Identification of the linkage group of the Z sex chromosomes of the sand lizard (Lacerta agilis,Lacertidae) and elucidation of karyotype evolution in lacertid lizards

The sand lizard (Lacerta agilis, Lacertidae) has a chromosome number of 2n?=?38, with 17 pairs of acrocentric chromosomes, one pair of microchromosomes, a large acrocentric Z chromosome, and a micro-W chromosome. To investigate the process of karyotype evolution in L. agilis, we performed chromosome banding and fluorescent in situ hybridization for gene mapping and constructed a cytogenetic map with 86 functional genes. Chromosome banding revealed that the Z chromosome is the fifth largest chromosome. The cytogenetic map revealed homology of the L. agilis Z chromosome with chicken chromosomes 6 and 9. Comparison of the L. agilis cytogenetic map with those of four Toxicofera species with many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) showed highly conserved linkage homology of L. agilis chromosomes (LAG) 1, 2, 3, 4, 5(Z), 7, 8, 9, and 10 with macrochromosomes and/or macrochromosome segments of the four Toxicofera species. Most of the genes located on the microchromosomes of Toxicofera were localized to LAG6, small acrocentric chromosomes (LAG11–18), and a microchromosome (LAG19) in L. agilis. These results suggest that the L. agilis karyotype resulted from frequent fusions of microchromosomes, which occurred in the ancestral karyotype of Toxicofera and led to the disappearance of microchromosomes and the appearance of many small macrochromosomes.     

Effects of sulfur amino acids, <Emphasis Type="SmallCaps">l</Emphasis>-methionine, <Emphasis Type="SmallCaps">l</Emphasis>-cystine and <Emphasis Type="SmallCaps">l</Emphasis>-cysteine on lipoprotein lipase and hormone-sensitive lipase in differentiated mouse 3T3-L1 adipocytes

Rats subcutaneously implanted with AH109A hepatoma cells show hyperlipidemia with high concentrations of serum triglyceride and nonesterified fatty acid, suppression of lipoprotein lipase (LPL), and elevation of hormone-sensitive lipase (HSL) activities during the growth of the hepatoma. Supplementation of the diet with sulfur amino acids such as l-methionine (Met) and l-cystine (Cys) improved hyperlipidemia by restoring LPL and HSL activities. In the present study, we have attempted to examine the effects of sulfur amino acids on the activity and mRNA level of LPL and the activity of HSL using 3T3-L1 cells, which are known to differentiate to adipocytes. The adipocytes were incubated with various concentrations of Met, Cys or l-cysteine (CysH) in the absence or presence of tumor necrosis factor-α (TNF-α). LPL activity was suppressed by TNF-α. In the absence of TNF-α, Met, Cys and CysH did not change the LPL activity. In the presence of TNF-α, Met and Cys significantly increased the LPL activity, and Met also enhanced the LPL mRNA level. HSL activity was also suppressed by TNF-α. In the absence of TNF-α, Met enhanced the HSL activity. In the presence of TNF-α, Met, Cys and CysH suppressed the HSL activity. Sulfur amino acids such as Met, Cys and CysH affected the LPL activity, mRNA level, and HSL activity in 3T3-L1 adipocytes. Some of these effects of sulfur amino acids were different between LPL and HSL, between the absence and the presence of TNF-α, and between 3T3-L1 adipocytes and the adipose tissue from rats.     

Near-full-length REV3L appears to be a scarce maternal factor in Xenopus laevis eggs that changes qualitatively in early embryonic development

REV3 is the catalytic subunit of DNA polymerase ζ (pol ζ), which is responsible for the damage-induced mutagenesis that arises during error-prone translesion synthesis in eukaryotes. The related REV3L genes in human and mouse encode proteins of approximately 350 kDa, twice as large as yeast REV3, but full-length REV3L has not been identified in any vertebrate cell. We report that Xenopus laevis REV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340 kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65 pM is much lower than those of other replication and repair proteins including the accessory pol ζ subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a pattern of regulation during development. These observations indicate the existence of REV3L as a scarce protein, of approximately the full predicted size, whose level may impose severe constraints on the assembly of pol ζ in X. laevis.     

