Natural variation in the appendicular skeleton of Triturus carnifex (Amphibia: Salamandridae)

Intraspecific variation in the appendicular skeleton of two geographically isolated populations of Triturus carnifex, one from northern Italy (Rosate, Milano) and one from central Italy (Bagnaia, Perugia), has been studied. A total of 1,746 forelimbs and 830 hindlimbs were examined. Forelimb skeletal variability was much greater in the Rosate than the Bagnaia population. Skeletal variants were present in 36.3% and 13.5% of the forelimbs, respectively, or in 54.7% and 22.7% of the netws (P < 0.0001). There were no predominant skeletal variants in Bagnaia, while in the Rosate population, the majority of the variants consisted of fusion of radiale and prepollicis and of phalangeal formula 1-2-3-2. Hindlimb skeletal variability was similar in the two populations and appeared to be much lower than that of the forelimb, with highly significant differences in the frequency of basipodium variants within the Rosate population and in the frequency of acropodium variants in both populations. Skeletal variants were present in about 9% of the hindlimbs, or in about 12% of the newts from either population. At present, no conclusion can be drawn about the mechanisms, genetic and/or epigenetic, underlying the skeletal variability observed in the Triturus carnifex from northern and central Italy. © 1996 Wiley-Liss, Inc.     

Susceptibility to Heavy Metals and Cadmium Accumulation in Aerobic and Anaerobic Thermophilic Microorganisms Isolated from Deep-Sea Hydrothermal Vents

Thirty thermophilic strains isolated from heavy metal-rich hydrothermal vent sites at Lau Basin were tested for their susceptibility to cadmium, zinc, cobalt, and nickel. The 14 aerobic spore formers belonging to the genus Bacillus, 6 anaerobic fermenters from the order Thermotogales, and 10 anaerobic sulfur reducers from the order Thermococcales could be clearly distinguished according to their metal susceptibilities. The Thermococcales were found to exhibit the highest resistance to cadmium and zinc, whereas Thermotogales were highly sensitive to these metals. In contrast, the Thermotogales displayed the highest resistance to cobalt ions. No clear distinction could be established between the metal susceptibilities of these strains and seven reference organisms used for comparative studies. Cadmium resistance, slightly inducible in some cadmium-resistant bacilli, was not plasmid mediated. The amount of cadmium immobilized by the Thermotogales was related to their level of resistance to this metal. Received: 24 February 2000 / Accepted: 28 March 2000     

Genetic analysis of isometric growth control mechanisms in the zebrafish caudal Fin

The body and fins of the zebrafish grow rapidly as juveniles and slower as they reach maturation. Throughout their lives, the fins grow isometrically with respect to the body. Growth of individual fin rays is achieved by the distal addition of bony segments. We have investigated the genetic control of mechanisms that initiate new segments or control size of newly initiated segments. We find that both segment initiation and segment length are regulated during fin growth in wild-type fish. We examined the growth properties of lof and sof fin length mutants for effects on the number and length of fin ray segments. Fins of lof mutants continue to grow rapidly even after wild-type fin growth slows, resulting in positive allometric growth and additional fin ray segments. We suggest that lof mutants bypass mechanisms that limit segment initiation. Isometric growth is retained in sof mutants, resulting in shorter fins one-half the length of wild-type fins. The primary defect in sof mutants is that fin ray segments are shorter than wild-type segments, although segment number is also diminished. Double mutants for sof;lof reveal that segment length and segment number are controlled in different pathways. Our findings suggest that the lof gene product regulates segment initiation and the sof gene product regulates segment length.     

Serum and glucocorticoid-regulated kinase Sgk1 inhibits insulin-dependent activation of phosphomannomutase 2 in transfected COS-7 cells

Serum- and glucocorticoid-regulated kinase (Sgk1) is considered to be an essential convergence point for peptide and steroid regulation of ENaC-mediated sodium transport. We tried to identify molecular partners of Sgk1 by yeast two-hybrid screening. Yeast two-hybrid screening showed a specific interaction between Sgk1 and phosphomannomutase (PMM)2, the latter of which is an enzyme involved in the regulation of glycoprotein biosynthesis. The interaction was confirmed in intact cells by coimmunoprecipitation and colocalization detected using confocal microscopy. We were then able to demonstrate that Sgk1 phosphorylated PMM2 in an in vitro assay. In addition, we found that the enzymatic activity of PMM2 is upregulated by insulin treatment and that Sgk1 completely inhibits PMM2 activity both in the absence and in the presence of insulin stimulation. These data provide evidence suggesting that Sgk1 may modulate insulin action on the cotranslational glycosylation of glycoproteins. Sgk; protein glycosylation; CDGIa     

Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.     

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse

The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as alpha-helices and beta-sheets. However, the infrared spectra of the N. coriiceps metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to alpha-helices whose content resulted in 5% of the total secondary structure of the protein. The short alpha-helix found in N. coriiceps metallothionein showed an onset of denaturation at 30 degrees C and a T(m) at 48 degrees C. The data suggest that in N. coriiceps metallothionein a particular cysteine is involved in the alpha-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps metallothionein possesses a more flexible structure than mouse metallothionein. The spectroscopic data are discussed in terms of the biological function of the metallothioneins.     

Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish)

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT–PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.