Apparent association constants of tRNAs for the ribosomal A, P, and E sites.

Association constants for tRNA binding to poly(U) programmed ribosomes were assessed under standardized conditions with a single preparation of ribosomes, tRNAs, and elongation factors, respectively, at 15 and 10 mM Mg2+. Association constants were determined by Scatchard plot analysis (the constants are given in units of [10(7)/M] measured at 15 mM Mg2+): the ternary complex Phe-tRNA.elongation factor EF-Tu.GTP (12 +/- 3), Phe-tRNA (1 +/- 0.4), AcPhe-tRNA (0.7 +/- 0.3), and deacylated tRNA(Phe) (0.4 +/- 0.15) bind with decreasing affinity to the A site of poly(U)-programmed ribosomes. tRNA(Phe) (7.2 +/- 0.8) binds to the P site with higher affinity than AcPhe-tRNA (3.7 +/- 1.3). The affinity of the E site for deacylated tRNA(Phe) (1 +/- 0.2) is about the same as that of the A site for AcPhe-tRNA (0.7 +/- 0.3). At lower Mg2+ concentrations the affinity of the E site ligand becomes stronger relative to the affinities of the A site ligands. Phe-tRNA and ternary complexes can occupy the A site at 0 degrees C in the presence of poly(U) even if the P site is free, whereas, as already known, deacylated tRNA or AcPhe-tRNA bind first to the P site of programmed ribosomes. Hill plot analyses of the binding data confirm an allosteric linkage between A and E sites in the sense of a negative cooperativity.     

Pro-(carboxypeptidases A) from whole pig pancreas. Their mass, size, shape and solvation.

Sedimentation analysis and light-scattering measurements were made with the two forms of pig pancreas pro-(carboxypeptidase A), in order to determine some of their physical properties. The following values were found (the first value applies to the binary complex and the second one to the monomer). The A 1%/280.1 cm values were 19.9 +/- 0.3 and 16.3 +/- 0.3. The partial specific volumes v -0 were 0.707 +/- 0.016 cm3/g and 0.714 +/- 0.015 cm3/g. The sedimentation coefficients S 0/20,w were 4.90 +/- 0.15S and 3.75 +/- 0.15 S. The diffusion coefficients D 0/20,w were (5.8 +/- 0.1) X 10(-7) cm2/s and (6.95 +/- 0.15) X 10(-7) cm2/s. From these data the following values were calculated. Relative molecular masses Mr were 71 000 +/- 4000 and 46 000 +/- 3000. The frictional ratios f/fmin. were 1.37 +/- 0.06 and 1.31 +/- 0.07; assuming a value for the solvation of the molecules (delta = 0.5 g/g) the asymmetry values range from 3 to 5 for the binary complex and from 2 to 4 for the monomer. The Mr values found in the present work coincide with those found by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate [Martínez, Avilés, SanSegundo & Cuchillo (1981) Biochem. J. 197, 141-147]. Therefore the low values obtained by those authors when using gel-filtration chromatography must be the result of the interaction of the zymogens with the gel matrix, as the asymmetry is too small to justify the large discrepancies found.     

A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.

A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.     

Relationship between the "phospholipid effect" and calcium in the thyroid. I. - Effects of calcium ions, E.G.T.A., ionophore A 23187, verapamil and chlorpromazine on resting and stimulate thyroid slices.

The "phospholipid effect" which is the enhanced turnover of the phosphorylinositol group of phosphatidylinositol (PI) occurs in the thyroid of response to thyreostimulin (TSH). The possibility that Ca2+ ions are involved in this stimulation has been investigated with pig thyroid slices. Experiments performed in media without Ca2+ or containing E.G.T.A. (2 mM), indicate that it is not the extracellular Ca2+ which is implied, but rather the intracellular Ca2+. The ionophore A23187 (6.10(-6) M) increases the specific radioactivity of the acid soluble precursors, but has also a specific effect on the PI turnover, which is additive with the effect of a high concentration of TSH (50 mU/ml). Washing and loading of slices with various Ca2+ concentrations show that 0.9 mM restores the TSH phospholipid effect. Verapamil (10(-3) M) and Chlorpromazine (10(-3) M) redirect glycerolipid metabolism by increasing PI and phosphatidic acid (PA) synthesis at the expense of other glycerolipids, as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). These results suggest that the "phospholipid effect" is not a result of Ca2+ entry into the thyroid cells. On the contrary, it seems that this increased turnover of PI in "long term" incubations (3 hr). An additive and acute effect of TSH effect is more pronounced when Ca2+ movements     

A study of phosphoglycerate kinase in human erythrocytes. I. Enzyme isolation, purification and assay.

