C6ORF32 is upregulated during muscle cell differentiation and induces the formation of cellular filopodia

We have identified a gene by microarray analysis that is located on chromosome 6 (c6orf32), whose expression is increased during human fetal myoblast differentiation. The protein encoded by c6orf32 is expressed both in myogenic and non-myogenic primary cells isolated from 18-week old human fetal skeletal muscle. Immunofluorescent staining indicated that C6ORF32 localizes to the cellular cytoskeleton and filopodia, and often displays polarized expression within the cell. mRNA knockdown experiments in the C2C12 murine myoblast cell line demonstrated that cells lacking c6orf32 exhibit a myogenic differentiation defect, characterized by a decrease in the expression of myogenin and myosin heavy chain (MHC) proteins, whereas MyoD1 was unaltered. In contrast, overexpression of c6orf32 in C2C12 or HEK293 cells (a non-muscle cell line) promoted formation of long membrane protrusions (filopodia). Analysis of serial deletion mutants demonstrated that amino acids 55-113 of C6ORF32 are likely involved in filopodia formation. These results indicate that C6ORF32 is a novel protein likely to play multiple functions, including promoting myogenic cell differentiation, cytoskeletal rearrangement and filopodia formation.     

Preliminary assessment of the risks associated with solar ultraviolet-A exposure

An approach is proposed to assess the periods of human skin exposure to solar ultraviolet-A (UV-A, 315–400 nm) irradiance in natural conditions that are able to yield doses found to trigger carcinogenesis in laboratory experiments. Weighting functions, adopted to perform such estimate are constructed, allowing for a comparison between environmental and laboratory doses. Furthermore, the impact of stratum corneum (SC) thickness on the studied environmental doses was investigated. Based on laboratory studies, it was found that exposure periods of less than a month, at mid-latitudes, could provide irradiance doses capable of causing tumor formation. The duration of these exposure periods closely depends on the exposure regime, atmospheric conditions and SC thickness. It is believed that the presented evaluations could provide a useful preliminary estimation of the risk associated with environmental UV-A exposure prior to the formulation of the corresponding action spectra and determination of the threshold doses.     

Examination of variables in the vaccination of mice against cutaneous leishmaniasis using living avirulent cloned lines and killed promastigotes of Leishmania major

Mitchell G. F., Handman E. and Spithill T. W. 1985. Examination of variables in the vaccination of mice against cutaneous leishmaniasis using living avirulent cloned lines and killed promastigotes of Leishmania major. International Journal for Parasitology15: 677–684. In vaccination experiments not involving adjuvants, genetically-susceptible mice were injected with living avirulent cloned promastigotes of Leishmania major or killed promastigotes prior to cutaneous challenge with virulent cloned promastigotes. Emphasis was placed on aspects that may contribute to marked variability between experiments and between laboratories in vaccination of mice against cutaneous leishmaniasis. One variable, the challenge promastigotes, was shown to be important in that cloned virulent parasites (V121) were less pathogenic in terms of rate of cutaneous lesion development, than the parental isolate LRC-L137 when low doses of promastigotes were used and particularly when harvested from the log phase of culture. It is likely that avirulent parasites in mixed isolates can increase the rate of lesion development after cutaneous deposition. As reported previously, intraperitoneal, and more particularly intravenous injections of living avirulent cloned parasites (A12) increase resistance in mice. Most importantly, a difference has been demonstrated in the vaccinating efficacy of killed promastigotes of various isolates injected intravenously. This implies that certain isolates of L. major (e.g. the “Moshkovsky strain”) express “host-protective antigens” at higher levels, or in a qualitatively different manner, than other isolates (e.g. LRC-L137). The finding will greatly facilitate the identification of vaccine antigens in this system using immunochemical and gene cloning approaches.     

Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperature-sensitive mutant altered in DNA primase-polymerase complex stability

Summary The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2. Temperature-sensitive mutants in POL1 have been obtained by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the mutated copy. Physiological and biochemical characterization of one temperature-sensitive mutant (pol1-1) shows that cells shifted to the non-permissive temperature can complete one round of cell division and DNA replication before they arrest. Analysis of DNA polymerase I in crude extracts and in partially purified preparations indicates that the pol1-1 mutation results in a conformational change and affects the stability of the DNA primase-polymerase complex.     

Inhibition of α-, β-, γ-, and δ-carbonic anhydrases from bacteria and diatoms with N′-aryl-N-hydroxy-ureas

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N’-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with K_Is in the range of 97.5?nM – 7.26?µM and 52.5?nM – 1.81?µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (K_Is of 4.75 – 8.87?µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (K_Is?>?10?µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (K_Is of 59.8?nM – 6.42?µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (K_Is of 33.3?nM – 8.74?µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (K_Is?>?10?µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.     

Effect of cell therapy with allogeneic osteoblasts on bone repair of rat calvaria defects

Background aims

Regenerative medicine strategies based on cell therapy are considered a promising approach to repair bone defects. The aims of this study were to evaluate the effect of subculturing on the osteogenic potential of osteoblasts derived from newborn rat calvaria and the effect of these osteoblasts on bone repair of rat calvaria defects.

Search for mutations in pancreatic sufficient cystic fibrosis Italian patients: detection of 90% of molecular defects and identification of three novel mutations

A cohort of 31 cystic fibrosis patients showing pancreatic sufficiency and bearing an unidentified mutation on at least one chromosome was analyzed through denaturing gradient gel electrophoresis of the whole coding region of the cystic fibrosis transmembrane conductance regulator gene, including intron-exon boundaries. Three new and 19 previously described mutations were detected. The combination of these with known mutations detected by other methods, allowed the characterization of mutations on 56/62 (90.3%) chromosomes. Among those identified, 17 can be considered responsible for pancreatic sufficiency, since they were found in patients carrying a severe mutation on the other chromosome. Among these presumed mild mutations, eight were detected more than once, R352Q being the most frequent in this sample (4.83%). Intragenic microsatellite analysis revealed that the six chromosomes still bearing unidentified mutations are associated with five different haplotypes. This may indicate that these chromosomes bear different mutations, rarely occurring among cystic fibrosis patients, further underlying the molecular heterogeneity of the genetic defects present in patients having pancreatic sufficiency.