Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species

Two species of Pseudomonas capable of utilizing nitroglycerin (NG) as a sole nitrogen source were isolated from NG-contaminated soil and identified as Pseudomonas putida II-B and P. fluorescens I-C. While 9 of 13 laboratory bacterial strains that presumably had no previous exposure to NG could degrade low concentrations of NG (0.44 mM), the natural isolates tolerated concentrations of NG that were toxic to the lab strains (1.76 mM and higher). Whole-cell studies revealed that the two natural isolates produced different mixtures of the isomers of dinitroglycerol (DNG) and mononitroglycerol (MNG). A monomeric, flavin mononucleotide-containing NG reductase was purified from each natural isolate. These enzymes catalyzed the NADPH-dependent denitration of NG, yielding nitrite. Apparent kinetic constants were determined for both reductases. The P. putida enzyme had a Km for NG of 52 +/- 4 microM, a Km for NADPH of 28 +/- 2 microM, and a Vmax of 124 +/- 6 microM x min(-1), while the P. fluorescens enzyme had a Km for NG of 110 +/- 10 microM, a Km for NADPH of 5 +/- 1 microM, and a Vmax of 110 +/- 11 microM x min(-1). Anaerobic titration experiments confirmed the stoichiometry of NADPH consumption, changes in flavin oxidation state, and multiple steps of nitrite removal from NG. The products formed during time-dependent denitration reactions were consistent with a single enzyme being responsible for the in vivo product distributions. Simulation of the product formation kinetics by numerical integration showed that the P. putida enzyme produced an approximately 2-fold molar excess of 1,2-DNG relative to 1,3-DNG. This result could be fortuitous or could possibly be consistent with a random removal of the first nitro group from either the terminal (C-1 and C-3) positions or middle (C-2) position. However, during the denitration of 1,2-DNG, a 1.3-fold selectivity for the C-1 nitro group was determined. Comparable simulations of the product distributions from the P. fluorescens enzyme showed that NG was denitrated with a 4.6-fold selectivity for the C-2 position. Furthermore, a 2.4-fold selectivity for removal of the nitro group from the C-2 position of 1,2-DNG was also determined. The MNG isomers were not effectively denitrated by either purified enzyme, which suggests a reason why NG could not be used as a sole carbon source by the isolated organisms.     

Omental transposition flap for gross locally recurrent breast cancer

A randomized double-blind controlled trial was undertaken to study the efficacy of a cow udder ointment versus petroalatum alone. A total of 30 patients participated in the study. Sixteen patients completed the trial with 8 of 9 patients improving in the active group and 6 of 7 patients improving in the placebo group. This difference was not statistically significant. The use of a cow udder ointment for psoriasis cannot be supported, particularly with the potential for side effects.     

Apolipoprotein B and AI distributions in the United States, 1988-1991: results of the National Health and Nutrition Examination Survey III (NHANES III)

Serum apolipoproteins (apo) B and AI were measured in a probability sample of the noninstitutionalized US civilian population, ages > or = 4 years, which included non-Hispanic whites, non-Hispanic blacks, and Mexican-Americans. Apo B concentrations were the same in males and females, lower in black males than in other males, low in childhood (approximately 0.80 g/L) and increasing to approximately 1.2 g/L in adults, and higher in younger women on hormones. Apo AI was higher in females than males, higher in blacks than in others, remained constant from childhood to adulthood (approximately 1.35 g/L) in males, but increased with age (approximately 1.30 g/L to approximately 1.55 g/L) in females, and was higher in women taking hormones. These are the first national probability estimates of apo B and apo AI in the US and are referable to the WHO-IFCC First International Reference Materials for apo AI and B.     

Protein thermostability above 100 degreesC: a key role for ionic interactions

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100 degreesC. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100 degreesC and 88 degreesC, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104 degreesC. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104 degreesC over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.     

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.     

A 68-nucleotide sequence within the 3' noncoding region of simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins

The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.     

Benign seizures associated with mild diarrhea: clinical analysis of 20 cases

Even without electrolyte imbalance, dehydration and fever, seizures are found occasionally in the cases of mild diarrhea. Such patients have shown favorable outcome during long-term follow-up. Twenty cases (10 boys and 10 girls) of benign seizure with mild diarrhea have been diagnosed at the Pediatric Department of Cathay General Hospital from January 1987 to March 1991. Ages-of-onset ranged from 3 months to 43 months (mean 19 months). Most cases were found between December and March (16 cases, 80%). Most seizure episodes were from 2 to 5 times (10 cases, 50%); the durations of seizures were mostly within a 5-minute period (16 cases, 80%). All seizures were generalized and symmetric, including tonic-clonic (13 cases, 65%), tonic (5 cases, 25%), and clonic (2 cases, 10%). Six out of 11 cases (54.5%) showed Rotazyme (Latex agglutination) positive. After six month to four year follow up, no patient had suffered any type of epilepsy, and psychomotor development was normal in all. Further discussion of clinical features is provided.     

Oral administration of rapamycin and cyclosporine differentially alter intestinal function in rabbits

The immunosuppressive drugs rapamycin (Rap) and cyclosporine A (CsA) are used clinically to modify or abolish immune-mediated functions. This study examined the effect of orally administered regimens of Rap, CsA, and a combination of Rap/CsA on intestinal function in male New Zealand white rabbits. Animals received oral doses of CsA (15 mg/kg/body weight/day), low-dose (LD) and high-dose (HD) Rap (0.25 or 1 mg/kg/body wt/day, respectively), or Rap/CsA (0.25 and 5 mg/kg/body wt/day, or 0.5 and 5 mg/kg/body wt/day, respectively) for 20 days. We measured in vitro uptake of nutrients and permeability, and morphometric measurements in the jejunum and ileum were made. Animals receiving HD-Rap or HD-Rap/CsA had decreased food intake, body weight, and intestinal weight, when compared with LD-Rap, LD-Rap/CsA, CsA, or controls. The maximal transport rate (Vmax) for the active jejunal uptake of D-glucose was increased in HD-Rap and CsA, but not in the HD-Rap/CsA-treated animals. The jejunal Vmax of D-glucose in the LD-Rap- or -Rap/CsA-treated animals was no different from controls. In the HD-Rap- and HD-Rap/ CsA-treated animals, jejunal rates of uptake of stearic, linoleic, and linolenic acids were reduced when compared with controls. Jejunal and ileal permeability (as assessed by the passive uptake of L-glucose, tissue conductance, and mucosal-to-serosal flux of [3H]inulin) was increased in animals treated with HD-Rap or HD-Rap/CsA, when compared with CsA or controls. These parameters of permeability were no different at lower doses of Rap or Rap/CsA. The jejunal and ileal villous surface area was increased in CsA, but decreased in HD-Rap or HD-Rap/CsA animals. Thus, HD-Rap given alone or in combination with CsA reduced body weight gain, in part due to reduced food intake and malabsorption of lipids, which was due at least in part to reduced intestinal surface area. The relevance of these findings to patients undergoing chronic immunosuppressive drug therapy needs to be established.