The enzyme ATP-3-phospho-D-glycerate-1-phosphotransferase (EC 2.7.2.3) (phosphoglycerate kinase) has been isolated from human red cells in crystalline form by a modification of the method of Yoshida and Watanabe (1972) J. Biol. Chem. 247, 440-445). The crystalline enzyme was further purified by electrofocusing using carrier ampholytes (pH 7-9). The isoelectric point of phosphoglycerate kinase was estimated to be 8.75. The specific activity of purified phosphoglycerate kinase from electrofocusing was 2200 units per mg of protein at pH 8.3 (37 degrees C). Enzyme activity was assayed in the forward direction leading from 1,3-diphosphoglycerate to a 3-phosphoglycerate using a fluorimetric procedure for NAD-coupled enzymes for the measurement of the reaction rate at very low substrate concentrations. The auxiliary indicator enzymes were added in excess to yield true initial velocity kinetics, i.e. with no time lag upon addition of substrate (1,3-diphosphoglycerate). This was established theoretically using a mathematical model and confirmed experimentally. Further phosphoglycerate kinase was shown to be the rate-limiting step when the assay conditions were varied.     

A controlled study of Tourette syndrome. IV. Obsessions, compulsions, and schizoid behaviors.

To determine the frequency of obsessive, compulsive, and schizoid behaviors in Tourette syndrome (TS), we prospectively questioned 246 patients with TS, 17 with attention-deficit disorder (ADD), 15 with ADD due to a TS gene, and 47 random controls. The comparative frequency of obsessive, compulsive, and repetitive behaviors--such as obsessive unpleasant thoughts, obsessive silly thoughts, echolalia, palilalia, touching things excessively, touching things a specific number of times, touching others excessively, sexual touching, biting or hurting oneself, head banging, rocking, mimicking others, counting things, and occasional or frequent public exhibitionism--were significantly more common in TS patients than in controls. The frequency of each of these was much higher for grade 3 (severe) TS. Most of these behaviors also occurred significantly more often in individuals with ADD or in individuals with ADD secondary to TS (ADD 2(0) TS). When these features were combined into an obsessive-compulsive score, 45.4% of TS patients had a score of 4-15, whereas 8.5% of controls had a score of 4 or 5. These results indicate that obsessive-compulsive behaviors are an integral part of the expression of the TS gene and can be inherited as an autosomal dominant trait. Schizoid symptoms, such as thinking that people were watching them or plotting against them, were significantly more common in TS patients than in controls. Auditory hallucinations of hearing voices were present in 14.6% of TS patients, compared with 2.1% of controls (P = .02). These symptoms were absent in ADD patients but present in ADD 2(0) TS patients. These voices were often blamed for telling them to do bad things and were frequently identified with the devil. None of the controls had a total schizoid behavior score greater than 3, whereas 10.9% of the TS patients had scores of 4-10 (P = .02). This frequency increased to 20.6% in the grade 3 TS patients. These quantitative results confirm our clinical impression that some TS patients have paranoid ideations, often feel that people are out to get them, and hear voices.     

A heparin binding site in antithrombin III. Identification, purification, and amino acid sequence

