Mammalian mitochondrial and microsomal cytochromes b(5) exhibit divergent structural and biophysical characteristics

The only outer mitochondrial membrane cytochrome b(5) examined to date, from rat (rOM b(5)), exhibits greater stability than known mammalian microsomal (Mc) isoforms, as well as a much higher kinetic barrier for hemin dissociation and a more negative reduction potential. A BlastP search of available databases using the protein sequence of rOM b(5) as template revealed entries for analogous proteins from human (hOM b(5)) and mouse (mOM b(5)). We prepared a synthetic gene coding for the heme-binding domain of hOM b(5), and expressed the protein to high levels. The hOM protein exhibits stability, hemin-binding, and redox properties similar to those of rOM b(5), suggesting that they are characteristic of the OM b(5) subfamily. The divergence in properties between the OM and Mc b(5) isoforms in mammals can be attributed, at least in part, to the presence of two extended hydrophobic patches in the former. The biophysical properties characteristic of the OM proteins may be important in facilitating the two functions proposed for them so far, reduction of ascorbate radical and stimulation of androgen synthesis.     

Peripheral benzodiazepine receptors in potatoes (Solanum tuberosum)

The peripheral benzodiazepine receptor (PBR), an internal protein of the mammalian mitochondrial membrane, is involved in several metabolic functions such as steroidogenesis, oxidative phosphorylation, and regulation of cell proliferation. Here we report the presence of PBRs in parenchymal and meristematic tissues of potato (Solanum tuberosum). PBRs are heterogeneously distributed in potato and are highly expressed in meristematic cells. In particular the receptor protein is mainly localised in the meristematic nuclear subcellular preparation. This 30-36 kDa protein, which corresponds to PBR, is increased, indeed, in meristematic compared to the parenchymal tissue. This suggests an involvement of this receptor in the regulation of cell plant growth. In addition, the demonstration that PBRs are also present in vegetables supports the hypothesis of a highly conserved receptor system during phylogenesis.     

Human DNA methyltransferase 1 is required for maintenance of the histone H3 modification pattern

DNA methyltransferase 1 (DNMT1) plays an essential role in murine development and is thought to be the enzyme primarily responsible for maintenance of the global methylation status of genomic DNA. However, loss of DNMT1 in human cancer cells affects only the methylation status of a limited number of pericentromeric sequences. Here we show that human cancer cells lacking DNMT1 display at least two important differences with respect to wild type cells: a profound disorganization of nuclear architecture, and an altered pattern of histone H3 modification that results in an increase in the acetylation and a decrease in the dimethylation and trimethylation of lysine 9. Additionally, this phenotype is associated with a loss of interaction of histone deacetylases (HDACs) and HP1 (heterochromatin protein 1) with histone H3 and pericentromeric repetitive sequences (satellite 2). Our data indicate that DNMT1 activity, via maintenance of the appropriate histone H3 modifications, contributes to the preservation of the correct organization of large heterochromatic regions.     

Role of caspases in the regulation of apoptotic pancreatic islet beta-cells death

The homeostatic control of beta-cell mass in normal and pathological conditions is based on the balance of proliferation, differentiation, and death of the insulin-secreting cells. A considerable body of evidence, accumulated during the last decade, has emphasized the significance of the disregulation of the mechanisms regulating the apoptosis of beta-cells in the sequence of events that lead to the development of diabetes. The identification of agents capable of interfering with this process needs to be based on a better understanding of the beta-cell specific pathways that are activated during apoptosis. The aim of this article is fivefold: (1) a review of the evidence for beta-cell apoptosis in Type I diabetes, Type II diabetes, and islet transplantation, (2) to review the common stimuli and their mechanisms in pancreatic beta-cell apoptosis, (3) to review the role of caspases and their activation pathway in beta-cell apoptosis, (4) to review the caspase cascade and morphological cellular changes in apoptotic beta-cells, and (5) to highlight the putative strategies for preventing pancreatic beta-cells from apoptosis.     

Decreased expression of peroxisome proliferator activated receptor gamma in cftr-/- mice

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARgamma. We hypothesized that PPARgamma expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARgamma mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild-type and cftr-/- mice by quantitative RT-PCR. PPARgamma expression was decreased twofold in CFTR-regulated tissues (colon, ileum, and lung) from cftr-/- mice compared to wild-type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARgamma in ileum and colon revealed a predominantly nuclear localization in wild-type mucosal epithelial cells while tissues from cftr-/- mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARgamma expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARgamma/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr-/- mice. Treatment of cftr-/- mice with the PPARgamma ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARgamma expression in cftr-/- mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.     

