Isolation of Two Streptothricin-like Antibiotics,Nos. 6241-A and B,as Inhibitors of de novo Starch Synthesis and Their Herbicidal Activity

Two streptothricin-like antibiotics, Nos. 6241-A and B, were obtained as inhibitors of de novo starch synthesis in excised leaf segments of barnyard millet (Panicum crus-galli). No. 6241-B was identified as SF-701, and No. 6241-A was a new antibiotic, in which N,N-dimethylglycine was substituted for the N-methylglycine of No. 6241-B (SF-701). Both antibiotics also inhibited plant growth. The inhibitory activity of No. 6241-B (SF-701) was approximately ten times that of No. 6241-A. By foliar treatment, No. 6241-B (SF-701) showed remarkable herbicidal activity against barnyard millet at a concentration of more than 500 ppm with little phytotoxicity for the rice plant (Oryza sativa L. cv. Nihonbare).     

Interaction of sodium decyl sulfate with poly(L-ornithine) and poly(L-lysine) in aqueous solution

The binding isotherms of sodium decyl sulfate to poly(L -ornithine), poly(D ,L -ornithine), and poly(L -lysine) at neutral pH were determined potentiometrically. The nature of a highly cooperative binding in all three cases suggests a micelle-like clustering of the surfactant ions onto the polypeptide side groups. The hydrophobic interaction between the nonpolar groups overshadows the coulombic interaction between the charged groups. The titration curves can be interpreted well by the Zimm–Bragg theory. The average cluster size of bound surfactant ions is sufficiently large to promote the β-structure of (L -Lys)_n even at a very low binding ratio of surfactant to polypeptide residue, whereas the onset of the helical structure for (L -Orn)_n begins after about 7 surfactant ions are bound to two turns of the helix. The CD results are consistent with this explanation.     

DNA Polymorphism of the major histocompatibility class I genes and their association with serologically defined haplotypes

DNA of unrelated persons as well as members of families that were totally or partially homozygous or completely heterozygous on the loci of the major histocompatibility class I genes has been isolated from peripheral blood lymphocytes and blot hybridized with the class I pseudogene pHLA 12.4 probe. The autoradiographic DNA patterns were discussed and compared with well-defined serological features. Positive associations with serologically typed alleles had been demonstrated for HLA-A1,11 ; -A2; -A3; -B7; -B14; -B35;-Bw41; and -Cw5.     

Identification of bacterial clones encoding bovine caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by ¹²⁵I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-β-lactamase: casein proteins synthesized in Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine χ-casein, clone 3-H7 contained a cDNA fragment of β-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of α_si-casein.     

Investigation of the antibiotic complex produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> 194, Variant K

Phase variation in the culture of the environmental strain Lactococcus lactis subsp. lactis 194 resulted in the formation of two types of colonies differing by 15% in antibiotic activity. The active variant 194-K produced an antibiotic complex with a broad spectrum of antibacterial and antifungal activity. Five components (194-A, B, C, D, and E) were isolated from the complex by solid-phase extraction and thin-layer chromatography. Components 194-A and 194-B were hydrophobic neutral compounds soluble in organic solvents. Component 194-A possessed fungicidal activity, whereas component 194-B exhibited only bactericidal activity. Physicochemical studies of the isolated components 194-A and 194-B revealed that they had no analogs in the Berdy database of biologically active substances (BNPD) and appeared to be novel antibiotics. Component 194-C was a hydrophilic polar compound inhibiting growth of gram-positive and gramnegative bacteria. Component 194-D belonged to peptide antibiotics; it inhibited growth of only gram-positive bacteria and was similar to nisin A in biological properties but differed in electrophoretic mobility and molecular mass.     

