Transglycosylation of glycosyl residues to cyclic tetrasaccharide by Bacillus stearothermophilus cyclomaltodextrin glucanotransferase using cyclomaltodextrin as the glycosyl donor

Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as 4-mono-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], and sachharide D was 4,4'-di-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.     

Isolation and Characterization of Esters of Indole-3-Acetic Acid from the Liquid Endosperm of the Horse Chestnut (Aesculus species)

Esters of indole-3-acetic acid were extracted and purified from the liquid endosperm of immature fruits of various species of the horse chestnut (Aesculus parviflora, A. baumanni, A.pavia rubra, and A. pavia humulis). The liquid endosperm contained, at least 12 chromatographically distinct esters. One of these compounds was purified and characterized as an ester of indole-3-acetic acid and myo-inositol. A second compound was found to be an ester of indole-3-acetic acid and the disaccharide rutinose (glucosyl-rhamnose). A third compound was partially characterized as an ester of indole-3-acetic acid and a desoxyaminohexose.     

Enzymatic properties of recombinant kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus ATCC43494

One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP ≧ 4 than KP from Thermoanaerobacter brockii ATCC35047.     

Chlamydia pneumoniae multiply in neutrophil granulocytes and delay their spontaneous apoptosis

The obligate intracellular bacterial pathogen Chlamydia pneumoniae (Cp) is responsible for a range of human diseases, including acute respiratory infection. Although experimental intratracheal infection with Cp results in a massive recruitment of neutrophil granulocytes (polymorphonuclear neutrophils (PMN)), the role of these cells in the defense against Cp is unclear. In this study the interactions of PMN with Cp were investigated. In vitro coincubation experiments showed that human granulocytes were able to internalize Chlamydia in an opsonin-independent manner. Importantly, phagocytosed Cp were not killed; the ingested bacteria survived and multiplied within PMN. Although uninfected granulocytes became apoptotic within 10 h, infected PMN survived up to 90 h. Coincubation with Cp significantly decreased the ratio of apoptotic PMN, as detected by morphological analysis, annexin V, and TUNEL staining. The observed antiapoptotic effect was associated with a markedly lower level of procaspase-3 processing and, consequently, reduced caspase-3 activity in infected PMN. LPS was found as a major, but not exclusive, component responsible for the observed antiapoptotic effect. Chlamydia LPS affected PMN apoptosis both by acting directly on the cells and by inducing the autocrine production of the antiapoptotic cytokine IL-8. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, Cp can extend the life span of neutrophil granulocytes, making them suitable host cells for survival and multiplication within the first hours/days after infection.     

Afghan Hindu Kush: Where Eurasian Sub-Continent Gene Flows Converge

Despite being located at the crossroads of Asia, genetics of the Afghanistan populations have been largely overlooked. It is currently inhabited by five major ethnic populations: Pashtun, Tajik, Hazara, Uzbek and Turkmen. Here we present autosomal from a subset of our samples, mitochondrial and Y- chromosome data from over 500 Afghan samples among these 5 ethnic groups. This Afghan data was supplemented with the same Y-chromosome analyses of samples from Iran, Kyrgyzstan, Mongolia and updated Pakistani samples (HGDP-CEPH). The data presented here was integrated into existing knowledge of pan-Eurasian genetic diversity. The pattern of genetic variation, revealed by structure-like and Principal Component analyses and Analysis of Molecular Variance indicates that the people of Afghanistan are made up of a mosaic of components representing various geographic regions of Eurasian ancestry. The absence of a major Central Asian-specific component indicates that the Hindu Kush, like the gene pool of Central Asian populations in general, is a confluence of gene flows rather than a source of distinctly autochthonous populations that have arisen in situ: a conclusion that is reinforced by the phylogeography of both haploid loci.     

Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major

Macrophages are the major target cell population of the obligate intracellular parasites LEISHMANIA: Although polymorphonuclear neutrophil granulocytes (PMN) are able to internalize Leishmania promastigotes, these cells have not been considered to date as host cells for the parasites, primarily due to their short life span. In vitro coincubation experiments were conducted to investigate whether Leishmania can modify the spontaneous apoptosis of human PMN. Coincubation of PMN with Leishmania major promastigotes resulted in a significant decrease in the ratio of apoptotic neutrophils as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low m.w. DNA fragments, and annexin V staining. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in PMN. The inhibition of PMN apoptosis depended on viable parasites because killed Leishmania or a lysate of the parasites did not have antiapoptotic effect. L. major did not block, but rather delayed the programmed cell death of neutrophils by approximately 24 h. The antiapoptotic effect of the parasites could not be transferred by the supernatants, despite secretion of IL-8 by PMN upon coculture with L. major. In vivo, intact parasites were found intracellularly in PMN collected from the skin of mice 3 days after s.c. infection. This finding strongly suggests that infection with Leishmania prolongs the survival time of neutrophils also in vivo. These data indicate that Leishmania induce an increased survival of neutrophil granulocytes both in vitro and in vivo.     

Morphometric analysis of dendritic tree of cerebellar Purkinje-cells

Basic neural processes of sensorimotor adaptation can be observed by cellular-level studies on cerebellar cortex, both by electrophysiological and morphological means. In earlier studies we demonstrated double (sometimes triple or quadruple) rhythmic prespike activity patterns in dendritic microelectrode records taken from cerebellar Pc. By their active and passive interactions, the actual input pattern will turn into an arrhythmic output spiking, realizing a nonlinear and phase-sensitive integration. This curious complex spike-generating process can give rise to novel cerebellar functional models. The acting membrane dynamics rely not just upon the ionic current machinery but also upon the specific micromorphology of the dendritic tree, especially that of the branching areas. Although, statistical analyses on Pc dendrites generally followed the graph-theory, elaborated abstract parameters for complexity and hierarchy, and denied realistic geometry. Thus a novel specific morphometric study should be carried out, first applied to the main branching sites.     

Novel bioactive molecules from Lentzea violacea strain AS 08 using one strain-many compounds (OSMAC) approach

A new eudesmane sesquiterpenoid (1), and a new homologue of virginiae butanolide E (2) along with butyl isobutyl phthalate (3) were isolated from, actinomycete-Lentzea violacea strain AS08 isolated from north western Himalayas by stressing on modified one strain-many compounds (OSMAC) method. The structures of the new compounds were elucidated by extensive spectroscopic analyses including 1D, 2D NMR along with HR-ESI-MS and FT-IR data. Herein, a distinctive method was added for inspecting secretory profile of the strain by quantification of extract value of cell free supernatant in different types of culture media fallowed by HPLC profiling of respective extracts, which revealed a highly altered metabolic profile of the strain and formed the base for the selection of media. The compounds 1 and 2 showed moderate activity against Gram negative (MIC ～32–64 µg ml^?1) in comparison to Gram positive bacterial pathogens. Compound 1 exhibited significant activity in human cancerous cell lines (IC₅₀ ～19.2 µM).     

Double-standard isotope dilution assay: I. Quantitative assay of indole-3-acetic acid

Isotope dilution analysis for the quantitation of labile compounds has been limited in applicability by the amount of sample necessary to determine specific activity. A method is described for the analysis of radiolabeled compounds which allows the direct determination of specific activity by gas chromatography. It requires the availability of the radiolabeled internal standard, as is customarily used in an isotope dilution assay, and also requires a chemically related radiolabeled compound to serve as a second internal standard. It is this second internal standard, added in known amounts, that permits quantitation of the gas chromatography. The method is illustrated by assaying indole-3-acetic acid in plant extracts using [¹⁴C]indole-3-acetic acid as the internal standard and adding [¹⁴C]indole-3-butyric acid as the second internal standard for quantitation of the gas chromatographic procedures. Used with a nitrogen-specific thermionic detector the method is selective and is sensitive at the nanogram level. The synthesis of [2-ring-¹⁴C]indole-3-butyric acid is also described.