Preparation and some properties of stable and carbon-14 and tritium-labeled short-chain aliphatic hydroxamic acids

A simple and safe procedure has been described for the preparation of short-chain aliphatic hydroxamic acids in quantities as large as 1 mole and as small as 0.01 mole. The procedure is equally suitable for the preparation of isotopically labeled hydroxamates, as has been demonstrated in the case of 1-¹⁴C-acetohydroxamic acid and ³H-acetohydroxamic acid. Some physical and chemical characteristics, including infrared spectra of formo-, aceto-, propiono-, and isobutyro-hydroxamic acids prepared by this method have been described.     

The Aerogenic Response of Escherichia coli and Strains of Aerobacter in EC Broth and Selected Sugar Broths at Elevated Temperatures

Gas formation by 116 strains of Escherichia coli and 104 strains of Aerobacter was determined in a specially constructed and accurately controlled water bath employing EC, lactose, maltose, sucrose, glucose, levulose, and galactose broths at temperatures ranging from 44.5 to 46.5 C.

Greatest gas activity occurred in EC broth. In the range 44.9 to 45.5 C over 92% of the E. coli cultures formed gas, but the Aerobacter strains dropped from 68 to 2%. A natural point of separation of the two groups occurred at 45.5 C.

Inhibition of the gas-forming mechanism rather than death is the universal response of the Escherichia organisms to these temperatures. The inhibition increases with rising temperatures and is readily reversible. At 46.5 C, 64.5% of all the Escherichia cultures were inhibited and 69.1% of all the cultures were actually viable.

In EC broth it was found that as a group atypical E. coli (-+--) were the most resistant gas-positive types. Least resistant in EC broth was a group of known typical fecal isolates of E. coli (++--). Of intermediate resistance between the two groups was the large body of typical E. coli (++--) organisms.

Certain individual strains of E. coli excelled in the production of gas in the variety of sugar broths tested at elevated temperatures. The Aerobacter strains did not exhibit this property.

Finally it is suggested that elevated temperature incubation studies of this type be conducted in critically controlled water baths with an ascertained accuracy in the vicinity of 45.5 ± 0.1 C under full load.

     

Urease catalysis and structure: X. Alternate bonding-site isozymes of jackbean urease

Three previously uncharacterized, nongenetic urease isozymes have been analyzed by sucrose density gradient sedimentation, gel electrophoresis, and chemical reactivity. The full complement of isozymes could be reliably generated by choosing appropriate levels of NaCl, pH, and ethylene glycol, and was stable for several days in dilute solution. The three forms of interest were found to be quaternary isomers of other isozymes, but differed from them qualitatively in their bonding sites, with disulfide bonds being substituted for noncovalent bonds. The separation of these isomer-pairs during sedimentation and electrophoresis cannot be readily explained by differences in size or charge, but must rather arise from a difference in shape. A simple two-dimensional model can provide the appropriate molecular architecture to satisfy these requirements: Only one of the two half-units in each α-urease molecule undergoes disulfide bonding during polymerization, and it does so with two adjacent molecules, thus producing asymmetric polymers from symmetric starting components.     

The anomeric form of D-fructose 1,6-bisphosphate used as substrate in the muscle and yeast aldolase reactions.

From a series of rapid quench kinetic experiments, it has been demonstrated that muscle D-fructose bisphosphate aldolase catalyzes the cleavage of beta-D-fructose 1,6-bisphosphate but not that of the alpha anomer, although the alpha anomer may be tightly bound. Yeast D-fructose bisphosphate aldolase appears to utilize both alpha and beta anomers of the substrate, with yeast apoaldolase catalyzing the interconversion of the alpha and beta forms.     

Studies on histoplasmosis IV. Elution characteristics of the Histo-inhibitory factor (HIF)

Summary The histo-inhibitory factor (HIF) derived from homogenates of liver or kidney from hamsters infected withHistoplasma capsulatum has been fractionated by column chromatography. It shows maximum absorption at 280 mµ, has a molecular weight of 142,000 and can be eluted from DEAE-cellulose or DEAE-Sephadex A—50 with 0.02 M phosphate — 4 M sodium chloride (1 : 1)HIF can be eluted fromHistoplasma yeast cells at pH 10.0, thus a greater number of positive cultures from chronic histoplasmosis could be expected to result from pretreatment of clinical specimens in glycine buffer pH 10.0 prior to culture.     

Proteomic signatures of in vivo muscle oxidative capacity in healthy adults

Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23–87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by ³¹P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τ_PCr) in the vastus lateralis muscle, with smaller τ_PCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC‐MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.     

