Cloning,characterization and overproduction of nuclease S1 gene (nucS) from Aspergillus oryzae

The nuclease S1 gene (nucS) from Aspergillus oryzae was isolated using a polymerase-chain-reaction-amplified DNA fragment as a probe, and a 2.6-kb SalI-EcoRI fragment containing the nucS gene was sequenced. It was deduced that the nucS gene had two short introns, 49 and 50 nucleotides in lenght. The nucS gene had an open-reading frame of 963 base pairs and coded for a protein of 287 amino acid residues, comprising the signal peptide of 20 amino acids and a mature protein of 267 amino acids. The deduced amino acid sequence agreed well with the published amino acid sequence except for one substitution. Southern hybridization analysis showed that the nucS gene existed as a single copy in the A. oryzae chromosome. When the structural gene of nucS was fused with the promoter of the glaA gene and introduced into A. oryzae, the yield od secreted nuclease S1 increased about 100-fold compared with the recipient strain.     

Macrophage colony-stimulating factor induces thrombospondin I production by cultured human macrophages

The role of colony-stimulating factors (CSFs) in regulating the synthesis of thrombospondin 1 (TSP1) by cultured human macrophages is investigated. Macrophage (M)-CSF is shown rapidly and transiently to induce two predominant species of TSP1 mRNA. One of these species was 3.2 kb in size and appeared to be specific to M-CSF-stimulated macrophages. Adherent M-CSF-treated macrophages are also shown to express abundant surface cell-associated TSP rapidly when examined by indirect immunofluorescence staining. Granulocyte-macrophage (GM)-CSF induced TSP1 mRNA at a later time point, and this was attributable to the effects of endogenous M-CSF induced by the GM-CSF; the GM-CSF-treated cells did not display surface-associated TSP after 3 hr of treatment. Analysis of the TSP1 protein synthesised by the M-CSF-treated macrophages revealed the expected trimeric form of the molecule. In addition, an unidentified 95-kDa protein was found to be covalently associated with immunoreactive TSP1, and this appeared to be specific to the macrophages as it was not found in TSP1 precipitated from other cell types. It is suggested that the induction of TSP1 by M-CSF may play an important role in the major physiological functions of macrophages. © 1995 Wiley-Liss, Inc.     

Parametric sensitivity of stoichiometric flux balance models applied to wild-type Escherichia coli metabolism

The esterification of lauric acid with geraniol catalyzed by the commercially immobilized lipase preparation from Mucor miehei, Lipozyme(R), was studied in well-stirred flasks. The enzyme support was characterized in terms of its internal and external surface area, protein location, and protein content. It was found that the enzyme was mainly located on the external surface of the support, therefore, internal diffusional limitations were not important. It was also shown that the protein content of the support depends on the size of the particle, with smaller particles containing higher amounts of protein per unit weight. Under the conditions studied, the reaction was not under external mass transfer limitations, and the initial reaction rate depended on the size of the support particles. This was mainly due to the different protein contents on the support as a function of particle size and not to internal or external mass transfer limitations. Also, it was found that the inhibition exerted by water was predominantly a physical effect due to its accumulation around the enzyme. It was also found that the reaction was substrate inhibited by lauric acid, but not by geraniol. (c) 1995 John Wiley & Sons, Inc.     

