A rapid separation of bovine brain S-100a and S-100b proteins and related conformation studies

S-100 protein absorbs to the calmodulin antagonist W-7 coupled to epoxy-activated Sepharose 6B in the presence of Ca²⁺ and is eluted by ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffer. S-100a and S-100b were separated and isolated by Ca²⁺-dependent affinity chromatography on W-7 Sepharose. The Ca²⁺-induced conformational changes of S-100a and S-100b were examined using circular dichroism, ultraviolet difference spectra, and a fluorescence probe. Differences in Ca²⁺-dependent conformational changes between S-100a and S-100b became apparent. Circular dichroism studies revealed that both S-100a and S-100b undergo a conformational change upon binding of Ca²⁺ in the aromatic and far-uv range. In the presence or absence of Ca²⁺, the aromatic CD spectrum of S-100a differed completely from that of S-100b, possibly due to the single tryptophan residue of S-100a. Far-uv studies indicate that α-helical contents of both S-100a and S-100b decreased with addition of Ca²⁺. Ca²⁺-induced conformational changes of S-100a and S-100b were also detected by uv difference spectra. The spectrum of S-100a also differed from that of S-100b. Fluorescence studies using 2-p-toluidinylnaphthalene-6-sulfonate (TNS), a hydrophobic probe for protein, revealed a slight difference in conformational changes of these two components. The interaction of TNS and S-100b was observed with concentrations above 3 μm Ca²⁺; on the other hand, S-100a required concentrations above 8 μm. This finding was supported by the difference in the binding affinities of S-100a and S-100b to the W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide)-Sepharose column; both S-100a and S-100b bound the column in the presence of Ca²⁺ but S-100a was eluted prior to S-100b. These results suggest that S-100a and S-100b differ in their dependence on Ca²⁺ and that the affinity-chromatographic separation of S-100a from S-100b on the W-7-Sepharose column makes feasible a rapid purification of these two components.     

Synthesis and biological evaluations of A-ring isomers of 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3

The activated vitamin D3 derivative 26,27-F6-1alpha,25(OH)2D3 (2a), its three A-ring diastereomers (2b, 2c, 2d), and 5,6-trans isomer (2e) were prepared. Two analogues (2b, 2c) of these isomers were synthesized by a palladium catalyzed coupling reaction using vinyl bromide 5 and enynes (6a, 6b), which were derived from readily commercially available 2S-(+)-glycidyl p-toluenesulfonate 7, as a common starting material. Competitive vitamin D receptor (VDR) binding affinities of these diastereomers of 2a were evaluated. Interestingly, the stereochemical effects at C-1,3 of 2a were considerably more moderate than those of 1alpha,25(OH)2D3 (1). In particular, isomerization at the 5,6-double bond of 2a only slightly reduced VDR affinity, whereas 5,6-trans-1alpha,25(OH)2D3 had a significantly lower binding affinity than 1.     

Introduction of antigenic determining 2,4-dinitrophenyl residues into 4-thiouridine, N3-(3-L-amino-3-carboxypropyl) uridine and tRNA-Phe from E. coli.

The introduction of antigenic determining 2,4-dinitrophenyl residues into the rare ribonucleosides 4-thiouridine (1a), and N3-(3-L-amino-3-carboxypropyl) uridine (2) as well as into tRNA-Phe from E. coli has been investigated. Alkylation of 1a with omega-bromo-2,4-dinitroacetophenone (3b) gives S-(2,4-dinitrophenacyl)-4-thiouridine (5A). Applying the reaction to the 5'-monophosphate of 1a, 5b is formed, but this product decomposes at pH 7. However, acylation of 2 with 2,4-dinitrobenzoic acid N-hydroxysuccinimide ester (4b) leads to N3-[3-carboxy-3-L-(2,4-dinitrobenzamido)propyl]uridine (6) which is stable in aqueous solution. The latter reaction was used for the introduction of an antigenic determining 2,4-dinitrophenyl residue into tRNA-Phe from E. coli. The modified tRNA-Phe was isolated and by degradation of the molecule with RNase T2 and alkaline phosphatase the nucleoside derivative 6 was obtained and found to be identical with the synthetic product.     