A heparin-binding peptide within antithrombin III (ATIII) was identified by digestion of ATIII with Staphylococcus aureus V8 protease followed by purification on reverse-phase high pressure liquid chromatography using a C-4 column matrix. The column fractions were assayed for their ability to bind heparin by ligand blotting with 125I-fluoresceinamine-heparin as previously described (Smith, J. W., and Knauer, D. J. (1987) Anal. Biochem. 160, 105-114). This analysis identified at least three fractions with heparin binding ability of which the peptide eluting at 25.4 min gave the strongest signal. Amino acid sequence analysis of this peptide gave a partially split sequence which was consistent with regions encompassing amino acids 89-96 and 114-156. These amino acids are present in a 1:1 molar ratio which is consistent with a disulfide linkage between Cys-95 and Cys-128. High affinity heparin competed more effectively for the binding of 125I-fluoresceinamine-heparin to this peptide than low affinity heparin. Chondroitin sulfate did not block the binding of 125I-fluoresceinamine-heparin to the peptide. These data strongly suggest that the isolated peptide represents a native heparin-binding region within intact ATIII. Computer generation of a plot of running charge density of ATIII confirms that the region encompassing amino acid residues 123-141 has the highest positive charge density within the molecule. A hydropathy plot of ATIII was generated using a method similar to that of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). This plot indicates that amino acid residues 126-140 are exposed to the exterior surface of the molecule. Based on these data, we suggest that the region corresponding to amino acid residues 114-156 is a likely site for the physiological heparin-binding domain of ATIII. We also conclude that the proposed disulfide bridges within the protein are suspect and should be re-examined (Petersen, T. E., Dudek-Wojiechowska, G., Sottrup-Jensen, L., and Magnussun, S. (1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen, D., Wiman, B., and Verstaeta, M., eds) pp. 43-54, Elsevier Scientific Publishing Co., Amsterdam).     

Long-acting nifedipine versus metoprolol as monotherapy for essential hypertension. A randomized,controlled crossover study.

We assessed the efficacy of long-acting nifedipine as monotherapy in 52 patients with mild to moderate essential hypertension in a randomized, controlled crossover study. Good blood pressure control was achieved in 34 of 40 patients (85%) receiving nifedipine (mean daily dose, 52 mg in 2 divided doses) compared with 23 of 40 patients (58%) receiving metoprolol (mean daily dose, 155 mg in 2 divided doses). After treatment for 4 weeks, the mean blood pressures with nifedipine (149.7 +/- 16.6/88.7 +/- 11.1 mm of mercury) and metoprolol administration (163.9 +/- 23.3/94.2 +/- 10.2 mm of mercury) were significantly lower than with placebo (176.7 +/- 17.3/100.9 +/- 7.1 mm of mercury) (P less than .05). The mean systolic pressure during nifedipine treatment was 14.2 mm of mercury lower (95% confidence interval [CI], 3.9 to 24.5 mm of mercury) and mean diastolic pressure 5.5 mm of mercury (95% CI, 0.3 to 10.7 mm of mercury) lower than with metoprolol therapy. Both drugs were reasonably well tolerated, and intolerance requiring withdrawal was encountered in 3 of 45 (7%) patients receiving nifedipine, compared with 1 of 45 (2%) of those taking metoprolol and placebo, respectively. Adverse effects of nifedipine, most of which were transient, included palpitations, headache, facial flushing, and ankle edema. Long-acting nifedipine is a promising agent when given alone for mild to moderate hypertension and can be safely administered in clinical practice.     

Serum urate, metabolic syndrome, and cardiovascular risk factors. A population-based study

We studied the associations between serum urate levels (determined in 503 subjects from a population of 1,344 subjects living in northern Madrid) and both the metabolic syndrome (MS) (defined by the Adult Treatment Panel III criteria) and C-reactive protein (CRP, determined in 382 subjects). MS was diagnosed in 25% (95%CI, 21-28%) and was associated with hyperuricemia (p<0.001). There was a graded increase in serum urate levels with increasing number of MS components. Urate concentrations significantly correlated with waist circumference (r=0,455, p<0.01). Serum urate was not independently associated with CRP levels. This study shows that serum urate levels are associated with the presence of MS and each of its features.     

Species of coccidia (Apicomplexa: Eimeriidae) in shrews from Alaska, U.S.A., and northeastern Siberia, Russia, with description of two new species