GENETIC CONSTRAINTS ON MACROEVOLUTION: THE EVOLUTION OF HOST AFFILIATION IN THE LEAF BEETLE GENUS OPHRAELLA

We hypothesize that the evolution of an ecologically important character, the host associations of specialized phytophagous insects, has been influenced by limitations on genetic variation. Using as a historical framework a phylogenetic reconstruction of the history of host associations in the beetle genus Ophraella (Chrysomelidae), we have employed quantitative-genetic methods to screen four species for genetic variation in larval survival, oviposition (in one species only), and feeding responses to their congeners' host plants, in the Asteraceae. We here report results of studies of one species and evaluate the results from all four. Analysis of half-sib/full-sib families and of progenies of wild females of O. notulata, a specialist on Iva (Ambrosiinae), provided evidence of genetic variation in larval consumption of five of six test plants and in adult consumption of four of six. Larval mortality was complete on five plants; only on Ambrosia, a close relative of the natural host, was there appreciable, and genetically variable, survival. Oviposition on Ambrosia showed marginally significant evidence of genetic variation; a more distantly related plant elicited no oviposition at all. In compiling results from four Ophraella species, reported in this and two other papers, we found no evidence of genetic variation in 18 of 39 tests of feeding responses and 14 of 16 tests of larval survival on congeners' hosts. This result is consistent with the hypothesis that absence or paucity of genetic variation may constrain or at least bias the evolution of host associations. The lower incidence of genetic variation in survival than in feeding behavior may imply, according to recent models, that avoidance is a more common evolutionary response to novel plants than adaptation. The usually great disparity between mean performance on congeners' hosts and the species' natural hosts, and an almost complete lack of evidence for negative genetic correlations, argue against the likelihood that speciation has occurred by sympatric host shift. The presence versus apparent absence of genetic variation in consumption was correlated with the propinquity of relationship between the beetle species tested and the species that normally feeds on the test plant, suggesting that the history of host shifts in Ophraella has been guided in part by restrictions on genetic variation. It was also correlated with the propinquity of relationship between a test plant and the beetle's natural host. The contributions of plant relationships and insect relationships, themselves correlated in part, to the pattern of genetic variation, are not readily distinguishable, but together accord with phylogenetic evidence that these and other phytophagous insects adapt most readily to related plants. In this instance, therefore, the macroevolution of an ecologically important character appears to have been influenced by genetic constraints. We hypothesize that absence of the structural prerequisites for genetic variation in complex characters may affect genetic variation and the trajectory of evolution.     

Model for prediction of immobilizedAcetobacter cell number in calcium alginate gel droplets

The geometry of calcium alginate gel spheres is studied by fractal interpretation for prediction of number of cells to be immobilized. For alginate concentrations from 1 to 5% the simulated results are of 6.65×10⁸, 1.44×10⁸ and 5.27×10⁷ N cell mL⁻¹ for respectively 1.5, 2.5 and 3.5 mm gel sphere diameters. Simulated data are compared with those resulting from literature and particularly with experimental trials where from 10⁷ to 10⁸ N cell mL⁻¹, in 2% alginate beads with a diameter of 1.5 mm, is able to fulfil mechanical and swelling characteristics of alginate gel beads besides to supply good oxygenation.     

Efficacy of the alcohol use disorders identification test as a screening tool for hazardous alcohol intake and related disorders in primary care: a validity study.

OBJECTIVE: To determine the properties of the alcohol use disorders identification test in screening primary care attenders for alcohol problems. DESIGN: A validity study among consecutive primary care attenders aged 18-65 years. Every third subject completed the alcohol use disorders identification test (a 10 item self report questionnaire on alcohol intake and related problems) and was interviewed by an investigator with the composite international diagnostic interview alcohol use module (a standardised interview for the independent assessment of alcohol intake and related disorders). SETTING: 10 primary care clinics in Verona, north eastern Italy. PATIENTS: 500 subjects were approached and 482 (96.4%) completed evaluation. RESULTS: When the alcohol use disorders identification test was used to detect subjects with alcohol problems the area under the receiver operating characteristic curve was 0.95. The cut off score of 5 was associated with a sensitivity of 0.84, a specificity of 0.90, and a positive predictive value of 0.60. The screening ability of the total score derived from summing the responses to the five items minimising the probability of misclassification between subjects with and without alcohol problems provided an area under the receiver operating characteristic curve of 0.93. A score of 5 or more on the five items was associated with a sensitivity of 0.79, a specificity of 0.95, and a positive predictive value of 0.73. CONCLUSIONS: The alcohol use disorders identification test performs well in detecting subjects with formal alcohol disorders and those with hazardous alcohol intake. Using five of the 10 items on the questionnaire gives reasonable accuracy, and these are recommended as questions of choice to screen patients for alcohol problems.