Estimation of the circular dichroism exhibited by statistical coils of poly(L-alanine) and unionized poly(L-lysine) in water

The circular dichroism of Ac-(Ala)_x-OMe and H-Lys-(Lys)_x-OH with x = 1, 2, 3, and 4 has been measured in aqueous solutions. The oligomers with x = 4 show similar circular dichroism spectra in water when the lysyl amino groups are protonated, and they respond in similar fashion to heating and to sodium perchlorate. Both oligomers at 15°C exhibit a positive circular dichroism band at 217–218 nm, which is eliminated by the isothermal addition of 4 M sodium perchlorate or by heating. The positive circular dichroism of the lysine oligomer is also eliminated when the pH is elevated to deprotonate the amino groups. Positive circular dichroism is still observed for Ac-(Ala)₄-OMe at elevated pH. Circular dichroism spectra have been estimated for poly(L -alanine) and poly(L -lysine) as statistical coils under the above conditions, based on the trends established with the oligomers. Poly(L -lysine) and poly(L -alanine) are predicted to exhibit similar circular dichroism behavior in aqueous solution so long as the lysyl amino groups are protonated. The circular dichroism of the statistical coil of poly(L -lysine), but not poly(L -alanine), is predicted to change when the pH is elevated sufficiently to deprotonate the lysyl amino groups. These results suggest that the unionized lysyl side chains participate in interactions that are not available to poly(L -alanine). Hydrophobic interactions may occur between the unionized lysyl side chains. Protonation of the lysyl amino groups is proposed to disrupt these interactions, causing poly(L -alanine) and protonated poly(L -lysine) to have similar circular dichroism properties.     

Mn<Superscript>2+</Superscript> enhances theanine-forming activity of recombinant glutamine synthetase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0–100 mM) of Mn²⁺ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9-A (details are described in “Materials and methods”) and 0.1% (w/v) lactose were selected as culture medium and inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the specific activity of the recombinant GS is 6.2 U/mg which is approximate to that (6.4 U/mg) induced in LB in the presence of 10 mM Mn²⁺ at optimal pH 7.5. The activity is markedly higher activated by Mn²⁺ than that by other nine bivalent cations. Furthermore, M9-B (5 μM Mn²⁺ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and 13.1 g/L by paper chromatography and HPLC, respectively.     

Cloning and characterization of the ribosomal protein L3 (<Emphasis Type="Italic">RPL3</Emphasis>) gene family from <Emphasis Type="Italic">Triticum aestivum</Emphasis>

Plant pathogenic fungi of the genus Fusarium can cause severe diseases on small grain cereals and maize. The contamination of harvested grain with Fusarium mycotoxins is a threat to human and animal health. In wheat production of the toxin deoxynivalenol (DON), which inhibits eukaryotic protein biosynthesis, is a virulence factor of Fusarium, and resistance against DON is considered to be part of Fusarium resistance. Previously, single amino acid changes in RPL3 (ribosomal protein L3) conferring DON resistance have been described in yeast. The goal of this work was to characterize the RPL3 gene family from wheat and to investigate the potential role of naturally existing RPL3 alleles in DON resistance by comparing Fusarium-resistant and susceptible cultivars. The gene family consists of three homoeologous alleles of both RPL3A and RPL3B, which are located on chromosomes 4A (RPL3-B2), 4B (RPL3-B1), 4D (RPL3-B3), 5A (RPL3-A3), 5B (RPL3-A2) and 5D (RPL3-A1). Alternative splicing was detected in the TaRPL3-A2 gene. Sequence comparison revealed no amino acid differences between cultivars differing in Fusarium resistance. While using developed SNP markers we nevertheless found that one of the genes, namely, TaRPL3-A3 mapped close to a Fusarium resistance QTL (Qfhs.ifa-5A). The potential role of the RPL3 gene family in DON resistance of wheat is discussed.     