Vaccination in humans generates broad T cell cytokine responses

In recent years, the quantification of T cell responses to pathogens or immunogens has become a common tool in the evaluation of disease pathogenesis or vaccine immunogenicity. Such measurements are usually limited to enumerating IFN-gamma-producing cells after ex vivo stimulation with Ag, but little is known about the phenotype or complete functional repertoire of the Ag-specific cells. We used 12-color flow cytometry to characterize Ag-specific T cells elicited by vaccines or natural infection to determine lineage and differentiation status as well as the capacity to produce four cytokines (IFN-gamma, TNF-alpha, IL-2, and IL-4) and a chemokine (MIP1beta). As expected, responding cells had a typical memory phenotype; however, the cytokine profiles associated with the responses were highly complex. The pattern of cytokine coexpression in response to specific Ags was a skewed subset of the complete repertoire (revealed by polyclonal stimulation). We found significant differences in the patterns of cytokines elicited by vaccination (where IFN-gamma was by far a subdominant response) vs natural infection; in addition, there was fairly significant intersubject variation. Our findings illustrate the limitation of the evaluation of immune responses using single functional measurements (such as IFN-gamma); in fact, it is likely that sensitive evaluation of Ag-specific T cells will require the coordinate measurement of several cytokines. The presence and variability of these complex response profiles introduce the possibility that selective functional expression patterns may provide correlates for vaccine efficacy or disease progression.     

Similascarophis n. gen. n. spp. (Nematoda: Cystidicolidae) parasitizing marine fishes off the Chilean coast

Similascarophis (Cystidicolidae) n. gen. is proposed. In the mouth of specimens of this genus, submedial labia are absent and pseudolabia do not have any part projecting toward the central oral opening. These nematodes were obtained from the alimentary tract of 7 marine fish species along the coast of Chile: Bovichthys chilensis Regan, Eleginops maclovinus (Cuvier), Pinguipes chilensis (Valenciennes), Cilus gilberti (Abbott), Cheilodactylus variegatus Valenciennes, Girella laevifrons (Tschudi), and Graus nigra Philippi. Morphology and morphometry are compared between 2 new Similascarophis species: Similascarophis maulensis n. sp. and S. chilensis n. sp., which differ in the presence of sublabia and in the length of the glandular esophagus and left spicule. We also recorded Similascarophis sp. in 2 other host species, which showed some distinct proportional measurements, although these differences were not sufficiently clear to identify them as a new species.     

Species composition of assemblages of Ceratomyxa (Myxozoa), parasites of lings Genypterus (Ophidiidae) in the southeastern Pacific Ocean: an ecomorphometric approach

Studies on Myxozoa have emphasized their potential impact on aquatic animal health but less on ecological aspects. In this investigation, we assess the importance of host and latitudinal variations in the morphometry of spores of Ceratomyxa Théohan, 1892 (Myxozoa) from the gall bladder of 3 ling Genypterus (Pisces; Ophidiidae) species. Discriminant analyses indicated that several morphospecies of Ceratomyxa coexist in these hosts, despite the fact that the specific level of taxonomic resolution of each spore was not attempted. At least 4 species, i.e., Ceratomyxa hokarari Meglitsch, 1960; Ceratomyxa inversa Meglitsch, 1960; Ceratomyxa laxa Meglitsch, 1960; and Ceratomyxa elongata Meglitsch, 1960 occur in the study area, and at least 2 species co-occur in each host and geographical location. The most widespread pattern found is that spores are larger in golden ling (Genypterus blacodes) and spore size decreases with increasing latitude; this may be associated with the species composition of these assemblages, as well as with the habitat and diet segregation of the host species.     

Rhizochaete, a new genus of phanerochaetoid fungi

A new basidiomycete genus, Rhizochaete (Phanerochaetaceae, Polyporales), is described. Rhizochaete is characterized by a smooth to tuberculate, pellicular hymenophore and hyphal cords that turn red or violet in potassium hydroxide, monomitic hyphal system of simple or nodose septate hyphae, cystidia, and small, cylindrical to subglobose basidiospores. It morphologically is most similar to Phanerochaete. Analyses of nuclear ribosomal and internal-transcribed spacer region sequence data support a close relationship between Rhizochaete and Phanerochaete. The new taxon R. brunnea, from southern Argentina, is described and illustrated. In addition, the new combinations R. americana, R. borneensis, R. filamentosa, R. fouquieriae and R. radicata are proposed. A key to the species of Rhizochaete is provided.     

Identification of a γ-hexachlorocyclohexane dehydrochlorinase (LinA) variant with improved expression and solubility properties

Under aerobic conditions, the enzyme γ-hexachlorocyclohexane dechlorinase (LinA) from Sphingomonas paucimobilis UT26 catalyses the elimination of chlorine atoms from the molecule of γ-hexachlorocyclohexane (γ-HCH) or lindane, a recalcitrant pesticide that is still widely used. In its native metabolic context, LinA starts the biodegradation process of lindane by transforming γ-HCH to 1,2,4 trichlorobenzene (TCB), a less persistent chemical. In an attempt to generate an improved version of this enzyme to be used in lindane bioremediation schemes, we have run an experimental evolution procedure on LinA, using Escherichia coli as the surrogate host. One round of random mutagenesis and subsequent screening for improved dechlorination in vivo sufficed to yield one mutant enzyme (LinAT10), bearing a single substitution C132R, that displayed a two-fold enhanced expression and three-fold enhanced solubility of the enzyme compared to the wild type protein. This resulted in a biological product with a six-fold increase in dechlorination ability when expressed in E. coli. The potential of this protein and its expression system for in situ bioremediation is discussed.