Analogs of iso-azepinomycin as potential transition-state analog inhibitors of guanase: Synthesis,biochemical screening,and structure–activity correlations of various selectively substituted imidazo[4,5-e][1,4]diazepines

Guanase is an important enzyme of the purine salvage pathway of nucleic acid metabolism and its inhibition has beneficial implications in viral, bacterial, and cancer therapy. The work described herein is based on a hypothesis that azepinomycin, a heterocyclic natural product and a purported transition state analog inhibitor of guanase, does not represent the true transition state of the enzyme-catalyzed reaction as closely as does iso-azepinomycin, wherein the 6-hydroxy group of azepinomycin has been translocated to the 5-position. Based on this hypothesis, and assuming that iso-azepinomycin would bind to guanase at the same active site as azepinomycin, several analogs of iso-azepinomycin were designed and successfully synthesized in order to gain a preliminary understanding of the hydrophobic and hydrophilic sites surrounding the guanase binding site of the ligand. Specifically, the analogs were designed to explore the hydrophobic pockets, if any, in the vicinity of N1, N3, and N4 nitrogen atoms as well as O⁵ oxygen atom of iso-azepinomycin. Biochemical inhibition studies of these analogs were performed using a mammalian guanase. Our results indicate that (1) increasing the hydrophobicity near O⁵ results in a negative effect, (2) translocating the hydrophobicity from N3 to N1 also results in decreased inhibition, (3) increasing the hydrophobicity near N3 or N4 produces significant enhancement of inhibition, (4) increasing the hydrophobicity at either N3 or N4 with a simultaneous increase in hydrophobicity at O⁵ considerably diminishes any gain in inhibition made by solely enhancing hydrophobicity at N3 or N4, and (5) finally, increasing the hydrophilic character near N3 has also a deleterious effect on inhibition. The most potent compound in the series has a K_i value of 8.0 ± 1.5 μM against rabbit liver guanase.     

Thoracic vein ablation terminates chronic atrial fibrillation in dogs

The thoracic vein hypothesis of chronic atrial fibrillation (AF) posits that rapid, repetitive activations from muscle sleeves within thoracic veins underlie the mechanism of sustained AF. If this is so, thoracic vein ablation should terminate sustained AF and prevent its reinduction. Six female mongrel dogs underwent chronic pulmonary vein (PV) pacing at 20 Hz to induce sustained (>48 h) AF. Bipolar electrodes were used to record from the atria and thoracic veins, including the vein of Marshall, four PVs, and the superior vena cava. Radio frequency (RF) application was applied around the PVs and superior vena cava and along the vein of Marshall until electrical activity was eliminated. Computerized mapping (1,792 electrodes, 1 mm resolution) was also performed. Sustained AF was induced in 30.6 +/- 6.5 days, and ablation was done 17.3 +/- 8.5 days afterward. Before ablation, the PVs had shorter activation cycle lengths than the atria, and rapid, repetitive activations were observed in the PVs. All dogs converted to sinus rhythm during (n = 4 dogs) or within 90 min of completion of RF ablation. Rapid atrial pacing afterward induced only nonsustained (<60 s) AF in all dogs. Average AF cycle lengths after reinduction were significantly (P = 0.01) longer (183 +/- 31.5 ms) than baseline (106 +/- 16.2 ms). There were no activation cycle length gradients after RF application. We conclude that thoracic vein ablation converts canine sustained AF into sinus rhythm and prevents the reinduction of sustained AF. These findings suggest that thoracic veins are important in the maintenance of AF in dogs.     

Recurrence of hepatitis C after liver transplantation is associated with increased systemic IL-10 levels

BACKGROUND: Recurrence of hepatitis C after liver transplantation is an almost universal occurrence. T-cell derived cytokines have an important role in the development of liver damage associated with chronic hepatitis C, their post-transplant levels, however, have not been correlated with histologic recurrence of the disease. AIMS: We sought to analyze levels of TNF-alpha, soluble IL-2 receptor, IL-4 and IL-10 at 1 month, 6 months and 1 year after transplantation in 27 patients undergoing transplantation for hepatitis C related end-stage liver disease. METHODS: HCV RNA levels were monitored by a branched-chain DNA signal amplification assay. Diagnosis of recurrent hepatitis was based on 1-year protocol biopsies and on biopsies performed for liver enzyme elevations. RESULTS: Recurrent hepatitis C was detected in 52% (n=14) of the 27 patients. HCV RNA levels rose over time in all patients regardless of histologic recurrence. TNF-alpha, and IL-4 levels, although elevated, did not show specific patterns over time or in correlation with recurrence. Similarly, the early elevation followed by a gradual decrease over the first year in the amount of soluble IL-2 receptor was not related to histologic recurrence. We observed a significant increase in circulating IL-10 levels over the first year in patients with biopsy-proven recurrence, while patients with no signs of histologic recurrence displayed increased, but steady levels. CONCLUSIONS: These results suggest that while these cytokines are associated with post-transplant recurrence of hepatitis C, their production may be altered by additional factors.