals↗
PubMed1991

Inactivation of retroviruses in biologicals manufactured for human use.

Human immunodeficiency virus may occur in human plasma and mammalian retroviruses in established cell lines used for the production of monoclonal antibodies or recombinant proteins. To avoid any risk of retrovirus infections being transmitted to human patients by human plasma proteins or other biologicals obtained from established cell lines, these products must be free of contaminating retroviruses. This can be achieved by excluding contaminated source material and by establishing manufacturing procedures which inactivate and eliminate retroviruses. Some methods were investigated, and it was proved that these methods not only completely inactivate retroviruses, but that they are at the same time mild enough not to destroy the biological activities of the substances to be used as therapeutics. In addition, the clearance of retroviruses by the entire manufacturing process of an immunoglobulin, human plasminogen, a murine monoclonal antibody and a recombinant human protein were studied. In each case cumulative clearance factors of greater than 10(12) were achieved for the retroviruses used in these spiking experiments. It can be concluded that these high clearance factors contribute a very high margin of safety to the biologicals manufactured.

Animals↗
PubMed1991

[Retrospective analysis of the results of the treatment of children with histiocytosis].

The results of the treatment of 26 patients with histiocytosis hospitalized in the years 1970-1990 were evaluated. The age of the children at the time of diagnosis was from 4 months to 14 years, mean 32 months. In 4 cases the changes were restricted to the skeletal system, in the remaining 22 patients generalized histiocytosis was present with involvement of the skeleton and/or internal organs, and/or bone marrow. In the group with generalized histiocytosis 3 children had respiratory failure, 5 had hepatic failure, and 6 bone marrow failure. Full remission was obtained in 19 cases. Another 4 ones had persistent focal changes in the bones. Three children died of disease progression. Thus the probability of 10-year survival was for the whole group 0.86 +/- 0.13.

Adolescent↗
PubMed1990

Analogs of oxalacetate as potential substrates for phosphoenolpyruvate carboxykinase.

Structural analogs of the substrate oxalacetate were examined as potential substrates and inhibitors for chicken liver mitochondrial phosphoenolpyruvate (P-enolpyruvate) carboxykinase. Steady-state kinetics were employed to characterize the inhibitory effects of these substrate analogs with the enzyme. Assays were carried out in both carboxylation and decarboxylation reaction directions. Pyruvate, beta-hydroxypyruvate, beta-mercaptopyruvate, beta-fluoropyruvate, DL-lactate, glycolate, glycoaldehyde, glyoxylate, glyphosate, and DL-aspartate showed no inhibitory effects by steady-state kinetics. Oxalate, acetopyruvate, and DL-, D-, and L-glycerate exhibited weak noncompetitive inhibition of the P-enolpyruvate carboxykinase-catalyzed reaction. DL-3-Nitro-2-hydroxypropionic acid, 3-nitro-2-oxopropionic acid, DL-malate, malonate, tartronate, and alpha-ketobutyrate all show weak inhibition with estimated inhibition constants greater than 20 nM. Several of these compounds were investigated by 31P NMR to determine if they function as phosphoryl acceptors for GTP. None of the compounds tested act as phosphoryl acceptors in the enzyme-catalyzed reaction. Chicken liver mitochondrial phosphoenolpyruvate carboxykinase shows a remarkably high degree of specificity at the binding site of oxalacetate.

Animals↗
PubMed1990

Description of a procedure which allows isolation of viral nonstructural proteins from BHK vertebrate cells infected with the West Nile flavivirus in a state which allows their direct chemical characterization.

We have developed a procedure which allows isolation of virus-coded nonstructural (NS) proteins from BHK cells infected with the West Nile (WN) flavivirus in a state of purity which allows their chemical characterization. A crude membrane fraction proved to be a suitable starting material. Incubation of crude membranes in buffer containing 1.2 M guanidine hydrochloride (GH) allows isolation of weakly washed membranes. Incubation of weakly washed membranes in the presence of either 5 M GH or 8 M urea allows the isolation of stringently washed membranes, as well as a soluble protein-containing wash which is called the differential wash. Stringently washed membranes contain proteins with apparent molecular weights of 14, 19, 23, 29, and 50 kDa as predominant constituents. These proteins are of sufficient purity after SDS-PAGE to allow amino-terminal sequence determination. Together with the genome RNA sequence these analyses show that these molecules represent the virus-coded proteins NS 2b, NS 2a, pre M, NS 4b, and E, respectively. Similar analysis of the proteins present in the differential wash shows that the proteins NS 5, NS 3, and NS 1 are major constituents of this material. The carboxy-terminal sequences of NS 5 and NS 1 have also been determined.

