Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability

A series of plasmid-based expression vectors have been constructed allowing stable intracellular expression of recombinant proteins in Bacillus subtilis strains. These expression vectors are based on the recently described Escherichia coli-B. subtilis shuttle vector pMTLBS72 which replicates as theta circles. Besides the weak constitutive promoter P(lepA), we inserted three different controllable promoters: P(gsiB) which can be induced by heat and acid shock, and by ethanol, P(xylA) and P(spac) which respond to the addition of xylose and IPTG, respectively. The versatility of these expression vectors was demonstrated by fusing their promoters to a reporter gene and by overexpression of the HtpG protein with three of them. All recombinant vectors exhibited full structural stability.     

Isoflavones Daidzein and Genistein: Preparation by Acid Hydrolysis of Their Glycosides and the Effect on Phospholipid Peroxidation

A mixture of isoflavones was obtained by acid hydrolysis of isoflavone glycosides isolated from the products of soybean processing by successive extraction with aqueous acetone and methanol. The homogeneous isoflavones daidzein and genistein were isolated from the aglycone mixture by adsorption chromatography and identified by spectral and chromatographic methods. The effect of both isoflavones on lipid peroxidation of soy phospholipids in multilamellar vesicles was studied at various concentrations. These aglycones were found to inhibit the formation of lipid hydroperoxides and malonic dialdehyde at concentrations as low as 1 mM.     

Characterisation of cerebroside 3-sulphate dispersions

Galactosyl ceramide 3-sulphate (cerebroside 3-sulphate) was tritiated using [³H]NaBH₄ with PdCl₂ as catalyst. Quantitative purification of the three components of the product was achieved by chromatography on Florisil. Dispersions, prepared by either low energy sonication or by dilution from organic solvent, were compared by controlled pore glass chromatography, ultra-centrifugation and electron microscopy. Both dispersion techniques were shown to form unilamellar vesicles, the average size of vesicles produced by sonication being far larger than those produced by solvent dilution. The diameter of isolated vesicles produced by solvent dilution was in the range 10–80 nm.     

Structures of acidic O-linked polylactosaminoglycans on human skim milk mucins

O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz 1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins: 1) the size of the alditols varies from tri- to decasaccharides, 2) the core structure is of the ubiquitous type 2, 3) the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAc beta(1-3)Gal (major) or GlcNAc beta(1-6)Gal units (minor).     

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.     

Juniper Shade Enables Terricolous Lichens and Mosses to Maintain High Photochemical Efficiency in a Semiarid Temperate Sand Grassland

On a semiarid sand grassland (Festucetum vaginatae) colonised by juniper (Juniperus communis L.) shrubs terricolous lichens and mosses segregate strongly between microhabitats: certain species grow in the open grassland, others almost exclusively in the shade of junipers. The contrasting irradiances of these microhabitats influence much the metabolism of these organisms, and thus affect their small-scale distribution. This was confirmed by determining the efficiency of photochemical energy conversion by measuring chlorophyll a fluorescence parameters. In the open grassland maximum photochemical efficiency of photosystem 2 (PS2, F_v/F_m) declined from the humid spring to the hot and dry summer in all species, and this was caused by an increase in base fluorescence (F₀), but not by the decrease in fluorescence maximum (F_m). In summer, mosses and lichens growing in the open grassland generally possessed lower F_v/F_m than cryptogams growing in the shade cast by juniper shrubs. Thus mosses and lichens in the open grassland suffer lasting reduction in photochemical efficiency in summer, which is avoided in the shade of junipers. Juniper shrubs indeed influence the composition and small-scale spatial pattern of sympatric terricolous lichen and moss communities by—among others—providing a shelter against high light in summer.     

The hernandez and fernandez genes of Drosophila specify eye and antenna

The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of "eye-specifying" genes, among them eyeless, which can direct eye development ectopically. We report here the characterization of the hernandez and fernandez genes, expressed in the antennal and eye primordia of the eye-antenna imaginal disc. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: it can form distal antenna, activating genes like homothorax, spineless, and spalt, and it can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. Our results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically.     

Synthesis and Pharmacological Activity of Betulin Dinicotinate

The assignment of NMR resonances of lupane triterpenoids was refined by the example of 3O,28O-dinicotinoylbetulin, obtained by acylation of betulin. Hepatoprotective, antiulcer, antiinflammatory, reparative, and anti-HIV activities were found for the compound. In addition, it was demonstrated to have immunomodulatory activity, for the first time detected among lupane triterpenoids.     

