A novel β-mannanase from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> A021: gene cloning,expression, purification and characterization

This paper describes the construction of a genomic library from the bacterium Pantoea agglomerans A021 and the subsequent cloning and expression of a novel mannanase gene (man26P). The gene consists of 1,047 bp and encodes a peptide (Man26P) of 348 amino acids with a calculated molecular mass of 38.5 kDa. Man26P is 63% identical with mannanase from Pectobacterium carotovorum at protein level and considered to be a member of the glycoside hydrolase family 26 (GH26). Man26P was expressed efficiently in E.coli BL21 (DE3) after induction with isopropylthiogalactoside (IPTG) and purified with a GST Bind Purification Kit. Maximum activity of purified Man26P was 514 U mg⁻¹, which was seen at pH 6.0 and a temperature of 55°C. Man26P was stable on exposure to buffers ranging from pH 4.0–10.0, and tolerant of temperature below 60°C. Zn²⁺, Mg²⁺ and Co²⁺ enhanced the activity, while Mn²⁺, Cu²⁺ and Hg⁺ had a negative effect. β-mercaptoethanol (1%) increased the activity twofold, while SDS (1%) inhibited it significantly. The enzyme showed optimal activity in a NaCl solution. The properties make it a candidate for various industrial applications.     

BDNF Val66Met and cognition: all,none, or some? A meta‐analysis of the genetic association

The Val66Met, G196A (rs6265) polymorphism in the brain-derived neurotrophic factor gene, BDNF, located at 11p13, has been associated with a wide range of cognitive functions. Yet, the pattern of results is complex and conflicting. In this study, we conducted a meta-analysis that included 23 publications containing 31 independent samples comprised of 7095 individuals. The phenotypes that were examined in this analysis covered a wide variety of cognitive functions and included indicators of general cognitive ability, memory, executive function, visual processing skills and cognitive fluency. The meta-analysis did not establish significant genetic associations between the Val66Met polymorphism and any of the phenotypes that were included.     

Δ<Superscript>6</Superscript>-Desaturase from <Emphasis Type="Italic">Mortierella alpina</Emphasis>: cDNA cloning,expression, and phylogenetic analysis

The open reading frame of the Δ⁶-desaturase gene was isolated from Mortierella alpina W15 and the gene was cloned into a pPIC3.5K vector. The vector was transformed into Pichia pastoris GS115 and expression was induced with methanol. The Δ⁶-desaturase expressed in P. pastoris GS115 catalyzed the conversion of linoleic acid to γ-linolenic acid but not the conversion of α-linolenic acid to octadecatetraenoic acid. The results indicate that the Δ⁶-desaturase gene from M. alpina W15 has substrate specificity in different organisms. Phylogenetic analysis revealed that Δ⁶-desaturase genes can be divided into four monophyletic groups. This work paves the way for further study of the functions of Δ⁶-desaturase in fatty acid metabolism and its three-dimensional structure.     

Characteristics of shell microstructure and growth analysis of the Antarctic bivalve <Emphasis Type="Italic">Laternula elliptica</Emphasis> from Lützow-Holm Bay,Antarctica

Growth performance of the Antarctic bivalve Laternula elliptica was examined both by shell microstructural observation and by applying a fluorescent substance, tetracycline, as a shell growth marker. The shell was composed of two calcareous layers: the thick outer layer was homogeneous or granular in structure and the thin inner layer was nacreous. The architecture of Antarctic L. elliptica was different from that of temperate L. marilina, and the ratio of thickness between the outer and inner layers appeared to be different. The growth rate of the nacreous layer was analyzed to be very low. High correlations were found between the major axis of chondrophore and both shell length and shell dry weight, respectively. It is suggested that the chondrophore is an appropriate growth indicator, and combining the information of growth increments with the fluorescent method may be useful in estimating the bivalve growth performance in the Antarctic sea.     