Fecal samples (n = 636) from 10 species of shrews collected in Alaska (n = 540) and northeastern Siberia (n = 96) were examined for the presence of coccidia (Apicomplexa: Eimeriidae). Five distinct oocyst morphotypes were observed. Three types were consistent with oocysts of previously recognized coccidia species from other shrew hosts. These were Eimeria inyoni, E. vagrantis, and Isospora brevicauda, originally described from the inyo shrew (Sorex tenellus), dusky shrew (S. monticolus), and northern short-tailed shrew (Blarina brevicauda), respectively. We found 5 new host records for E. inyoni, 3 for E. vagrantis, and 3 for I. brevicauda. The 2 additional oocyst morphotypes, both from the tundra shrew (Sorex tundrensis), are putative new species. Sporulated oocysts of Eimeria beringiacea n. sp. are subspheroidal, 17.7 x 15.6 microm (14-24 x 13-20 microm) with a length (L)/width (W) ratio of 1.1 (1.0-1.4); these lack a micropyle (M), an oocyst residuum (OR), and a polar granule (PG). Sporocysts are ellipsoidal, 10.3 x 6.1 microm (7-14 x 4-8 microm), with a L/W ratio of 1.7 (1.3-2.3) and have a Stieda body (SB), Substieda body (SSB), and sporocyst residuum (SR). Oocysts of Eimeria tundraensis n. sp. are spheroidal to subspheroidal, 24.8 x 23.5 microm (23-26 x 22-25 microm), with a L/W ratio of 1.1 (1.0-1.2); these lack a M and OR, but a single PG is present. Sporocysts are elongate ellipsoidal, 15.4 x 8.3 microm (13-17 x 7-9 microm), with a L/W ratio of 1.9 (1.4-2.1) and have a SB, SSB, and SR.     

Grommets,tonsillectomies, and deprivation in Scotland.

OBJECTIVE--To see whether there is a relation between grommet insertion operation and tonsillectomy rates, otolaryngology services, and deprivation scores in Scotland. DESIGN--Analysis of routine 1990 NHS data on grommet insertions and tonsillectomies in Scottish children aged 0-15 years compared with data on general practitioner and otolaryngology services and Carstairs deprivation scores. SETTING--All 15 Scottish health boards. SUBJECTS--All children aged 0-15 (1,021,933). RESULTS--Tonsillectomy was more common than grommet insertion operations in Scotland (6182:4850). Health boards with high grommet insertion rates were more likely to have low tonsillectomy rates (Spearman''s rank correlation -0.59; 95% confidence interval -0.87 to -0.03). Grommet insertion rates varied fourfold (from 2.4/1000 to 9.2/1000) and tonsillectomy rates twofold (from 3.6/1000 to 8.0/1000) across Scottish health boards. Variation between health boards had changed over the 15 years 1975-90. Variation in grommet insertion rates did not reflect variation in the supply of otolaryngology consultants (Spearman''s rank correlation -0.25). There was a non-significant tendency for high general practitioner referral rates to be associated with high grommet insertion rates, low tonsillectomy rates, and less deprived areas (Spearman''s rank correlation coefficients 0.50, -0.53, and -0.43). Deprivation (measured by Carstairs scoring for each health board) was associated with higher tonsillectomy rates (Spearman''s rank correlation 0.41; 95% confidence interval -0.22 to 0.80) and significantly lower grommet insertion rates (-0.73; -0.92 to -0.28). CONCLUSION--Social factors as well as differences in disease prevalence and medical practice need to be considered when studying variation in childhood grommet insertion and tonsillectomy rates.     

The protein phosphatases involved in cellular regulation. 2. Purification, subunit structure and properties of protein phosphatases-2A0, 2A1, and 2A2 from rabbit skeletal muscle

Protein phosphatases-2A0, 2A1 and 2A2 have been purified to homogeneity from rabbit skeletal muscle. Approximately 1 mg of phosphatase-2A0 and 2A1, and 0.5 mg of phosphatase-2A2, was isolated from 4000 g muscle within 10 days. Protein phosphatases-2A0 and 2A1 each comprised three subunits, termed A, B' and C (2A0) or A, B and C (2A1), while phosphatase-2A2 contained only two subunits, A and C. The A and C components of phosphatases-2A0, 2A1 and 2A2 had indistinguishable mobilities on sodium dodecyl sulphate/polyacrylamide gels and identical peptide maps. By these criteria, the C component was also identical to the catalytic subunit of phosphatase-2A purified from ethanol-treated muscle extracts. The electrophoretic mobilities of the B and B' subunits were slightly different, and their peptide maps were distinct. The molecular masses of the native enzymes determined by sedimentation equilibrium centrifugation were 181 +/- 6 kDa (2A0), 202 +/- 6 kDa (2A1) and 107 +/- 5 kDa (2A2), while those of the subunits estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis were 60 kDa (A), 55 kDa (B), 54 kDa (B') and 36 kDa (C). These values, in conjunction with molar ratios estimated by densitometric analyses of the gels, suggest that the subunit structures of the enzymes are AB'C2 (2A0), ABC2 (2A1) and AC (2A2). Protein phosphatase-2A2 appears to be derived from 2A0 and/or 2A1 during purification through degradation or dissociation of the B' and/or B subunits. Protein phosphatases-2A0, 2A1 and 2A2 were the only phosphorylase phosphatases in rabbit skeletal muscle that were activated by the basic proteins, protamine (A0.5 = 0.25 microM), histone H1 (A0.5 = 0.3 microM) and polylysine (A0.5 = 0.04 microM). Activation by protamine varied over 5-20-fold for phosphatase-2A0 and 5-7-fold for phosphatases-2A1 and 2A2. The dephosphorylation of glycogen synthase was activated by basic proteins in a similar manner to the phosphorylase phosphatase activity. The isolated C subunit was also stimulated by histone H1 and protamine, but 5-10-fold higher concentrations were required, and with phosphorylase as substrate, maximum activation was only about 2-fold. Activation by basic proteins appears to involve their interaction with the A and/or C subunits, but not with the B or B' subunits, or substrates phosphorylase and glycogen synthase.     

