The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein.

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.     

Xerotolerant Cladosporium sphaerospermum Are Predominant on Indoor Surfaces Compared to Other Cladosporium Species

Indoor fungi are a major cause of cosmetic and structural damage of buildings worldwide and prolonged exposure of these fungi poses a health risk. Aspergillus, Penicillium and Cladosporium species are the most predominant fungi in indoor environments. Cladosporium species predominate under ambient conditions. A total of 123 Cladosporium isolates originating from indoor air and indoor surfaces of archives, industrial factories, laboratories, and other buildings from four continents were identified by sequencing the internal transcribed spacer (ITS), and a part of the translation elongation factor 1α gene (TEF) and actin gene (ACT). Species from the Cladosporium sphaerospermum species complex were most predominant representing 44.7% of all isolates, while the Cladosporium cladosporioides and Cladosporium herbarum species complexes represented 33.3% and 22.0%, respectively. The contribution of the C. sphaerospermum species complex was 23.1% and 58.2% in the indoor air and isolates from indoor surfaces, respectively. Isolates from this species complex showed growth at lower water activity (≥ 0.82) when compared to species from the C. cladosporioides and C. herbarum species complexes (≥ 0.85). Together, these data indicate that xerotolerance provide the C. sphaerospermum species complex advantage in colonizing indoor surfaces. As a consequence, C. sphaerospermum are proposed to be the most predominant fungus at these locations under ambient conditions. Findings are discussed in relation to the specificity of allergy test, as the current species of Cladosporium used to develop these tests are not the predominant indoor species.     

Purification and Characterization of an l-Aminopeptidase from Pseudomonas putida ATCC 12633

An l-aminopeptidase of Pseudomonas putida, used in an industrial process for the hydrolysis of d,l-amino acid amide racemates, was purified to homogeneity. The highly l-enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and alpha-H amino acid amides, e.g., l-phenylglycine amide.     

Periconceptional parental conditions and perimembranous ventricular septal defects in the offspring

Background: The perimembranous ventricular septal (pVSD) defect is the most common congenital heart disease phenotype. Several parental factors are associated with pVSD risk in the offspring. To contribute to the future prevention of pVSDs, we investigated associations with nongenetic parental conditions. Methods: In a case–control study with standardized data collection at 17 months after birth, 115 parents of a child with pVSD and 484 parents of a healthy child completed questionnaires about periconceptional nongenetic conditions. Univariable and multivariable logistic regression analyses were used to estimate odds ratios (OR) with 95% confidence intervals (95% CI). Results: Complete data were available for 588 families (98%). Maternal risk conditions associated with pVSD offspring were a positive family history of congenital heart disease (OR, 2.61; 95%CI, 0.98–6.91), medication use (OR, 1.80; 95%CI, 1.13–2.85) and advanced age (OR, 1.07; 95%CI, 1.02–1.12). Exposure to phthalates (OR, 1.93; 95%CI, 1.05–3.54) was the only paternal risk condition associated with pVSD offspring. Conclusion: Four periconceptional parental conditions contributed to pVSD risk in the offspring. Couples planning pregnancy should be counseled on these risk conditions which are partially modifiable to contribute to the future prevention of pVSDs. Birth Defects Research (Part A) 100:944–950, 2014. © 2014 Wiley Periodicals, Inc.     

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.     

Transient Marker System for Iterative Gene Targeting of a Prototrophic Fungus

Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of pyrG. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed, pyrG function can be rapidly returned to wild type by a simple selection scheme.     

Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected cell lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of alpha-synuclein is regulated by phosphorylation/dephosphorylation.