Critical Amino Acid Residues for Nicotine 5＇ -Hydroxylation in Human CYP2A Enzymes

Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK） and aflatoxin BI（AFB1） carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods： A series of reciprocally substituted mutants of CYP2A6lle^300→ Phe, CYP2A6Gly^301aAla, CYP2A6Ser^369 → Gly, CYP2A13Phe^300→ Ile, CYP2A13Ala^301 → Gly and CYP2A13Gly^369 → Set were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5＇hydroxylatin by wild type and mutant CYP2A proteins was performed. Results：All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile^300→ Phe and CYP2A6Gly^301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe^300→Ile and CYP2A13Ala^301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substitution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser^369 → Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly^369 → Ser. Conclusion：#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5＇ -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Set369 to Gly substitution indirectly affected nicotine binding by     

Algorithms for identification of Guillain–Barré Syndrome among adolescents in claims databases

Background

Health insurance claims databases can provide data for studies of vaccine-related Guillain–Barre’ Syndrome (GBS), but not all patients with a diagnostic ICD-9-CM code for GBS have the disease. The objective of this study was to evaluate the positive predictive values (PPVs) of claims-based algorithms for identifying GBS cases in 4 claims database environments.

Methods

Potential cases were adolescents ages 11–21 with at least one claim for GBS (ICD-9-CM code 357.0). Medical record reviews by a panel of 3 neurologists were conducted for case confirmation. Claims data considered for inclusion in the case-ascertainment algorithm included coding position, physician specialty, visit type, diagnostic tests. PPVs were used to assess the contribution of study factors in predicting case status.

Results

Among 361 individuals with a GBS diagnosis code, 106 were confirmed overall (PPV = 0.29), varying from 0.24 to 0.56 across the 4 sites. Requiring the GBS code to be associated with a neurologist visit (PPV = 0.53) or to be in a primary position on an inpatient claim (0.56) improved the performance. A composite algorithm including a primary inpatient GBS code and a neurologist visit associated with any GBS code gave the highest PPV (0.70). Incorporating claims for diagnostic testing had little impact on the PPV. Findings were generally similar across study sites.

Conclusions

Algorithms were able to identify GBS cases better than the single occurrence of the diagnostic code for GBS, and these algorithms may perform similarly in different claims environments.     

Effect of long‐term depletion of plasma methionine on the growth and survival of human brain tumor xenografts in athymic mice

Depletion of plasma methionine is expected to inhibit or reverse growth of methionine‐dependent tumors; however, modulation of methionine and other sulfur amino acids is not a trivial task in experimental animals. l‐Methioninase from Pseudomonas putida at 1,000 U/kg causes acute reduction of plasma methionine by 80% in mice, but recovery occurs within 14 hours. Restriction of dietary choline and replacement of dietary methionine with homocystine results in 50% chronic reduction of plasma methionine. A >70% reduction can be accomplished with a diet deficient in methionine, homocystine, and choline, but ultimately this diet is lethal. Plasma methionine can be lowered to a steady state of <5 μM in mice with a combination of dietary restriction of methionine, homocysteine, and choline and synchronous treatments with intraperitoneal injections of 1,000 U/kg L‐methioninase and 25–50 mg/kg homocystine, each administered at 12‐hour intervals. Modulation of plasma methionine by this means causes no weight loss or pathologies in liver or pancreas, and it does not markedly alter levels of cysteine, homocysteine, or glutathione in plasma or in hepatic tissue. When this procedure is applied to athymic mice bearing human medulloblastoma (Daoy) tumors subcutaneously, tumor growth is inhibited. Methionine deprivation arrests mitosis by blocking the cell cycle in G2 and induces apoptosis. Tumor stasis was achieved in 100% of treated animals within 4 days of treatment, and regression was seen in one‐third of animals after a 10‐day period. These data strongly support the use of methionine‐depleting regimens for tumor treatments.     

A novel tetrameric gp350 as a potential Epstein–Barr virus vaccine

Infectious mononucleosis and B-cell transformation in response to infection with Epstein–Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on B-cells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1–470) gp350 protein (gp350^1–470). Tetrameric gp350^1–470 induced ∼20-fold higher serum titers of gp350^1–470-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp350^1–470. Further, epidermal immunization with plasmid DNA encoding gp350^1–470 tetramer induced 8-fold higher serum titers of gp350^1–470-specific IgG relative to monomer. Tetrameric gp350^1–470 binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp350^1–470 had no effect on the gp350^1–470-specific IgG response in naïve mice, and resulted in suppressed gp350^1–470-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp350^1–470 is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.     

Preservation of alpha-tocopherol in sunflower oil by herbs and spices

The ability of some commercially available herb and spice extracts to preserve alpha-tocopherol in sunflower oil during heating at 85-105°C was assessed using sunflower oil as a model system. The Rancimat® was evaluated for the heating stage and was used throughout as it was shown to be viable: α-tocopherol did not evaporate under the test conditions. The delay in the onset of rancidity was found to be directly related to the initial α-tocopherol concentration (P < 0.01). Rosemary, thyme, turmeric, sage, oregano and cumin extracts (2000 mg.kg^?1) delayed rancidity (P < 0.01) and preserved α-tocopherol (P < 0.01). Some preservation was observed with clove extract but coriander and cardamom extracts were pro-oxidants. With thyme extract, the log of the induction time (as an indicator of the delay in rancidity) was directly proportional to the temperature (85-100°C). The ethyl acetate, hexane and methanol extracts of fresh sage were effective for preserving α-tocopherol (P < 0.01). With thyme, rosemary and sage extracts, the increase in the preservation of α-tocopherol was directly related to the concentration of the herb extract (P < 0.01) and was quite effective even at 100 mg.kg^?1. The increased delay in the onset of rancidity was due directly to the improved preservation of α-tocopherol (P < 0.01). In further experiments, the preservative effect of turmeric was shown not to be due to its reported major antioxidant, curcumin, even though it delayed rancidity. When herb/spice extracts were examined mixed with thyme, bay and turmeric showed synergism (P < 0.01) whereas bay alone was slightly inhibitory. The mode of action appeared to be due to free radical activity rather than through singlet oxygen generation.     

Absorption of Pesticides in a Chronic Skin Disease

The case of a 64-year-old pesticide formulator is presented. Despite the fact that the patient had no more exposure to dieldrin and several other organochlorine pesticides than his occupational associates, and that he used protective clothing, he, nonetheless, developed relatively high levels of these chemicals in his blood and tissues. The high levels are thought to be the result of increased absorption of these materials through his atrophic, ulcerated, crusted skin. These skin lesions were caused by the disease scleroderma. The findings suggest that persons with chronic skin diseases should not be employed to formulate pesticides, or at least that they use extra precautions while working at this occupation.     

The Diagnosis of Acute Poliomyelitis

Unfortunately, in the past, the medical attitude toward hemiplegia has been one of hopelessness and helplessness. If a dynamic approach is used, however, the hemiplegic is not a lost cause. Spot checks have shown that 90 per cent can be taught ambulation, self-care and urinary and fetal continence, and 40 per cent can be taught to do gainful work.