Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.     

Transfection of epithelioma papulosum cyprini (EPC) carp cells

The variables involved in the transfection of epithelioma papulosum cyprini (EPC) cells (a representative carp fish cell line) with the genes for -galactosidase from E. coli, for luciferases from firefly or renilla, for the G protein of viral haemorrhagic septicemia virus or for green fluorescent protein under the cytomegalovirus, the SV40 or the T7 polymerase promoters have been studied. Fugene was selected among 10 transfection different reagents because it is simpler to use and it induced maximum efficiences of transfection of 37% equivalent to 10–15 ng -galactosidase per 500000 EPC cells.     

Synthesis and antiparasitic activity of 1H-benzimidazole derivatives

Compounds 1-18 have been synthesized and tested in vitro against the protozoa Giardia lamblia, Entamoeba histolytica and the helminth Trichinella spiralis. Inhibition of rat brain tubulin polymerization was also measured and compared for each compound. Results indicate that most of the compounds tested were more active as antiprotozoal agents than Metronidazole and Albendazole. None of the compounds was as active as Albendazole against T. spiralis. Although only compounds 3, 9 and 15 (2-methoxycarbonylamino derivatives) inhibited tubulin polymerization, these were not the most potent antiparasitic compounds.     

Comparative Molecular Evolution of Primary (<Emphasis Type="BoldItalic">Buchnera</Emphasis>) and Secondary Symbionts of Aphids Based on Two Protein-Coding Genes

A+T content, phylogenetic relationships, codon usage, evolutionary rates, and ratio of synonymous versus non-synonymous substitutions have been studied in partial sequences of the atpD and aroQ/pheA genes of primary (Buchnera) and secondary symbionts of aphids and a set of selected non-symbiotic bacteria, belonging to the five subdivisions of the Proteobacteria. Compared to the homologous genes of the last group, both genes belonging to Buchnera behave in a similar way, showing a higher A+T content, forming a monophyletic group, a loss in codon bias, especially in third base position, an evolutionary acceleration and an increase in the number of non-synonymous substitutions, confirming previous results reported elsewhere for other genes. When available, these properties have been partly observed with the secondary symbionts, but with values that are intermediate between Buchnera and free living Proteobacteria. They show high A+T content, but not as high as Buchnera, a non-solved phylogenetic position between Buchnera, and the other γ-Proteobacteria, a loss in codon bias, again not as high as in Buchnera and a significant evolutionary acceleration in the case of the three atpD genes, but not when considering aroQ/pheA genes. These results give support to the hypothesis that they are symbionts at different stages of the symbiotic accommodation to the host.     

New pinch-porphyrin complexes with quantum mixed spin ground state S=3/2,5/2 of iron (III) and their catalytic activity as peroxidase

New complexes of the pinch-porphyrin family were obtained from the dimethylester of (proto-, meso-, and deutero-porphyrinato)iron(III) with the ligand [N,N'-bis-pyridin-2-ylmethyl-propane-1,3-diamine] 1-3 and with the ligand [N-pyridin-2-ylmethyl-N'-[3-[(pyridin-2-ylmethyl)-amino]-propyl]-propane-1,3-diamine] 4-6. The UV/VIS studies of 1-6 indicate an increase in the distortion of the ligand field excited state. The 1H NMR spectra of 1-6 at RT and over the range 223-328 K show iron(III)-complexes with quantum mixed spin state (qms) S=5/2, S=3/2. The chemical shifts of the meso protons are consistent with qms state S=3/2, S=5/2, where the S=3/2 spin state is lowest in energy. For methyl-heme the chemical shifts are also consistent with a qms state but now the S=5/2 ground state is lowest in energy. ESR spectra of 1-6 show two different species, B and C, of iron(III) with qms, S=5/2, S=3/2 consistent with the 1H NMR results. Species B with 70% of S=5/2 and species C with 72.5% of S=3/2. The catalytic activity as peroxidase of 1-6 was quantified by guaiacol test; their theoretical maximum rate constants were k(cat) approximately 10(2)-10(3) M(-1) s(-1). A quantitative empirical correlation is found: the higher the 32 spin contribution to the qms state and the higher proportion of this species into the samples, the higher the peroxidase activity. Such a correlation was also obtained for pinch-porphyrins already reported.     

Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking

The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast. Both proteins assemble into large (∼350 kDa) cytosolic protein complexes and we show that the yeast complex contains another class E Vps protein, Vps28p. tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady-state distribution of the mannose-6-phosphate receptor. Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells. We propose that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments. One consequence of this endosomal trafficking defect is the delayed down-regulation/degradation of activated cell surface receptors, resulting in prolonged signaling. This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts.     

Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro

Nicotine is a major component of cigarette smokeand has been postulated to play an important role in atherogenesis andmalignancy. Endothelial cell growth may be regulated by nicotine, yetoperative mechanisms at the endothelial level are poorly understood. We studied the effects of nicotine(10¹⁴-10⁴M) on endothelial DNA synthesis, DNA repair, proliferation, and cytotoxicity by using cultures of bovine pulmonary artery endothelial cells. Assays were performed on cells incubated with nicotine in thepresence and absence of hydroxyurea (an inhibitor of scheduled DNAsynthesis), serum, human platelet-poor plasma, and platelet-derived growth factor and endothelial cell growth factor (PDGF andPDECGF, respectively). Nicotine significantly stimulatedendothelial cell DNA synthesis and proliferation at concentrationslower than those obtained in blood after smoking(<10⁸ M). The stimulatoryeffects of nicotine were enhanced by serum (0.5%) and PDECGF and wereblocked by the nicotinic-receptor antagonist hexamethonium. Theresponse to nicotine was bimodal because cytotoxicity was observed athigher concentrations(>10⁶ M). This study hasimplications for understanding cellular mechanisms of nicotine action.The results may be important in tumor angiogenesis, atherogenesis, andvascular dysfunction in smokers.

     

Association of SNAREs and Calcium Channels with the Borders of Cytoskeletal Cages Organizes the Secretory Machinery in Chromaffin Cells

In chromaffin cells, SNARE proteins, forming the basic exocytotic machinery are present in membrane clusters of 500–600 nm in diameter. These microdomains containing both SNAP-25 and syntaxin-1 are dynamic and the expression of altered forms of SNAREs modifies not only their motion but also the mobility of the associated granules. It is also clear that SNARE microdomain location defines the place for individual vesicle fusion and that the alteration of cluster dynamics affects the fusion process itself. Interestingly, these SNARE patches colocalize with the borders of F-actin cages forming the cytoskeletal cortical network, and these borders also contain clusters of L- and P/Q type calcium channels. The organization of the secretory machinery in association with the borders of cytoskeletal cages seems to be an effective way to promote fast coupling between calcium entry and catecholamine release as demonstrated with the use of mathematical secretory models.