Long-term specific immune responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine: characterization of CD8+-T-cell epitopes recognized

We studied the effect of booster injections and the long-term immune response after injections of an anti-human immunodeficiency virus type 1 (HIV-1) lipopeptide vaccine. This vaccine was injected alone or with QS21 adjuvant to 28 HIV-uninfected volunteers. One month later, after a fourth injection of the vaccine, B- and T-cell anti-HIV responses were detected in >85% of the vaccinated volunteers. One year after this injection, a long-term immune response was observed in >50% of the volunteers. At this point, a positive QS21 effect was observed only in the sustained B-cell and CD4(+)-T-cell responses. To better characterize the CD8(+)-T-cell response, we used a gamma interferon enzyme-linked immunospot method and a bank of 59 HIV-1 epitopes. For the six most common HLA molecules (HLA-A2, -A3, -A11, -A24, -B7 superfamily, and -B8), an average of 10 (range, 3 to 15) HIV-1 epitopes were tested. CD8(+)-T-cell responses were evaluated according to the HLA class I molecules of the volunteers. Each assessment was based on 18 HIV-1 epitopes in average. We showed that 31 HIV-1 epitopes elicited specific CD8(+)-T-cell responses after vaccination. The most frequently recognized peptides were Nef 68-76 (-B7), Nef 71-79 (-B7), Nef 84-92 (-A11), Nef 135-143 (-B7), Nef 136-145 (-A2), Nef 137-145 (-A2), Gag 259-267 (-B8), Gag 260-268 (-A2), Gag 267-274 (-A2), Gag 267-277 (-B7), and Gag 276-283 (A24). We found that CD8(+)-T-cell epitopes were induced at a higher number after a fourth injection (P < 0.05 compared to three injections), which indicates an increase in the breadth of HIV CD8(+)-T-cell epitope recognition after the boost.     

Cross-stimulation of antigens under separate histocompatibility-linkedIr gene control

The antibody response of rats to the related polypeptide antigens (T,G)-A —L, (H,G)-A -L, and (Phe,G)-A -L is controlled byIr gene(s) linked to the major histocompatibility genes. When lymph node cells of rats primed with one of these polypeptides were cultured with the homologous antigen, a proliferative secondary response was induced with cells from high, but not from low responder strains. Thus, responsiveness as defined by antibody production is clearly reflected in this cellular in vitro assay.In contrast to the antipolypeptide antibodies which crossreact extensively, no crossreaction could be detected on the cellular level among the three polypeptides except for one combination: (T,G)-A -L-primed cells were strongly cross-stimulated by (Phe,G)-A -L, but the opposite was not true. The two antigens, however, show a distinct response pattern in various rat strains. The unilateral cross-stimulation may be explained by the special chemical relationship of determinants containing tyrosine and phenylalanine residues. It could occur on the level of the T cell receptor or, if distinct from it, on that of theIr gene product.Abbreviations used in this paper are as follows GT linear copolypeptide, poly (L-Glu, L-Tyr) - (T,G)-A-L the branched synthetic polypeptides poly(L-Tyr, L-Glu)-poly-DL-Ala-polyL-Lys - (H,G)-A -L the branched synthetic polypeptides poly(L-His, L-Glu)-poly-DL-Ala-poly-L-Lys - (Phe,G)-A-L poly(L-Phe, L-Glu)-poly-DL-Ala — poly-L-Lys - i.m. intramuscularly - LDH_A lactate dehydrogenase, isozyme A - MBSA methyl ester of bovine serum albumin - PHA phytohemagglutinin ofPhaseolus vulgaris     