Cytochrome b2, an electron carrier between flavocytochrome b2 and cytochrome c. Rapid kinetic characterization of the electron-transfer parameters with ionic-strength-dependence.

The oxidation-reduction properties of free cytochrome b2 isolated by controlled proteolysis from flavocytochrome b2, i.e. the flavodehydrogenase-bound cytochrome b2, were investigated by using stopped-flow spectrophotometry. The rapid kinetics of the reduction of cytochrome b2 by flavocytochrome b2 in the presence of L-lactate are reported. The self-exchange rate constant between reduced cytochrome b2 bound to the flavodehydrogenase and free cytochrome b2 was determined to be 10(5) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. The specific electron-transfer reaction between reduced cytochrome b2 and cytochrome c was also studied, giving an apparent second-order rate constant of 10(7) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. This electron-exchange rate is slightly modulated by ionic strength, following the Debye-Hückel relationship with a charge factor Z1Z2 = -1.9. Comparison of these data with those for the reduction of cytochrome c by flavodehydrogenase-bound cytochrome b2 [Capeillère-Blandin (1982) Eur. J. Biochem. 128, 533-542] leads to the conclusion that the intramolecular electron exchange between haem b2 and haem c within the reaction complex occurs at a rate very similar to that determined experimentally in presence of the flavodehydrogenase domain. The low reaction rate observed with free cytochrome b2 is ascribed to the low stability of the reaction complex formed between free cytochrome b2 and cytochrome c.     

Enthalpy change on mixing a couple of S- and R-enantiomers of some chiral compounds at 298.15 K

Enthalpy change on the mixing of R- and S-enantiomers of chiral liquid compounds such as dimethyl malate (1), methyl 3-hydroxylbutanoate (2), 2-butanol (3), ethyl 4-chloro-3-hydroxylbutanoate (4), 1,3,3-trimethylbicycle-[2.2.1]heptan-2-one (5), 3,7-dimethyl-6-octenal (6), and 8-bromo-2,6-dimethyl-2-octene (7) is measured over the entire range of mole fractions at 298.15 K, albeit very small values. The mixing of chiral liquids of R-1 + S-1, R-2 + S-2, R-3 + S-3, R-6 + S-6, and R-7 + S-7 produces enthalpic destabilization over the entire range of mole fractions, while that of R-4 + S-4 and R-5 + S-5 shows enthalpic stabilization over entire compositions. Enthalpy change on mixing at an equimolar concentration and the intermolecular interaction obtained by the molecular mechanics calculations show a linear correlation, except for a few compounds measured.     

Alpha-pyrones and a 2(5H)-furanone from Hyptis pectinata

Three pyrones and a 2(5H)-furanone, designated pectinolides D-G, have been isolated from the dichloromethane extract of Hyptis pectinata. The metabolites were characterized on the basis of 1D and 2D NMR spectroscopic techniques. The pyrones were identified as 6S-[3S,6S-(diacetoxy)-5R-hydroxy-1Z-heptenyl]-5S-hydroxy-5,6-dihydro-2H-pyran-2-one (1)- pectinolide D, 6S-[3S,5R,6S-(triacetoxy)-1Z-heptenyl]-5S-acetoxy-5,6-dihydro-2H-pyran-2-one (2)- pectinolide E and 6S-[3S,5R,6S-(triacetoxy)-1Z-heptenyl]-5S-acetoxy-4R-methoxy-3,4,5,6-tetrahydro-4H pyran-2-one (3)- pectinolide F. The furanone was identified as [2'Z,5(1')Z] 5-(4'S,6'R,7'S-triacetoxy-2-octenylidene)-2(5H)-furanone (4)-pectinolide G.     