Nucleic acid vibrational circular dichroism, absorption, and linear dichroism spectra. II. A DeVoe theory approach.

The DeVoe polarizability theory is used to calculate vibrational circular dichroism (VCD) and infrared (IR) absorption spectra of four polyribonucleotides: poly(rA) x poly(rU), poly(rU) x poly(rA) x poly(rU), poly(rG) x poly(rC), and poly(rC+) x poly(rI) x poly(rC). This is the first report on the use of the DeVoe theory to calculate VCD, oriented VCD, IR absorption, and IR linear dichroism (LD) spectra of double- and triple-stranded polyribonucleotides. Results are reported for DeVoe theory calculations--within the base-stretching 1750-1550 cm(-1) spectral region--on several proposed multistranded polyribonucleotide geometries. The calculated spectra obtained from these proposed geometries are compared with previously reported measured and calculated VCD and IR spectral results. Base-base hydrogen-bonding effects on the frequencies and magnitudes of the base carbonyl stretching modes are explicitly considered. The good agreements found between calculated and measured spectra are proposed to be further evidence of the usefulness of the DeVoe theory in drawing three-dimensional structural conclusions from measured polyribonucleotide VCD and IR spectra.     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

A slicer for polyacrylamide gels of 3-, 6-, and 18-mm diameter.

A new gel sectioning device for polyacrylamide gel electrophoresis (PAGE) utilizing an iris diaphragm type of concentric cutting blade has been recently described (1). Although gel cylinders of 5- or 6-mm diam are most often used, cylinders of other diameters are often utilized for purposes of sample economy or ease of preparation. The versatile single cutting device described here enables sectioning of PAGE cylinders having 3-, 6-, or 18-mm diam; it can be easily adapted to accept gels with diameters less than 18 mm.     

Genetics of Plant Development by L.A. Lutova,N.A. Provorov,O.N. Tikhodeev,I.A. Tikhonovich,L.T. Khodzhaiova,and S.O. Shishkova

Russian Journal of Genetics -     

Development of enzyme technology for Aspergillus oryzae,A. sojae,and A. luchuensis,the national microorganisms of Japan

This paper describes the modern enzymology in Japanese bioindustries. The invention of Takadiastase by Jokiti Takamine in 1894 has revolutionized the world of industrial enzyme production by fermentation. In 1949, a new γ-amylase (glucan 1,4-α-glucosidase, EC 3.2.1.3) from A. luchuensis (formerly designated as A. awamori), was found by Kitahara. RNase T₁ (guanyloribonuclease, EC 3.1.27.3) was discovered by Sato and Egami. Ando discovered Aspergillus nuclease S₁ (single-stranded nucleate endonuclease, EC 3.1.30.1). Aspergillopepsin I (EC 3.4.23.18) from A. tubingensis (formerly designated as A. saitoi) activates trypsinogen to trypsin. Shintani et al. demonstrated Asp76 of aspergillopepsin I as the binding site for the basic substrate, trypsinogen. The new oligosaccharide moieties Man₁₀GlcNAc₂ and Man₁₁GlcNAc₂ were identified with α-1,2-mannosidase (EC 3.2.1.113) from A. tubingensis. A yeast mutant compatible of producing Man₅GlcNAc₂ human compatible sugar chains on glycoproteins was constructed. The acid activation of protyrosinase from A. oryzae at pH 3.0 was resolved. The hyper-protein production system of glucoamylase was established in a submerged culture.     