Genetic control of IgM responses to (T,G)-A — L

The primary antibody response to aqueous immunization with a low molecular-weight lot of (T,G)-A — L (#420) was studied in congenic pairs of inbred mouse strains. Two new genetic controls were identified, both of which quantitatively regulate the production of IgM anti-(T,G)-A — L antibody. Testing of F₁ and F₂ progeny demonstrated that one of these genes is linked to the major histocompatibility (H-2) complex, and that high response is dominant over low response. Whether this gene is identical toIr-1A is not yet known. The other gene, designatedIg-TGAL _M , is linked to the immunoglobulin heavy-chain allotype locus (Ig-1) and is expressed in a genedose dependent manner. Following secondary challenge with (T,G)-A — L 420, quantitative differences in IgG antibody response were observed inIr-1A high-responder congenics differing only at theIg-1 locus. Breeding studies, however, failed to demonstrate any linkage between this locus and the quantitative control of IgG anti-(T,G)-A — L antibody. These data demonstrate thatH-2-linked immune response genes can regulate IgM as well as IgG antibody responses, that genetic control of the IgM response to (T,G)-A — L is linked toIg-1, and that bothH-2-linked andIg-1-linked genes may simultaneously affect an IgM antibody response to the same antigen.Abbreviations used in this paper are (T,G)-A — L poly-l-(Tyr,Glu)-poly-d,l-Ala-poly-l-Lys - NMS normal mouse serum - SRBC sheep red blood cells - i.p. intraperitoneal - PBS phosphate-buffered saline - RAMG polyvalent rabbit anti-mouse globulin - 2-Me 2-Mercaptoethanol - 2-MeS 2-Me-sensitive - PFC plaque-forming cells - ABC antigen-binding cells     

The Effects of Cerium Valence States at Cerium Oxide Coatings on the Responses of Bone Mesenchymal Stem Cells and Macrophages

Ideal orthopedic coatings should trigger good osteogenic response and limited inflammatory response. The cerium valence states in ceria are associated with their anti-oxidative activity and anti-inflammatory property. In the study, we prepared two kinds of plasma sprayed CeO₂ coatings with different Ce⁴⁺ concentrations to investigate the effects of Ce valence states on the response of bone mesenchymal stem cells (BMSCs) and macrophage RAW264.7. Both the coatings (CeO₂-A and CeO₂-B) were characterized via XRD, SEM, and X-ray photoelectron spectroscopy. The CeO₂ coatings enhanced osteogenic behaviors of BMSCs in terms of cellular proliferation, alkaline phosphatase (ALP) activity and calcium deposition activity in comparison with the Ti substrate. In particular, the CeO₂-B coating (higher Ce⁴⁺ concentration) elicited greater effects than the CeO₂-A coating (higher Ce³⁺ concentration). RT-PCR and western blot results suggested that the CeO₂-B coating promoted BMSCs osteogenic differentiation through the SMAD-dependent BMP signaling pathway, which activated Runx2 expression and subsequently enhanced the expression of ALP and OCN. With respect to either CeO₂-A coating or Ti substrate, the CeO₂-B coating exerted greater effects on the macrophages, increasing the anti-inflammatory cytokines (IL-10 and IL-1ra) expression and suppressing the expression of the pro-inflammatory cytokines (TNF-α and IL-6) and ROS production. Furthermore, it also upregulated the expression of osteoinductive molecules (TGF-β1 and BMP2) in the macrophages. The regulation of cerium valence states at plasma sprayed ceria coatings can be a valuable strategy to improve osteogenic properties and alleviate inflammatory response.     

Association of CHRNA5-A3-B4 Variation with Esophageal Squamous Cell Carcinoma Risk and Smoking Behaviors in a Chinese Population

Background

CHRNA5-A3-B4, the gene cluster encoding nicotinic acetylcholine receptor subunits, is associated with lung cancer risk and smoking behaviors in people of European descent. Because cigarette smoking is also a major risk factor for esophageal squamous cell carcinoma (ESCC), we investigated the associations between variants in CHRNA5-A3-B4 and ESCC risk, as well as smoking behaviors, in a Chinese population.

Methods

A case-control study of 866 ESCC patients and 952 healthy controls was performed to study the association of polymorphisms (rs667282 and rs3743073) in CHRNA5-A3-B4 with cancer risk using logistic regression models. The relationships between CHRNA5-A3-B4 polymorphisms and smoking behaviors that can be quantified by cigarettes smoked per day (CPD) and pack-years of smoking were separately estimated with Kruskal-Wallis tests among all 840 smokers.