Regional distribution of S-100a0 (αα), S-100a (αβ) and S-100b (ββ) in the bovine central nervous tissue determined with a sensitive enzyme immunoassay system

A sensitive sandwich-type enzyme immunoassay system for separate measurement of 3 forms of bovine S-100 protein, S-100a₀ (αα), S-100a (αβ) and S-100b (ββ), was developed by the use of purified antibodies to the α or the β subunit of bovine S-100 protein. The assay system consisted of polystyrene balls with immobilized antibody (anti-α for S-100a₀ and S-100a assays, and anti-β for S-100b assay) F(ab′)₂ fragments and antibody (anti-α for S-100a, assay, and anti-β for S-100a and S-100b assays) Fab′ fragments labeled with β-d-galactosidase from Escherichia coli. The minimum measurable sensitivity of each assay was less than 10 pg/assay tube. The assay system for S-100a cross-reacted little with S-100a₀ and S-100b. The assay systems for S-100a₀ and S-100b cross-reacted (10 and 17%, respectively) with S-100a which contains α and β subunits in the molecule. However, levels of S-100a₀, S-100a and S-100b in the soluble extract of bovine brain could be determined by correcting the cross-reacted S-100a to the assays of S-100a₀ and S-100b. Various regions of bovine central nervous tissue were found to contain 0.3–1 μg of S-100a₀, 4–14 μg of S-100a, and 8–30 μg of S-100b per mg soluble protein. The percent concentrations of three forms of S-100 protein in the cerebral cortex were about 3, 38, and 59, for S-100a₀, S-100a, and S-100b, respectively, and those in the cerebellar cortex were 2, 21 and 77, respectively. Purified S-100a and S-100b preparations from human and rat brains were also reactive with the respective assay system for bovine S-100 protein, suggesting that the present assay system is applicable to the assay of three forms of S-100 protein in human and rat tissues.     

Copper Sorption Behavior of Selected Soils of the Oasis in the Middle Reaches of Heihe River Basin,China

The mobility and bioavailability of copper (Cu) depends on the Cu sorption capacity of soil and also on the chemical form of Cu in soils. Laboratory batch experiments were carried out to study the sorption and distribution of Cu in nine soils differing in their physicochemical properties from the oasis in the middle reaches of Heihe river basin, China: desert soil (S-1), agricultural soils (S-2, S-3, S-8, and S-9), marshland soil (S-4), and hungriness shrub soils (S-5 and S-6). Copper sorption behavior was studied using the sorption isotherm and sequential extraction procedure. In general, the sorption capacity for Cu decreased in the order: S-4 > S-9 > S-2 > S-8 > S-3 > S-6 > S-5 > S-7 > S-1. The correlation results suggest that soils with higher CEC, silt, clay, CaCO3, and organic matter will retain Cu more strongly and in greater amounts than soils that are sandy with lower CEC, CaCO3, and organic matter. pH is not an important impact factor to Cu sorption in experimental soil samples because pH in soils used in this study had a narrow range. The distribution of sorbed Cu varied between nine soils studied and depended on both soil properties and initial added Cu concentration. There are significant differences in the distribution of Cu in each soil with the increase of initial Cu concentration. The predominance of Cu associated with the available fraction, which was over 50% of the total sorbed Cu in most cases, indicates that the change of geochemical conditions might promote the release of Cu back into soil solution thus impacting organisms in the soils. The added Cu has also the tendencies to locate in the residual fraction, which was larger than 5% of the total amount extracted from the four fractions in most soils.     