Amino Acid Sequence↗
PubMed1990

Catatonia and burns.

Two case reports describing symptoms of catatonia associated with thermal injury are reported. The incidence of catatonia in a burn unit was found to be about three times that in a general hospital.

Adult↗
PubMed1990

[Changes in RCBF and edema after transient cerebral ischemia--ischemic threshold of postischemic hypoperfusion].

Brain damage after transient cerebral ischemia may be related to changes in postischemic cerebral blood flow and brain edema. In this study, the relationship between postischemic cerebral blood flow and edema was evaluated in the gerbil. Bilateral carotid occlusion (for 1, 1.5, 5, 15, or 30 min) was carried out in 110 female gerbils (50-70 g) under anesthesia with 2% halothane in 30% O2 and 70% NO2. Cerebral blood flow was measured by a [14C]-iodoantipyrine method modified slightly for use in small animals, and brain edema was evaluated by a specific gravity method. The threshold duration of ischemia which gives rise to subsequent hypoperfusion or edema was also established. In another 52 female gerbils under the same anesthesia, we investigated the effect of ischemia of variable duration on postischemic blood pressure and blood gas. Throughout all experiments, rectal temperature was maintained at 37-38 degrees C with a heating pad. Student's t test was used to calculate statistical significance. Neither blood pressure nor blood gas did vary significantly at any time following recirculation. Each brain region showed the same pattern of blood flow change, one almost independent of duration of occlusion. Namely, after the release of occlusion, transient recovery of blood flow was observed. But the flow then fell to 30-40% of the normal value at 1 h, after which it returned up to normal at 6 h. The severity of postischemic hypoperfusion was also independent of occlusion time. Interestingly, we did not observe postischemic hyperemia in any experimental groups except mild hyperemia in hippocampus after 5 and 15 min ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A histidine residue at the active site of avian liver phosphoenolpyruvate carboxykinase.

The histidine-selective reagents diethylpyrocarbonate (DEPC) and dimethylpyrocarbonate were used to study active site residues of phosphoenolpyruvate carboxykinase. Both reagents show pseudo first-order inhibition of enzyme activity at 22 +/- 1 degree C with calculated second-order rate constants of 2.8 and 4.6 M-1 s-1, respectively. The inhibition appears partially reversible. Substrates affect the rate of inhibition: KHCO3 enhances the rate, Mn2+ has little effect, and phosphoenolpyruvate decreases the rate. The best protection is obtained by IDP or IDP and Mn2+. The kinetic studies show that modification of histidine is specific and leads to loss of enzymatic activity. Two histidines per enzyme are modified by DEPC, as measured by an absorption change at 240 nm, in the absence of substrate, leading to loss in activity. One histidine per molecule is modified in the presence of KHCO3, giving inactivation. Cysteine and lysine residues are not affected. A study of the inhibition rate constant as a function of pH gives a pKa of 6.7. Enzyme modified by DEPC in the absence of substrate (1% remaining activity) shows no binding of ITP or of phosphoenolpyruvate to the enzyme.Mn2+ complex as studied by proton relaxation rates. When enzyme is modified in the presence of KHCO3 (44% remaining activity), ITP and KHCO3 bind to the enzyme.Mn2+ complex similarly to the binding to native enzyme. Phosphoenolpyruvate binding to modified enzyme.Mn results in an enhancement of proton relaxation rates rather than the decrease observed with native enzyme.Mn. The CD spectra of histidine-modified enzyme show a decrease in alpha-helical and random structure with an increase in anti-parallel beta-sheet structure compared to native enzyme. These results show that avian phosphoenolpyruvate carboxykinase has 2 histidine residues which are reactive with DEPC and dimethylpyrocarbonate, and one of the 15 histidine residues in the protein is at or near the phosphoenolpyruvate binding site and is involved in catalysis.

Animals↗