Results

CHRNA5-A3-B4 rs667282 TT/TG genotypes were associated with significantly increased risk of ESCC [adjusted odds ratio (OR) = 1.32, 95% confidence interval (CI) = 1.03 – 1.69, P = 0.029]. The increased ESCC risk was even higher among younger subjects (≤60 years) (OR = 1.44, 95% CI = 1.04 – 1.98, P = 0.024). These effects were not found in another polymorphism rs3743073. No evident association between the two polymorphisms and smoking behaviors was observed.

Conclusions

These results support the hypothesis that CHRNA5-A3-B4 is a susceptibility gene cluster for ESCC. The relationship between CHRNA5-A3-B4 and smoking behaviors in a Chinese population needs further investigation.     

Block copolymers of amino acids. I. Synthesis and structure of copolymers of L-alanine or L-phenylalanine with D,L-lysine-d7 or D,L-lysine

Water-soluble block copolymers of the type (A)_m-(B)_n-(A)_p, where (A)_m,p was either poly(D ,L -lysine-α,β,β,γ,γ,δ,δ-d₇) or poly(D ,L -lysine) and (B)_n was either poly(L -alanine) or poly(L -phenylalanine), were synthesized for conformational studies by proton magnetic resonance spectroscopy. Analytical determination of the amount of the initiator fragment (n-hexylamine) at the C-terminus of the copolymers was used to obtain the number-average degrees of polymerization, DP _n, and thereby, together with the amino acid composition, to establish the covalent structures of the polymers. The values of DP _n were found to be much lower than those deduced from sedimentation equilibrium or form viscosity measurements. These deviations, which also are thought to have arisen in similar studies reported in the literature, are attributable to intermolecular aggregation; the relation of such aggregation to covalent structure (and its effect on the polymerization reaction) is discussed in terms of the conditions and mechanism of synthesis of block copolymers of amino acids.     

Biooxidation of 2-phenylethanol to phenylacetic acid by whole-cell Gluconobacter oxydans biocatalyst immobilized in polyelectrolyte complex capsules

A high-performance biocatalyst in the form of encapsulated cells of Gluconobacter oxydans have been developed for production of phenylacetic acid (PAA) as a natural flavor component. Polyelectrolyte complex (PEC) capsules consisting of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl₂, and NaCl were used for highly controlled and mild encapsulation of cells. Utilization of encapsulated G. oxydans cells was a significant improvement on existing data on operational stability of cells and cumulative product concentration during biocatalytic production of PAA from 2-phenylethanol. Concerning operational stability, encapsulated cells were active over 12 cycles with a high biotransformation rate, while free cells were inactive after 7 cycles of use. The biocatalytic properties of encapsulated G. oxydans were tested in a bubble column reactor over 7 days with a final cumulative product concentration of 25 g/L. High cell viability (90%) was observed within PEC capsules by confocal laser scanning microscopy, performed before and after repetitive PAA production in the bubble column reactor. The surface microstructure of fully hydrated capsules with and without G. oxydans cells was investigated and compared using an environmental scanning electron microscope.     

The beta conformation of polypeptides of valine, isoleucine, and threonine in solution and solid-state: optical and infrared studies.

Water-soluble polypeptides of L -valyl and L -isoleucyl residues flanked with DL -lysyl blocks [poly(DL Lys · HCl)_x–poly(L Val)_y–poly(DL Lys · HCl)_x, poly(DL Lys · HCl)_x–poly-(L Ile)_y–poly(DL Lys · HCl)_x] and homopoly(L -threonine) were prepared. The β conformation of these polymers in water, as well as in aqueous methanol, was confirmed by infrared spectroscopy and circular dichroism studies. The optical properties of these valyl and isoleucyl polypeptides were quite different from those of previously reported synthetic homopolypeptides in the β structure. Their differences could be explained by the presence of a “single extended β chain” without either intra- or interchain association.     