Competitive interaction between two functional S-haplotypes confer self-compatibility on tetraploid Chinese cherry (Prunus pseudocerasus Lindl. CV. Nanjing Chuisi)

Self-incompatibility (SI) has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility (GSI). In the present study, four PpsS-haplotypes (Prunus pseudocerasus S-haplotypes) comprising at least two genes, i.e., PpsS-RNase (P. pseudocerasus S-RNase) and PpsSFB (P. pseudocerasus S-haplotype-specific F-box) have been successfully isolated in tetraploid P. pseudocerasus Lindl. CV. Nanjing Chuisi ("NC") which exhibited self-compatibility (SC), and its S-genotype was determined as S-1/S-3'/S-5/S-7. These PpsS-RNases, which were expressed exclusively in style, shared the typical structural features with S-RNases from other Prunus species exhibiting GSI. All PpsSFBs showed similar structure characteristics of SFBs from other Prunus species, and matched with the necessary conditions for pollen S-determinant. No mutations leading to dysfunction of S-haplotype were found in their full-length c-DNA sequences, except for PpsS-3'-haplotype which was not amplified by PCR. These four S-haplotypes complied with tetrasomic inheritance. Diploid pollen grains with S-genotypes S-7/S-1, S-7/S-5 and S-1/S-5 can grow the full length of the style after self-pollination, while pollen grains with S-3'/S-7, S-3'/S-1 and S-3'/S-5 cannot. These results suggest that PpsS-haplotypes-1, -5 and -7 are functional, and that competitive interaction between two of them confer self-compatibility on cultivar "NC". Furthermore, in terms of recognition specificity, diploid pollen grains carrying PpsS-3'-haplotype are equal to monoploid pollen grains carrying the other functional S-haplotype.     

Synthesis and crystal structure of a complex between lanthanum nitrate and a bicyclic polyether

Reaction between lanthanum nitrate hexahydrate and a macrobicyclic polyether in ethanol has yielded a product of overall stoichiometry 3:2. The ligand, 21R, 26S, 29R, 34S-21, 22, 23, 24, 25, 26, 29, 30, 31,32,33, 34-dodecahydro-1,4,7,14,17,20,28,35-octaoxa (2^3,29syn2^18,34syn) (7.7) orthocyclophane, L, provides 8 oxygen donor atoms. Crystals were obtained from MeOH/EtOH (50/50). Crystal structure determination on 9590 observations, R=0.059, has shown the triclinic unit cell, a = 26.562(6), b = 13.486(3), c = 12.154(3) Å, α=63.9(1), β = 100.0(1), γ = 102.0(1)° space group P , V = 3806 Å to contain, as the asymmetric unit, two complex cations (La(NO₃)₂L)⁺ and one complex anion (La- (NO₃)₅MeOH)²⁻. The lanthanum is 11-coordinated in the anion and one of the cations, in which there is one bidentate and one monodentate nitrate anion and 12-coordinated in the other cation. For the monodentate nitrate La---O = 2.448(9) Å, all other nitrate ions are bidentate (La---O = 2.594(9)−2.743(10) Å). Most La---O bonds are shorter in the 11 than in the 12-coordinated cation. There are large differences in La---O bond lengths according to the nature of the carbon atoms to which the oxygen is attached. The methanol molecule forms a hydrogen bond to one oxygen atom of the monodentate nitrate group.     

Purification and characterization of adipose tissue S-100b protein

We have purified S-100 protein from bovine brain using Ca2+-dependent affinity chromatography on N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)-Sepharose (Endo, T., Tanaka, T., Isobe, T., Kasai, H., Okuyama, T., and Hidaka, H. (1981) J. Biol. Chem. 256, 12485-12489). By essentially the same procedure, W-7-Sepharose binding protein has been purified to apparent homogeneity from bovine abdominal adipose tissue. Electrophoretically, the purified protein from adipose tissue co-migrated with brain S-100b protein both in the presence and absence of sodium dodecyl sulfate and the protein was indistinguishable from brain S-100b region in terms of amino acid composition, two-dimensional tryptic peptide mapping and reactivity with anti-brain S-100b serum. Immunohistochemical analysis confirmed the existence of S-100b protein in the adipose cell where the protein seems to be located in both the nucleus and cytoplasm. Thus, the results indicate that the adipose cells contain the protein possibly identical with brain S-100b protein. In addition, the contents of S-100b protein in various rat tissues were measured by enzyme immunoassay method using the anti-bovine brain S-100b serum. Significant amounts of S-100b protein were found not only in the adipose tissue but also in the peripheral tissue such as trachea and skin. These observations suggest that S-100b protein should no longer be considered as a protein specific to nervous tissues.     