Fine structure of the vomeronasal organ in the grass lizard, Takydromus tachydromoides

The squamates are composed of many taxa, among which there is morphological variation in the vomeronasal organ (VNO). To elucidate the evolution of chemoreception in squamate reptiles, morphological data from the VNO from a variety of squamate species is required. In this study, the morphology of the VNO of the grass lizard Takydromus tachydromoides was examined using light and electron microscopy. The VNO consists of a pair of dome-shaped structures, which communicate with the oral cavity. There are no associated glandular structures. Microvilli are present on the apical surfaces of receptor cells in its sensory epithelium, as well as on supporting cells, and there are centrioles and ciliary precursor bodies on the dendrites. In addition to ciliated cells and basal cells in the non-sensory epithelium, there is a novel type of non-ciliated cell in T. tachydromoides. They have constricted apical cytoplasm and microvilli instead of cilia, and are sparsely distributed in the epithelium. Based on these results, the variation in the morphology of the VNO in scincomorpha, a representative squamate taxon, is discussed.     

Crystal structure of scytalidoglutamic peptidase with its first potent inhibitor provides insights into substrate specificity and catalysis

Scytalidoglutamic peptidase (SGP) from Scytalidium lignicolum is the founding member of the newly discovered\ family of peptidases, G1, so far found exclusively in fungi. The crystal structure of SGP revealed a previously undescribed fold for peptidases and a unique catalytic dyad of residues Gln53 and Glu136. Surprisingly, the beta-sandwich structure of SGP is strikingly similar to members of the carbohydrate-binding concanavalin A-like lectins/glucanases superfamily. By analogy with the active sites of aspartic peptidases, a mechanism employing nucleophillic attack by a water molecule activated by the general base functionality of Glu136 has been proposed. Here, we report the first crystal structures of SGP in complex with two transition state peptide analogs designed to mimic the tetrahedral intermediate of the proteolytic reaction. Of these two analogs, the one containing a central S-hydroxyl group is a potent sub-nanomolar inhibitor of SGP. The inhibitor binds non-covalently to the concave surface of the upper beta-sheet and enables delineation of the S4 to S3' substrate specificity pockets of the enzyme. Structural differences in these pockets account for the unique substrate preferences of SGP among peptidases having an acidic pH optimum. Inhibitor binding is accompanied by a structuring of the region comprising residues Tyr71-Gly80 from being mostly disordered in the apoenzyme and leading to positioning of crucial active site residues for establishing enzyme-inhibitor contacts. In addition, conformational rearrangements are seen in a disulfide bridged surface loop (Cys141-Cys148), which moves inwards, partially closing the open substrate binding cleft of the native enzyme. The non-hydrolysable scissile bond analog of the inhibitor is located in the active site forming close contacts with Gln53 and Glu136. The nucleophilic water molecule is displaced and a unique mode of binding is observed with the S-OH of the inhibitor occupying the oxyanion binding site of the proposed tetrahedral intermediate. Details of the enzyme-inhibitor interactions and mechanistic interpretations are discussed.     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.     

Characterization of Glial Populations in the Aging and Remyelinating Mouse Corpus Callosum

Cells in the white matter of the adult brain have a characteristic distribution pattern in which several cells are contiguously connected to each other, making a linear array (LA) resembling pearls-on-a-string parallel to the axon axis. We have been interested in how this pattern of cell distribution changes during aging and remyelination after demyelination. In the present study, with a multiplex staining method, semi-quantitative analysis of the localization of oligodendrocyte lineage cells (oligodendrocyte progenitors, premyelinating oligodendrocytes, and mature oligodendrocytes), astrocytes, and microglia in 8-week-old (young adult) and 32-week-old (aged) corpus callosum showed that young adult cells still include immature oligodendrocytes and that LAs contain a higher proportion of microglia than isolated cells. In aged mice, premyelinating oligodendrocytes were decreased, but microglia continued to be present in the LAs. These results suggest that the presence of microglia is important for the characteristic cell localization pattern of LAs. In a cuprizone-induced demyelination model, we observed re-formation of LAs after completion of cuprizone treatment, concurrent with remyelination. These re-formed LAs again contained more microglia than the isolated cells. This finding supports the hypothesis that microglia contribute to the formation and maintenance of LAs. In addition, regardless of the distribution of cells (LAs or isolated cells), astrocytes were found to be more abundant than in the normal corpus callosum at 24 weeks after cuprizone treatment when remyelination is completed. This suggests that astrocytes are involved in maintaining the functions of remyelinated white matter.