Putrescine-modified catalase with preserved enzymatic activity exhibits increased permeability at the blood-nerve and blood-brain barriers

Much evidence exists in support of the hypothesis that free radicals contribute to the pathogenesis of several neurodegenerative disorders and that mechanisms of free radical generation occur both intracellularly and extracellularly. Previous studies in this laboratory have shown that covalent modification of growth factors and antioxidant enzymes with the naturally occurring polyamine, putrescine, increases their permeability at the blood-nerve and blood-brain barriers (BNB and BBB), but does not significantly inhibit bioactivity. Furthermore, putrescine-modified superoxide dismutase (SOD) was shown to reduce neurodegeneration in a rat model of global cerebral ischemia. The purpose of the present study was to modify the antioxidant enzyme, catalase (CAT), with putrescine (PUT) at carboxylic acid groups whose ionization, and hence reactivity, was controlled with pH and investigate the effects on permeability and enzymatic activity. Modification of CAT with PUT increased its permeability 2-3-fold and preserved 67% of its enzymatic activity compared to native CAT and 137% compared to lyophilized CAT. The results of this study indicate that modification of CAT with putrescine increases its permeability while preserving enzymatic activity. PUT-SOD administered in combination with PUT-CAT may eliminate both the superoxide radical and the H2O2 produced from the dismutation of superoxide, respectively, and thus prevent the formation of hydroxyl radicals. This combination may exhibit increased neuroprotective effects, compared to native enzymes, following systemic administration for the treatment of free radical associated neurodegenerative disorders.     

Microbiological evaluation of glycerolized cadaveric donor skin

Record linkage was carried out between the national Registry of AIDS and 13 Cancer Registries (CRs) covering, in 1991, about 15% of the Italian population. Observed and expected numbers of cancers and standardized incidence ratios (SIRs) were assessed in 6067 persons with AIDS, for a total of 25,759 person-years. Significantly increased SIRs were found for Hodgkin's disease [8.9, 95% confidence interval (CI) 4.4-16.0], in which seven of 11 cases were of mixed cellularity type; invasive carcinoma of the cervix uteri (15.5; 95% CI 4.0-40.1); and non-melanomatous skin cancer (3.0, 95% CI 1.3-5.9), in which five of eight cases were basal cell carcinoma. An excess was also seen for brain tumours, but this may be partly due to misdiagnosis of brain non-Hodgkin's lymphoma or other brain diseases occurring near the time of the AIDS diagnosis. The risk for all cancer types, after exclusion of Kaposi's sarcoma (KS) and non-Hodgkin's lymphoma (NHL), was approximately twice the general population risk. An increased SIR for Hodgkin's disease in persons with AIDS is thus confirmed, though it is many times smaller than that for NHL. An association with invasive carcinoma of the cervix is also shown at a population level. The excess of non-melanomatous skin cancer seems to be lower than in transplant recipients.     

Blood transfusion. An independent risk factor for postinjury multiple organ failure

OBJECTIVE: To determine if blood transfusion is a consistent risk factor for postinjury multiple organ failure (MOF), independent of other shock indexes. DESIGN: A 55-month inception cohort study ending on August 30, 1995. Data characterizing postinjury MOF were prospectively collected. Multiple logistic regression analysis was performed on 5 sets of data. Set 1 included admission data (age, sex, comorbidity, injury mechanism, Glasgow Coma Scale, Injury Severity Score, and systolic blood pressure determined in the emergency department) plus the amount of blood transfused within the first 12 hours. In the subsequent 4 data sets, other indexes of shock (early base deficit, early lactate level, late base deficit, and late lactate level) were sequentially added. Additionally, the same multiple logistic regression analyses were performed with early MOF and late MOF as the outcome variables. SETTING: Denver General Hospital, Denver, Colo, is a regional level I trauma center. PATIENTS: Five hundred thirteen consecutive trauma patients admitted to the trauma intensive care unit with an Injury Severity Score greater than 15 who were older than 16 years and who survived longer than 48 hours. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The relationship of blood transfusions and other shock indexes with the outcome variable, MOF. RESULTS: A dose-response relationship between early blood transfusion and the later development of MOF was identified. Despite the inclusion of other indexes of shock, blood transfusion was identified as an independent risk factor in 13 of the 15 multiple logistic regression models tested; the odds ratios were high, especially in the early MOF models. CONCLUSIONS: Blood transfusion is an early consistent risk factor for postinjury MOF, independent of other indexes of shock.     

Threonine formation via the coupled activity of 2-amino-3-ketobutyrate coenzyme A lyase and threonine dehydrogenase

The enzymes L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A (CoA) lyase are known to catalyze the net conversion of L-threonine plus NAD+ plus CoA to NADH plus glycine plus acetyl-CoA. When homogeneous preparations of these two enzymes from Escherichia coli were incubated together for 40 min at 25 degrees C with glycine, acetyl-CoA, and NADH, a 36% decrease in the level of glycine (with concomitant NADH oxidation) was matched by formation of an equivalent amount of threonine, indicating that this coupled sequence of enzyme-catalyzed reactions is reversible in vitro. Several experimental factors that affect the efficiency of this conversion in vitro were examined. A constructed strain of E. coli, MD901 (glyA thrB/C tdh), was unable to grow unless both glycine and threonine were added to defined rich medium. Introduction of the plasmid pDR121 (tdh+kbl+) into this strain enabled the cells to grow in the presence of either added glycine or threonine, indicating that interconversion of these two amino acids occurred. Threonine that was isolated from the total pool of cellular protein of MD901/pDR121 had the same specific radioactivity as the [14C]glycine added to the medium, establishing that threonine was formed exclusively from glycine in this strain. Comparative growth rate studies with several strains of E. coli containing plasmid pDR121, together with the finding that kcat values of pure E. coli 2-amino-3-ketobutyrate CoA lyase favor the cleavage of 2-amino-3-ketobutyrate over its formation by a factor of 50, indicate that the biosynthesis of threonine is less efficient than glycine formation via the coupled threonine dehydrogenase-2-amino-3-ketobutyrate lyase reactions.     