Somatostatin-28 regulates GLP-1 secretion via somatostatin receptor subtype 5 in rat intestinal cultures

Five somatostatin receptors (SSTRs) bind somatostatin-14 (S-14) and somatostatin-28 (S-28), but SSTR5 has the highest affinity for S-28. To determine whether S-28 acting through SSTR5 mediates inhibition of glucagon-like peptide-1 (GLP-1), fetal rat intestinal cell cultures were treated with somatostatin analogs with relatively high specificity for SSTRs 2-5. S-28 dose-dependently inhibited GLP-1 secretion stimulated by gastrin-releasing peptide more potently than S-14 (EC(50) 0.01 vs. 5.8 nM). GLP-1 secretion was inhibited by an SSTR5 analog, BIM-23268, more potently than S-14 and nearly as effectively as S-28. The SSTR5 analog L-372,588 also suppressed GLP-1 secretion equivalent to S-28, but a structurally similar peptide, L-362,855 (Tyr to Phe at position 7), was ineffective. An SSTR2-selective analog was less effective than S-28, and an SSTR3 analog was inactive. Separate treatment with GLP-1-(7-36)-NH(2) increased S-28 and S-14 secretion by three- and fivefold; BIM-23268 abolished S-28 without altering S-14, whereas the SSTR2 analog was inactive. The results indicate that somatostatin regulation of GLP-1 secretion occurs via S-28 through activation of SSTR5. GLP-1-stimulated S-28 secretion is also autoregulated by SSTR5 activation, suggesting a feedback loop between GLP-1 and S-28 modulated by SSTR5.     

The effects of halothane on hepatic microsomal electron transfer.

1. The effects of halothane (CF3CHBrCl), a volatile anaesthetic agent, on electron transfer in isolated rat liver microsomal preparations were examined. 2. At halothane concentrations achieved in tissues during clinical anaesthesia (1-2mM), halothane shifts the redox equilibrium of microsomal cytochrome b5 in the presence of NADPH towards the oxidized form. Halothane accelerates stoicheiometric consumption of NADPH and O2, increases the rate of reoxidation of NADH-reduced microsomal ferrocytochrom b5, but does not affect NADPH- or NADH-cytochrome c reductase activity. The enhanced microsomal electron flow seen in the presence of halothane is not diminished by CO nor is it increased by pretreatment of the animals with phenobarbital. 3. The effects of halothane are maximum in microsomal preparations isolated from animals fed on a high-carbohydrate diet to induce stearate desaturase activity. Changes in microsomal electron transfer caused by halothane are in all cases abolished by low concentrations (1-2mM) of cyanide. Microsomal stearate desaturase activity is unaffected by halothane. 4. The first-order rate constant for oxidation of membrane-bound ferrocytochrome b5 in the absence of added substrate (k1 equals 1.5 times 10(-3)A-1) is similar to that for autoxidation of purified ferrocytochrome b5(k1 equals 7 times 10(-3)S-1) the rate of autoxidation of soluble ferrocytochrome b5 is unaffected by halothane. 5. It is concluded that the effects of halothane on microsomal electron transfer are not related to cytochrome P-450 linked metabolism but rather arise from the interaction of halothane with the cyanide-sensitive factor of the stearate desaturase pathway.     

Synthesis and some reactions of 6-bromoandrogens: potential affinity ligand and inactivator of estrogen synthetase.