Taxonomic revision of Aegista subchinensis (M?llendorff, 1884) (Stylommatophora,Bradybaenidae) and a description of a new species of Aegista from eastern Taiwan based on multilocus phylogeny and comparative morphology

Aegista subchinensis (Möllendorff, 1884) is a widely distributed land snail species with morphological variation and endemic to Taiwan. Three genetic markers (partial sequence of the mitochondrial cytochrome c oxidase subunit I [COI], the 16S rDNA and the nuclear internal transcribed spacer 2 [ITS2]) were analysed to infer phylogenetic relationships and genetic divergence of closely related species of the genus Aegista, Aegista vermis (Reeve, 1852) and Aegista oculus (Pfeiffer, 1850). A new species from Aegista subchinensis has been recognized on the basis of phylogenetic and morphological evidences. The nominal new species, Aegista diversifamilia sp. n. is distinguished from Aegista subchinensis (Möllendorff, 1884) by its larger shell size, aperture and apex angle; wider umbilicus and flatter shell shape. The northernmost distribution of Aegista diversifamilia sp. n. is limited by the Lanyang River, which is presumed to mark the geographic barrier between Aegista diversifamilia sp. n. and Aegista subchinensis.     

Complexes prepared from protein A and human serum,IgG, or Fcγ fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion

Summary Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of ¹²⁵I-protein A and human ¹³¹I-IgG alone or in serum, and ¹³¹1-Fc fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess ¹³¹I-IgG or ¹³¹1-Fc, all the ¹²⁵I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess ¹²⁵I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.Abbreviations SpA protein A of Staphyloccus aureus - VBS EDTA gel, 0.0055 M veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin, pH 7.4 - PBS 0.01 M phosphate buffered saline, pH 7.4     

Purification,substrate specificity,and N-terminal amino acid sequence analysis of a β-lactamase-free penicillin amidase from <Emphasis Type="Italic">Alcaligenes</Emphasis> sp.

A -lactamase-free penicillin amidase from Alcaligenes sp. active against various -lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.     

Cry3Aa11: A New Cry3Aa δ-Endotoxin from a Local Isolate of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A local isolate of Bacillus thuringiensis Mm2 had insecticidal activity against the larvae of Melolontha melolontha, Agelastica alni, Leptinotarsa decemlineata and Amphimallon solstitiale and produced a 65 kDa protein. SDS-PAGE profile of B. thuringiensis Mm2 was compared with those of 29 different Cry3Aa producers which verified Cry3Aa biosynthesis by the isolate. The cry3Aa gene of Mm2 was cloned, sequenced and the deduced amino acid sequence was compared with the cry3Aa sequences of ten different quaternary ranks. Its identity to these sequences ranged between 97.4% and 99.2%. The gene was next cloned into E. coli-Bacillus shuttle vector pNW33N and expressed at a low level in B. subtilis 168.     

R1 and R2 retrotransposons of German cockroach <Emphasis Type="Italic">Blatella germanica</Emphasis>: A comparative study of 5′-truncated copies integrated into the genome

For the first time, extended fragments (5′-truncated copies) of R1 and R2 retrotransposons integrated into the Blattella germanica genome were identified, cloned, and sequenced. Structural comparison of the clones revealed two distinct R1 subfamilies. However, all R1 clones had two common features: poly(T) tails and similar target site duplications. R1 retrotransposons are the first known mobile elements with poly(T) tails on the 3′-ends. The structure and nucleotide sequences of five sequenced R2 fragments were similar to each other. Nucleotide sequence analysis of R2 retrotransposons revealed typical deletions at the 3′ ends of the target sites and the lack of homopolynucleotide tails.     

Mössbauer spectroscopy of the iron cores in human liver ferritin, ferritin in normal human spleen and ferritin in spleen from patient with primary myelofibrosis: preliminary results of comparative analysis

Comparative study of human liver ferritin and spleen tissues from healthy human and patient with primary myelofibrosis was carried out using Mössbauer spectroscopy with a high velocity resolution at 295 and 90 K and with a low velocity resolution at 20 K. The results obtained demonstrated that the iron content in patient’s spleen in the form of iron storage proteins was about ten times larger than that in normal tissue. However, in the case of patient with primary myelofibrosis the magnetic anisotropy energy barrier differed from that in normal case and, probably, the iron core size was supposed to be slightly larger than that in both normal spleen tissue and normal human liver ferritin in contrast to well-known data for iron overload in patients with thalassemia accompanied by the iron-core size increase. Therefore, the iron overload in the case of patient with primary myelofibrosis may be related to increase in the ferritin content mainly. It was also found that Mössbauer hyperfine parameters for normal and patient’s spleen and normal human liver ferritin demonstrated some small differences related, probably, to some small structural variations in the ferritin iron cores of patient’s spleen.