Purification and characterization of a novel neutral β-glucanase and an alkaline β-glucanase from an alkaliphilic Bacillus isolate

A relatively high β-glucanase-producing strain Bacillus sp. III-3 was isolated from soda lakes in Neimenggu, China. This alkaliphilic strain was found to produce two thermostable β-glucanases including a novel neutral β-glucanase III-3-A and an alkaline β-glucanase III-3-B. The β-glucanases were purified to homogeneity from the culture supernatant with a two-step column chromatographic procedure. III-3-A and III-3-B had molecular mass of approximately 45 and 85 kDa, respectively. Mass spectrometry analysis indicated that III-3-A was probably different from the β-glucanases reported, whereas III-3-B showed high homology with those of family A5 alkaline β-glucanases from alkaliphilic bacilli. The optimum pH of III-3-A was about 7.0, while that of III-3-B was 8.0–10.0. Both enzymes exhibited maximum activity at 45 °C and were stable up to 50 °C. Ca²⁺ ion stimulated the activity of III-3-A but enhanced the thermostability of III-3-B. The two enzymes were resistant to most metal ions and reagents examined.     

Protective role of astaxanthin against u.v.-B irradiation in the green alga Haematococcus pluvialis

Cyst cells of the green alga Haematococcus pluvialis accumulate astaxanthin with maturation of the resting stage. To study the protective role of astaxanthin against u.v. damage, both immature (astaxanthin-poor) and mature (astaxanthin-rich) cyst cells were exposed to u.v.-A or u.v.-B irradiation, and the residual cell viability and astaxanthin levels were determined. u.v.-B decreased both cell viability and astaxanthin level of cyst cells to a greater extent than u.v.-A. Tolerance of mature cyst cells to u.v.-B was 6-fold higher than that of immature cyst cells. These results indicated that astaxanthin in cyst cells functions as a protective agent against u.v.-B irradiation.     

The Purification and properties of some less common allotypes of the fourth component of human complement

Human complement component C4 is coded by two genes situated between HLA-D and HLA-B. Both genes are highly polymorphic; C4-A gene products normally carry the blood group antigen Rodgers and C4-B proteins usually carry the Chido antigen. Using a monoclonal antibody which binds Rodgers-positive and Chido-positive proteins with different affinities, we have purified a number of less common C4 allotypes and compared their properties. All C4-B allotypes tested have similar specific hemolytic activities and binding efficiencies to small molecules. All C4-A proteins tested had similar binding to small molecules and hemolytic activities except for the C4-A6 proteins from two individuals with different extended haplotypes, both of which had identical hemolytic activities and much lower ones than other C4-A allotypes. Two allotypes, C4 Al, Rodgers-negative but Chido-positive, and C4-B5, Chido-negative but probably Rodgers-positive, were found to behave as typical C4A and C4-B proteins, respectively, apart from the switch in their antigenic properties.Deceased     

Heat capacities of solid poly(amino acid)s. II. The remaining polymers

In the first paper heat capacities C_p, of polyglycine, poly(L -alanine), and poly (L -valine) were analyzed using approximate group vibrations and fitting the C_p contributions of the skeletal vibrations to a two-parameter Tarasov function. In this second paper all other poly (amino acid) s are similarly analyzed. Heat capacities were measured by differential scanning calorimetry in the temperature range of 230–390 K for poly(L -leucine), poly(L -serine), poly (sodium-L -aspartate), poly(sodium-L -glutamate), poly(L -asparagine), poly(L -phenylalanine), poly(L -tyrosine), poly(L -methionine), poly (L -tryptophane), poly(L -proline), poly(L -lysine · HBr), poly(L -histidine), poly(L -histidine- HCl), and poly(L -arginine · HCl). Good agreement exists between experiment and calculation. Predictions of heat capacities were made for all not-measured poly (amino acid) s. Enthalpies, entropies, and Gibbs functions for the solid state have been derived. © 1993 John Wiley & Sons, Inc.