The synthesis of epimeric 6-bromo-4-androstene-3,17-dione (1a and 1b), 6-bromotestosterone (2a and 2b) and its acetate (3a and 3b), and 6-bromo-16 alpha-acetoxy-4-androstene-3,17-dione (5a and 5b), and 6 beta-bromo-16 alpha-hydroxy-4-androstene-3,17-dione (4) is described. The interconversions among compounds 1, 2, and 3 are also studied. The 6 beta-isomer (1b, 2b, and 3b) was epimerized to the 6 alpha-isomer (1a, 2a and 3a) in carbon tetrachloride or chloroform-methanol (9:1) and the 6 alpha-isomer was isolated by fractional crystallization from the epimeric mixture. 6 alpha-Bromo isomer 1a was also epimerized back to 6 beta-bromo isomer 1b in chloroform-methanol (9:1). Two polymorphic forms of 6 beta-bromotestosterone acetate (3b) were isolated (mp. 114--117 degrees and 138--141 degrees). The 6 beta-bromo isomers were found to be unstable in methanol and decomposed to give 5 alpha-androstane-3,6-dione derivative (6). The results of irreversible inactivation of human placental androgen aromatase with some of these 6-bromoandrogens are discussed.     

Mechanistic studies with 2-C-methyl-D-erythritol 4-phosphate synthase from Escherichia coli

The mechanism of the reaction catalyzed by 2-C-methyl-d-erythritol 4-phosphate (MEP) synthase from Escherichia coli has been studied by steady-state and single-turnover kinetic experiments for the 1-deoxy-d-xylulose 5-phosphoric acid (DXP) analogues, 1,1,1-trifluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(3)-DXP), 1,1-difluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(2)-DXP), 1-fluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF-DXP), and 1,2-dideoxy-d-hexulose 6-phosphate (Et-DXP). CF(3)-DXP, CF(2)-DXP, and Et-DXP were poor inhibitors, most likely because of the increase in steric bulk at C1 of DXP. The three analogues were also poor substrates for the enzyme. In contrast, CF-DXP was a good substrate (k(cat)(CF)(-)(DXP) = 37 +/- 2 s(-)(1), K(m)(CF)(-)(DXP) = 227 +/- 25 microM) for MEP synthase when compared to DXP (k(cat)(DXP) = 29 +/- 1 s(-)(1), K(m)(DXP) = 45 +/- 4 microM). A primary deuterium isotope effect was observed under single-turnover conditions when CF-DXP was incubated with 4S-[(2)H]NADPH ((H)k/(D)k = 1.34 +/-0.01), whereas no isotope effect was observed upon incubation with DXP and 4S-[(2)H]NADPH ((H)k/(D)k = 1.02 +/- 0.02). The reaction did not exhibit burst kinetics for either substrate, indicating that product release is not rate-limiting. These studies suggest that positive charge does not develop at C2 of DXP during catalysis. In addition, the isotope effect with CF-DXP and 4S-[(2)H]NADPH but not DXP indicates that the rearrangement step, which precedes hydride transfer, is rate-limiting for DXP but becomes partially rate-limiting for CF-DXP. Thus, rearrangement appears to be enhanced by substitution of a hydrogen atom in the methyl group of DXP by fluorine. These observations are consistent with a retro-aldol/aldol mechanism for the rearrangement during conversion of DXP to MEP.     

Studies of human hemoglobin intermediates. The double mixing method for studying the reactions of the species Hb4(CO) and Hb4(CO)2

Using the double mixing method we have studied the reactions of the partially liganded species (Hb4, Hb4L1, Hb4L2, Hb4L3) of normal human hemoglobin with carbon monoxide. In the first mixing, oxygen is removed from the species Hb4(O2) chi (CO) gamma and at the second mixing the species Hb4(CO) gamma reacts with CO. At 90% saturation of oxyHb with CO the main intermediate species are Hb4(CO)3 and Hb4(CO)2, and at 10% saturation Hb4 and Hb4(CO). The four CO-combination rate constants determined are: l'1 = 1 X 10(5) M-1 S-1, l'2 = 7 X 10(5) M-1 S-1, l'3 = 2 X 10(5) M-1 S-1 and l'4 = 4.8 X 10(6) M-1 S-1. The results indicate that there is no monotonic increase in the successive CO-combination rate constants. It is difficult to explain these results on the basis of the two-state model (Monod et al., 1965) or the stereochemical model of Perutz (1970).     