Reversed-field pinch studies in the Madison Symmetric Torus

Studies of large-size (R=1.5 m,a=0.5 m), moderate current (I <750 kA) reversed-field pinch (RFP) plasmas are carried out in the Madison Symmetric Torus in order to evaluate and improve RFP confinement, study general toroidal plasma MHD issues, determine the mechanism of the RFP dynamo, and measure fluctuation-induced transport and anomalous ion heating. MST confinement scaling falls short of the RFP scaling trends observed in smaller RFPs, although the plasma resistance is classical. MHD tearing modes with poloidal mode numberm=1 and toroidal mode numbersn=5–7 are prevalent and nonlinearly couple to produce sudden relaxations akin to tokamak sawteeth. Edge fluctuation-induced transport has been measured with a variety of insertable probes. Ions exhibit anomalous heating, with increases of ion temperature occurring during strong MHD relaxation. The anomalous heating fraction decreases with increasing density, such that ion temperatures approach the lower limit given by electron-ion friction. The RFP dynamo has been studied with attention to various possible mechanisms, including motion-EMF drive, the Hall effect, and superthermal electrons. The toroidal field capacity of MST will be upgraded during Summer 1993 to allow low-current tokamak operation as well as improved RFP operation.     

Children''s rights and the biological bias in biological parent versus third-party custody disputes

Hydrophobically modified carboxymethylpullulan (HMCMP) samples were obtained by reaction of small amounts of C16 alkylamine on carboxylic groups of the corresponding polyacid. The molar contents of alkyl chains ranged from 1.3 to 6.8% with respect to the anhydroglucose units (AGU) and the degree of substitution (DS) of carboxylic groups varied from 0.76 to 0.84. Solution properties of the sodium salt of HMCMPs were studied mainly by viscometric and size-exclusion chromatography/light-scattering methods. The low-shear viscosity of modified pullulans in 0.1 M NaCl solutions drastically increases with the hydrophobic content and polymer concentrations and the 6.8% modified sample has a quite pseudoplastic behaviour. These data showed that the polymers aggregated intermolecularly and displayed a compact globular structure in dilute solution. Furthermore, addition of NaCl or ethanol induced a decrease in viscosity although the molecular weights remained approximately constant. These results are consistent with a collapse of the polysaccharide aggregates.     

Occurrence of homologs of the Escherichia coli lytB gene in gram-negative bacterial species

The Escherichia coli LytB protein regulates the activity of guanosine 3',5'-bispyrophosphate synthetase I (RelA). A Southern blot analysis of chromosomal DNA with the E. coli lytB gene as a probe revealed the presence of lytB homologs in all of the gram-negative bacterial species examined but not in gram-positive species. The lytB homologs from Enterobacter aerogenes and Pseudomonas fluorescens complemented the E. coli lytB44 mutant allele.     

Radiography of the medial elbow ligaments

The anterior, posterior, and transverse bundles of the medial collateral ligaments of fresh cadaveric elbows were painted with a special radiopaque tantalum cyanoacrylic preparation. The extent of each ligament and their origins and insertions were examined with radiography. This study provided radiographic correlation for these important soft tissue structures and an anatomic understanding of elbow fractures with ligament avulsion.     

L-threonine dehydrogenase. Purification and properties of the homogeneous enzyme from Escherichia coli K-12

L-Threonine dehydrogenase, which catalyzes the conversion of L-threonine to aminoacetone + CO2 presumably via the intermediate formation of alpha-amino-beta-ketobutyrate, has been purified to apparent homogeneity from extracts of a mutant of Escherichia coli K-12 which has constitutively derepressed levels of the enzyme. Three fractionation steps were used including controlled heat denaturation, DEAE-Sephadex chromatography, and blue dextran-Sepharose affinity chromatography. The purified enzyme migrated as a single band, coincident with dehydrogenase activity, when electrophoresed on polyacrylamide gels at pH 8.0 and 9.5. Electrophoresis in 1% sodium dodecyl sulfate also showed one band and a single schlieren peak was seen during sedimentation velocity centrifugation. The enzyme has an apparent molecular weight of 140,000 +/- 4,000 as determined by sucrose density and sedimentation equilibrium centrifugation. Based on electrophoresis in 1% sodium dodecyl sulfate, sedimentation equilibrium centrifugation in 6 M guanidine.HCl, and cross-linking with dimethyl suberimidate, the molecule is a tetramer consisting of identical (or nearly identical) subunits with Mr approximately equal to 35,000. L-Threonine dehydrogenase is specific for NAD+ or NAD+ analogs and utilizes L-threonine, D-allothreonine, or L-threonine amide as the best substrates. In 50 mM Tris.HCl buffer (pH 8.4) and 37 degrees C, the Km values for L-threonine and NAD+ are 1.43 and 0.19 mM, respectively. The enzyme has a pH optimum of 10.3, is activated by Mn2+, and shows a substantial loss of activity when treated with certain sulfhydryl-reacting reagents.