An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans

Summary An acetyl esterase was purified from Trichoderma reesei by cation and anion exchange chromatography. The enzyme had a molecular weight of 45 000 as determined by SDS-electrophoresis, or 67 000 as determined by gel filtration. In chromatofocusing the enzyme was shown to consist of two isoenzymes with isoelectric points of 6.8 and 6.0. The enzyme showed activity towards naphthyl acetate, triacetin and glucose-and xylose acetates. However, it liberated acetic acid from acetylated xylo-oligomers only to a small extent. The liberation of acetic acid from the oligomeric substrate was enhanced by addition of endoxylanase and -xylosidase.     

Identification of Pneumocystis carinii in immunodeficient mice

Various procedures were utilized to determine the most sensitive, cost and labor effective techniques for detection of Pneumocystis carinii in immunologically compromised mice. Immunoperoxidase staining techniques that utilized polyclonal antibodies directed against purified rat or mouse P. carinii were more sensitive and specific than staining with Gomori's methenamine silver. Indirect immunofluorescence microscopy on frozen sections was comparable to immunoperoxidase staining, but lacked fine cytologic detail. Impression smears were of limited value when stained with Diff-Quik Stain, Harleco's Hemacolor, Wright-Giemsa or Wright-Leishman stains. However, cysts could be detected consistently in imprints stained with Gomori's methanamine silver. Transmission electron microscopy showed ultrastructural detail of P. carinii, but this technique was too costly and time consuming for routine use. Thus, because of its sensitivity and specificity, immunohistochemistry on paraffin sections was the most satisfactory method for screening and identifying P. carinii in lungs of immunocompromised mice.     

Patterns of trace metal accumulation in Swedish marine nemerteans

Total body burdens of Zn, Pb, Al and Cu were measured in Swedish marine nemerteans sampled from an area close to a major source of pollution. Accumulation patterns varied both seasonally and spatially. The results suggest that nemerteans may be useful bioindicator species in environmental monitoring programmes.     

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.     

Central neural peptides and catecholamines in spontaneous and DOCA/salt hypertension

Hypothalamic and neurophypophyseal levels of catecholamines and peptides were measured in spontaneous and deoxycorticosterone (DOCA)/salt hypertension. Catecholamines, norepinephrine, epinephrine and dopamine were measured by electrochemical detection while the peptides, vasopressin, oxytocin, luteinizing hormone-releasing hormone (LHRH), the enkephalins and somatostatin (SRIF) were measured by radioimmunoassay. Blood pressure was significantly elevated in both groups as compared to their controls. Marked changes in central neural peptides were observed in the SHR, while no differences were seen in DOCA/salt hypertension. Hypothalamic vasopressin, oxytocin, LHRH and SRIF were significantly decreased. In the posterior pituitary, enkephalins were increased twofold in the SHR. With regard to catecholamines, there was no change in hypothalamic content. However, a dramatic decrease in neurohypophyseal dopamine was observed in SHR. Plasma levels of vasopressin were significantly elevated in both types of hypertension while oxytocin was increased only in the DOCA/salt model. These result show that (1) a wide spectrum of neuroendocrine changes are associated with genetic hypertension, (2) there are CNS differences between DOCA/salt and spontaneous hypertension, and (3) central aminergic changes may be involved in th neuroendocrine alterations seen in the SHR.     

苎麻疫霉激发蛋白基因断裂现象初探

苎麻疫霉（PhytophthoraboehmeriaeSaw．）可分泌具有诱抗作用的激发蛋白（α－elicihn）,根据α－elicitin第24～30和56～63位保守区氨基酸推导的寡核苷酸引物序列,对苎麻疫霉基因组DNA进行特异PCR扩增反应,发现其扩增的DNA片段大于预计的片段。回收纯化的特异扩增DNA,并进行克隆和测序分析,结果表明特异扩增的elicihn基因亚克隆DNA为570hp,大于预计的117bp。在特异片段中,存在3个内含子将基因断裂成4个阅读框架,即ORF1、ORF2、ORF3和ORF4,其中ORF1和ORF4含有与引物相同的序列,但与其它序列与已克隆的elicihn基因无同源性。因此,芒麻疫霉基因组中的elicitin基因可能存在断裂现象。     

A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.