Primary biliary cirrhosis,dark adaptometry,electro-oculography,and vitamin A state.

Twenty five patients with primary biliary cirrhosis were studied for vitamin A state. In nine patients found to have low circulating vitamin A concentrations no abnormality was found on electro-oculography or in dark adaptation. A positive correlation was found between retinol binding protein and vitamin A values (r = +0.88; p less than 0.001) and between serum albumin and vitamin A values (r = +0.75; p less than 0.001). A weaker and negative correlation was found between serum bilirubin (r = -0.47; p less than 0.05) and vitamin A values. Patients with primary biliary cirrhosis should not receive regular parenteral or even oral vitamin A supplementation unless dark adaptometry or electrooculography yields an abnormal result.     

A molecularly cloned, pathogenic, neutralization-resistant simian immunodeficiency virus, SIVsmE543-3.

An infectious molecular clone of simian immunodeficiency virus SIVsm was derived from a biological isolate obtained late in disease from an immunodeficient rhesus macaque (E543) with SIV-induced encephalitis. The molecularly cloned virus, SIVsmE543-3, replicated well in macaque peripheral blood mononuclear cells and monocyte-derived macrophages and resisted neutralization by heterologous sera which broadly neutralized genetically diverse SIV variants in vitro. SIVsmE543-3 was infectious and induced AIDS when inoculated intravenously into pig-tailed macaques (Macaca nemestrina). Two of four infected macaques developed no measurable SIV-specific antibody and succumbed to a wasting syndrome and SIV-induced meningoencephalitis by 14 and 33 weeks postinfection. The other two macaques developed antibodies reactive in Western blot and virus neutralization assays. One macaque was sacrificed at 1 year postinoculation, and the survivor has evidence of immunodeficiency, characterized by persistently low CD4 lymphocyte subsets in the peripheral blood. Plasma samples from these latter animals neutralized SIVsmE543-3 but with much lower efficiency than neutralization of other related SIV strains, confirming the difficulty by which this molecularly cloned virus is neutralized in vitro. SIVsmE543-3 will provide a valuable reagent for studying SIV-induced encephalitis, mapping determinants of neutralization, and determining the in vivo significance of resistance to neutralization in vitro.     

A new oxygenase, 2-nitropropane dioxygenase of Hansenula mrakii. Enzymologic and spectrophotometric properties.

2-Nitropropane dioxygenase, purified to homogeneity from Hansenula mrakii (IFO 0895), has a molecular weight of approximately 62,000 and consists of two subunits nonidentical in molecular weight (39,000 and 25,000). Stoichiometrical studies and the results obtained with 18O2 showed that 2 atoms of molecular oxygen are incorporated into 2 molecules of acetone formed from 2-nitropropane. In addition to 2-nitropropane, nitroethane, 3-nitro-2-pentanol, and 1-nitropropane are oxidatively dentrified. The enzyme, which exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm, contains 1 mol of FAD and 1 g atom of non-heme iron per mol of enzyme. The enzyme-bound FAD is reduced by 2-nitropropane under anaerogic conditions, but the enzyme-bound Fe3+ is not affected. The introduction of oxygen to the reduced form of enzyme causes reoxidation of the enzyme. The bound FAD and Fe3+ are reduced by the addition of nitromethane, which is not a substrate, under anaerobic conditions. The aerobic dialysis of the enzyme treated with nitromethane causes reoxidation of only the Fe2+. Sodium dithionite also reduces both the enzyme-bound FAD and Fe3+ under anaerobic conditions. When the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appeared with concomitant restoration of approximately 80% of the activity. The enzyme activity is significantly inhibited by pyrocatechol-3,5-disulfonate disodium salt, 8-hydroxyquinoline, reducing agents such as 2-mercaptoethanol, and HgCl2. The Michaelis constants are as follows: 2-nitropropane (2.13 X 10(-2) M), nitroethane (2.43 X 10(-2) M), 3-nitro-2-pentanol (6.8 X 10(-3) M), 1-nitropropane (2.56 X 10(-2) M), and oxygen (3.03 X 10(-4) M, with 2-nitropropane).