Thioglycosides as glycosylating agents in oligosaccharide synthesis

A review is given of the use of thioglycosides as glycosyl donors in oligosaccharide synthesis. Both indirect use, by conversion of the thioglycoside into a glycosyl halide and direct use, by electrophilic activation of the thioglycoside, are discussed.Abbreviations DMTST dimethyl(methylthio)sulfonium trifluoromethanesulfonate - Bz benzoyl - Bn benzyl - pNBz p-nitrobenzoyl - Phth phthallyl - Ph phenyl     

Isolation of Phytate-Hydrolysing Microbial Strains From Traditional Waste Water of Rice Fermentation and Liquid Cattle Feeds

Traditional fermentation systems employed by rural people of India were initially screened for phytate-hydrolysing microbes on solid medium supplemented with calcium phytate. It was followed by enzyme assay of culture filtrates to differentiate the phytase-producing microbes. Three new microbial genera are added to the list of phytase-producing organisms.     

Fungal Thyroiditis: An Overview

The authors review the epidemiology, clinical manifestations, diagnosis, and treatment of fungal thyroiditis cases previously reported in the medical literature. Aspergillus was by far the most common cause of fungal thyroiditis. Immunocompromised patients, such as those with leukemia, lymphoma, autoimmune diseases, and organ-transplant patients on pharmacological immunosuppression were particularly at risk. Fungal thyroiditis was diagnosed at autopsy as part of disseminated infection in a substantial number of patients without clinical manifestations and laboratory evidence of thyroid dysfunction. Local signs and symptoms of infection were indistinguishable from other infectious thyroiditis and included fever, anterior cervical pain, thyroid enlargement sometimes associated with dysphagia and dysphonia, and clinical and laboratory features of transient hyperthyroidism due to the release of thyroid hormone from follicular cell damage, followed by residual hypothyroidism. Antemortem diagnosis of fungal thyroiditis was made by direct microscopy and culture of a fine-needle aspirate, or/and biopsy in most cases. Since most patients with fungal thyroiditis had disseminated fungal infection with delay in diagnosis and treatment, the overall mortality was high.     

The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays

A general method is described for the assay of glycosyltransferase activity, which makes use of synthetic glycoside acceptors attached to hydrophobic aglycones. The products formed by incubation of an enzyme with acceptor and radiolabelled sugarnucleotide can then be rapidly (one minute) separated from interfering radioactivity by adsorption on to reverse-phase C-18 cartridges. After aqueous washing, products are easily isolated by elution with methanol. The utility of the method for the assay of (1–4)galactosyltransferase, (1–2)fucosyltransferase andN-acetylglucosaminyltransferase I and V is demonstrated.     

Oriole pollination ofErythrina breviflora (Leguminosae): Evidence for a polytypic view of ornithophily

Erythrina breviflora is visited by large numbers of passerine birds of which orioles (Icterus: Icteridae) are the primary pollinators. The flowers produce large quantities of nectar but they are rarely visited by hummingbirds. Inflorescence and floral morphology, and low levels of sucrose in the nectar probably explain the rarity of foraging hummingbirds. A comparison of Old WorldErythrina and their pollinators with New World species pollinated by orioles and hummingbirds suggests that parallel evolution has occurred. When the comparison is expanded to include other species pollinated by orioles, it is clear that various New WorldIcteridae, Thraupidae, etc. are ecological equivalents of Old WorldOriolidae, Pycnonotidae, Sturnidae, etc. and that flowers pollinated by these birds have similar characteristics.     

Effects of surfactants on cell growth and pigment production in suspension cultures ofPerilla frutescens

The effects of surfactants, adecanol LG-294 and silicone A, on anthocyanin accumulation and the growth ofPerilla frutescens cells in suspension cultures were studied. Production of the red pigment was remarkably reduced from about 1.9 g/l to 0.4 g/l by adecanol LG-294 at 0.06 ml/l but not by silicone A up to 0.4 ml/l. Several repeated shake-flask cultures also demonstrated no adverse effects of silicone A on the metabolite accumulation by the suspended cells. Furthermore, the addition of silicone A to a culture in a stirred bioreactor produced a three-fold higher growth rate and a seven-fold increase in anthocyanin compared with surfactant-free cultures. The improvement was due to the substantial reduction or prevention of foaming and of cell adhesion to the bioreactor wall.     