Growth,vitamin B12, and photopigment formations of Rhodopseudomonas sphaeroides S growing on propionate media under dark and light conditions

Summary The effects of light illumination and dissolved oxygen concentration (DO) on the growth characteristics of Rhodopseudomonas sphaeroides S and intracellular accumulations of vitamin B₁₂ and photopigments were studied in continuous cultures of aerobic-dark (S-1, DO>5 mg l^–1), aerobic-light (S-2, DO>5 mg l^–1, 4.5 klux), microaerobic-light (S-3, DO0,4.5 klux) and anaerobic-light (S-4, 4.5 klux) conditions using propionate media. Growth yields from propionic acid determined in S-3 and 4 were 1.5 to twofold greater than in S-1 and 2. Carbon dioxide evolution observed in S-3 and 4 was 0.05 to 0.1 times that in S-1 and 2. Overall carboxylase activity was maximal in S-4. Intracellular accumulations of bacteriochlorophyll and carotenoid were maximal in S-4, followed by S-3 and almost nil in S-1 and 2. The high growth yields observed in S-3 and 4 could be accounted for the high level of activity of carbon dioxide fixation and by the additional or effective utilization of light energy.Intracellular accumulation of vitamin B₁₂ in S-2, 3 and 4 was 1.5 to 1.8 times that in S-1. The maximum content of the vitamin was 74 g-B₁₂ g-cell^–1. The maximum productivity of vitamin B₁₂, g-B₁₂ l^–1 h^–1, was 6.6 in S-3 since specific growth rate, growth yield and the vitamin content of the cells were maximal in S-3.     

Synthesis and enzymic hydrolysis of cyclic peptides containing an anthranilic acid residue

Two cyclic peptides cyclo (Phe-MeAnt-Glyn) with MeAnt = 5-methyl-anthranilic acid residue, n = 4 (3b) and n = 6 (4b), have been synthesized in solution and their reaction with alpha-chymotrypsin analyzed. The polyglycyl chain was prepared by the phosphazo method; cyclization at the Gly-Phe site occurred in good yield using the azide method. Catalysis of the hydrolysis of peptides 3b and 4b by alpha-chymotrypsin was characterized at 37 degrees by the apparent second-order rate constants kcat/Km 0.12 and 1.15 M-1 S-1, respectively, in agreement with the usual acceleration observed upon enlargement of the size of the peptidic ring in cyclic peptides. alpha-Chymotrypsin specifically split the Phe-MeAnt amide bond in cyclopeptide 4b. This specific orientation suggests that analogous structures with a functionalized methylene group instead of the methyl substituent can be used in the design of suicide substrates for serine proteases.     

Immunocytochemical characterization of S-100 beta-positive human T-lymphocytes by a double immunostaining method

The antigenic properties of S-100 beta-positive human T-lymphocytes (S-100 beta+ T-cells) were investigated by a double immunostaining technique employing an indirect immunoperoxidase method for cytoplasmic S-100 beta subunit and an immunoalkaline phosphatase method for cell surface antigens detected by various monoclonal antibodies to human lymphocytes. S-100 beta+ T-cells recognized by their diffuse intracytoplasmic immunoperoxidase reaction, also expressed CD2, CD3, CD8 antigens demonstrated by surface blue alkaline phosphatase reactivity, but not CD4, CD1, CD25 (interleukin-2 receptor), or HLA-DR antigens. However, they displayed a blastic change to T-cell mitogens, such as Concanavalin A(Con-A) and PHA, followed by the expression of CD25 and HLA-DR antigens. Under normal conditions, S-100 beta+ T-cells comprised approximately 5-22.8% of CD8+ cells amongst human peripheral blood mononuclear cells.