Molecular mapping of rice chromosomes

Summary We report the construction of an RFLP genetic map of rice (Oryza sativa) chromosomes. The map is comprised of 135 loci corresponding to clones selected from a PstI genomic library. This molecular map covers 1,389 cM of the rice genome and exceeds the current classical maps by more than 20%. The map was generated from F₂ segregation data (50 individuals) from a cross between an indica and javanica rice cultivar. Primary trisomics were used to assign linkage groups to each of the 12 rice chromosomes. Seventy-eight percent of the clones assayed revealed RFLPs between the two parental cultivars, indicating that rice contains a significant amount of RFLP variation. Strong correlations between size of hybridizing restriction fragments and level of polymorphism indicate that a significant proportion of the RFLPs in rice are generated by insertions/delections. This conclusion is supported by the occurrence of null alleles for some clones (presumably created by insertion or deletion events). One clone, RG229, hybridized to sequences in both the indica and javanica genomes, which have apparently transposed since the divergence of the two cultivars from their last common ancestor, providing evidence for sequence movement in rice. As a by product of this mapping project, we have discovered that rice DNA is less C-methylated than tomato or maize DNA. Our results also suggest the notion that a large fraction of the rice genome (approximately 50%) is single copy.     

Comparison between the allergenic airborne pollen in Trieste and at Lozzo di Cadore (Italy) in 1989

Summary A comparative study was carried out between aeroallergenic pollen spectra in Trieste and at Lozzo di Cadore. The two localities were found to be different on a five-day running mean basis as to the 8 pollen taxa monitored by the National Aerobiological Monitoring Network managed by the Italian Association of Aerobiology (AIA). In the zone of Lozzo the pollination curve of the spring flowering taxa showed a lag of about one month due to the colder climate. Lozzo di Cadore showed a poor airborne pollen content,Corylus and Graminaceae being prevailing. Trieste has a higher airborne pollen diversity and longer pollination times because of its higher floristic and vegetational complexity. In Trieste the allergenic pollen spectrum showed great quantities ofOstrya, Quercus, Oleaceae, Graminaceae and of ruderal taxa, widespread over the area, due to man's influence.     

Apoptotic Regulation of Caspase Activity

Intracellular cysteine aspartate-specific proteases (caspases) play both signaling and effector roles in realizing the program of cell death. Caspases function as proteolytic cascades unique for each cell type and signal triggering apoptosis. All parts of the proteolytic cascades are duplicated and controlled by feedback signals. Amplification cycles between pairs of caspases (the third and the sixth, the ninth and the third, the twelfth and the sixth, and others) help multiply the initial apoptotic signal. The presence of physiological inhibitors of apoptosis that directly interact with caspases creates a multilevel regulatory network of apoptosis in cell. The caspase proteolytic cascades are also regulated by sphingolipid secondary messengers, among them ceramide, sphingosine, and their phosphates. Moreover, an association of the caspase signaling with ubiquitin-dependent proteolysis is shown in cells. In particular, the use of extracellular activators and inhibitors of caspases allows irreversible activation of apoptosis in tumor cells or the prevention of apoptosis in cortical neurons under neurodegenerative diseases.     

Biosynthesis of glycoconjugates in mitochondrial outer membranes

The results reported in this paper show two distinct ways for the incorporation ofN-acetylglucosamine into mitochondrial outer membranes. The first one is the glycosylation of dolichol acceptors, which is indicated by the inhibition of the synthesis of these products by the inhibitors of the dolichol intermediates (tunicamycin and GDP). The second one is the incorporation ofN-acetylglucosamine into protein acceptors directly from UDP-N-acetylglucosamine. This second way of glycosylation is only localized in mitochondria outer membranes.The existence of a direct route forN-glycoprotein biosynthesis has been based on the following evidence. First, the synthesis of theN-acetylglucosaminylated protein acceptors was not inhibited by tunicamycin or GDP. Second, the addition of exogenous dolichol-phosphate did not change the rate of biosynthesis of glycosylated protein material. Third, the sequential incorporation ofN-acetylglucosamine and mannose from their nucleotide derivatives in the presence of GDP and tunicamycin led to the synthesis of glycosylated protein material which entirely bound to Concanavalin A-Sepharose. The oligosaccharide moiety of the glycosylated protein material resulting from the direct transfer of sugars from their nucleotide derivatives to the protein acceptor is of theN-glycan type. On sodium dodecylsulphate polyacrylamide gel electrophoresis, this glycosylated material migrated as a marker protein with a molecular weight between 45 000 and 63 000. HPLC chromatofocusing analysis revealed that the fraction studied was anionic. The oligosaccharide moiety of the glycoprotein material can only be elongated by the incorporation ofN-acetylglucosamine and galactose from their nucleotide derivatives.