Studies in vitro on the effects of 1H,2H,4H(5H)-octafluorocyclohexane and 1H,4H(2H)-nonafluorocyclohexane on enzymes and organelles

A comparison has been made of the effect of 1H,2H,4H(5H)-octafluorocyclohexane, which is highly toxic (LD(50) 17mg./kg. in rats), and of 1H,4H(2H)-nonafluorocyclohexane, which is relatively non-toxic (LD(50)>440mg./kg. in rats), on the respiration of rat liver homogenates and mitochondria in vitro. 1H,2H,4H(5H)-Octafluorocyclohexane strongly inhibited the respiration of both homogenates and mitochondria, but neither compound had any significant effect on glycolysis or on glutamate dehydrogenase or NADH-cytochrome c reductase activity. 1H,2H,4H(5H)-Octafluorocyclohexane, however, caused a very marked inhibition of cytochrome oxidase activity, causing an almost complete lesion in this region of the respiratory chain. 1H,4H(2H)-Nonafluorocyclohexane was without effect in this respect. A marked decrease in turbidity of mitochondrial suspensions at 520nm. was caused by addition of both compounds, the effect being greater with 1H,2H,4H(5H)-octafluorocyclohexane. ATP, Mg(2+) and bovine serum albumin did not reverse these changes. Mitochondrial adenosine triphosphatase activity was increased twofold by the toxic compound, but only slightly by the non-toxic compound. Electron-microscopic examination of mitochondria treated with 1H,2H,4H(5H)-octafluorocyclohexane revealed gross morphological damage, whereas the effect of 1H,4H(2H)-nonafluorocyclohexane appeared to be merely to cause swelling. The results obtained account, to some extent at any rate, for the toxic effects of 1H,2H,4H(5H)-octafluorocyclohexane.     

Immunoglobulin (IgG) allotypes of the American mink with Aleutian disease

Mink Aleutian disease (AD) is characterized by intensive proliferation of B-lymphocytes and hypergammaglobulinemia. Populational distribution of five genetic immunoglobulin markers (light chain allotype L1 and C gamma-allotypes H2, H3, H6 and H8) in minks of different coat color (Sapphire, Standard and Topaz) was studied. The groups of infected minks differed significantly from healthy ones in the distribution of the H3 allotype: the frequencies of some phenotypes--H3, H6, H8 and L1, H3, H6, H8 (Sapphire, Standard). H2, H3, H6, H8 and L1, H2, H3, H6, H8 (Sapphire) were increased significantly. At the same time, the frequencies of H6, H8; L1, H6, H8 and H2, H6, H8; L1, H2, H6, H8 were decreased in the AD population. The preferential stimulation of proliferation of the H3 + B-lymphocyte clones is suggested.     

The carboxyl-terminal domain of murine H1(0). Immunochemical and partial amino acid sequence comparisons with other H1(0)/H1/H5 histones

The carboxyl-terminal domain of murine H1(0) histone was compared with that of human H1(0), bovine H1(0) and other H1 and H5 histones. Two sets of antibodies were induced by murine H1(0). One set reacted with only the carboxyl-terminal domain of murine H1(0) and preferred the murine over the bovine and human proteins. The second set of antibodies reacted with the globular domain of murine H1(0) and did not distinguish among murine, bovine and human H1(0) species. There were five positions in the first 60 residues of the carboxyl-terminal domain in which the murine H1(0) differed from the human H1(0). In this region, the murine H1(0) had no more than 49% overall homology with other H1 and H5 histones; however, short sequences in the domain were very similar to short sequences that occur in rabbit H1.3, trout H1 and goose or chicken H5. In comparisons based on these and other published data, the carboxyl-terminal domain of H1(0) is found to be more variable among species than is the globular domain; the first two-thirds of the H1(0) carboxyl-terminal domain is largely unique and does not show great overall homology with H1 or H5, whereas the last third is again more conserved. As the first two-thirds of the domain is the only portion where the homology with H5 is less than 50%, it may be responsible for functional differences between H1(0) and H5.     

Individual Somatic H1 Subtypes Are Dispensable for Mouse Development Even in Mice Lacking the H10 Replacement Subtype

H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H1(0), the most divergent subtype, develop normally. Examination of chromatin in H1(0-/-) mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H1(0) to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H1(0) (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434-6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H1(0-/-) mice, each of the three H1 knockout mouse lines was bred with H1(0) knockout mice to generate H1c/H1(0), H1d/H1(0), or H1e/H1(0) double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.     

Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire

The antigen-binding site of the camel heavy-chain antibodies devoid of light chain consists of a single variable domain (V(H)H) that obviously lacks the V(H)-V(L) combinatorial diversity. To evaluate the extent of the V(H)H antigen-binding repertoire, a germline database was constructed from PCR-amplified V(H)H/V(H) segments of a single specimen of Camelus dromedarius. A total of 33 V(H)H and 39 V()H unique sequences were identified, encoded by 42 and 50 different genes, respectively. Sequence comparison indicates that the V(H)Hs evolved within the V(H) subgroup III. Nevertheless, the V(H)H germline segments are highly diverse, leading to a broad structural repertoire of the antigen-binding loops. Seven V(H)H subfamilies were recognized, of which five were confirmed to be expressed in vivo. Comparison of germline and cDNA sequences demonstrates that the rearranged V(H)Hs are extensively diversified by somatic mutation processes, leading to an additional hypervariable region and a high incidence of nucleotide insertions or deletions. These diversification processes are driven by hypermutation and recombination hotspots embedded in the V(H)H germline genes at the regions affecting the structure of the antigen-binding loops.     

Study of the spatial structure of the duplex d(pTGTTTGGC) d(pCCAAAC)A in aqueous solution by methods of uni- and two-dimensional (1)H-NMR spectroscopy and organic molecular mechanics

Structure of the complementary complex d(pTGTTTGGC) d(pCCAAAC)A in the aqueous solution has been investigated by one- and two-dimensional 1H NMR spectroscopy. The resonances of nonexchangeable protons of bases as well as methyl and deoxyribose 1', 2'a, 2'b, 3' and 4' protons have been assigned by means of two-dimensional J-correlated spectroscopy (COSY) and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY). Using one-dimensional NOE measurements, 62 interproton distances (intranucleotide: (H6/H8)i--(H1')t, (H6/H8)i--(H2'a)i, (H1')i--(H2'a)i, (H1')i--(H2'b)i; internucleotide: (H6/H8)i--(H1')i-1, (H6/H8)i--(H2'a)i-1, (H6/H8)i--(H2'b)i-1, (H5/CH3)i--(H6/H8)i-1, (H5/CH3)i--(H2'a/H2'b)i-1) have been determined for nearest-neighbour protons. Spin-coupling constant values for some sugar protons have been obtained from COSY spectra. The restrained molecular mechanics calculations have yielded the possible solution structures of duplex fitting the experimental set of interproton distances and coupling constants.     

Separation of rat tissue histone H1 subtypes by reverse-phase h.p.l.c. Identification and assignment to a standard H1 nomenclature.

H1 histones from rat liver and rat testis were separated by reverse-phase h.p.l.c. Within 40 min six subfractions (H1(0), H1b, H1a, H1d, H1e + H1c and H1c) and seven subfractions (H1(0), H1b, H1a, H1d, H1e + H1c, H1c and H1t) respectively were isolated by using a linear acetonitrile gradient. Each individual H1 subtype was identified either by comparing the H1 variants (contained in both tissues but in different quantities) or by SDS/PAGE and acetic acid/urea/PAGE. Moreover, all H1 variants were characterized by amino acid analyses. The amino acid compositions of rat histone subfractions H1(0), H1b and H1e were determined for the first time. It was possible to classify unambiguously the H1 subfractions obtained by h.p.l.c. by following the standardized H1 nomenclature for electrophoretic systems recommended by Lennox, Oshima & Cohen [(1982) J. Biol. Chem. 257, 5183-5189]. Incorrect assignments that have been made in various publications are discussed.     

Mechanism of arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae

Arsenate resistance is exhibited by the ericoid mycorrhizal fungus Hymenoscyphus ericae collected from As-contaminated mine soils. To investigate the mechanism of arsenate resistance, uptake kinetics for arsenate (H(2)AsO(4)(-)), arsenite (H(3)AsO(3)), and phosphate (H(2)PO(4)(-)) were determined in both arsenate-resistant and -non-resistant H. ericae. The uptake kinetics of H(2)AsO(4)(-), H(3)AsO(3), and H(2)PO(4)(-) in both resistant and non-resistant isolates were similar. The presence of 5.0 microM H(2)PO(4)(-) repressed uptake of H(2)AsO(4)(-) and exposure to 0.75 mM H(2)AsO(4)(-) repressed H(2)PO(4)(-) uptake in both H. ericae. Mine site H. ericae demonstrated an enhanced As efflux mechanism in comparison with non-resistant H. ericae and lost approximately 90% of preloaded cellular As (1-h uptake of 0.22 micromol g(-1) dry weight h(-1) H(2)AsO(4)(-)) over a 5-h period in comparison with non-resistant H. ericae, which lost 40% of their total absorbed H(2)AsO(4)(-). As lost from the fungal tissue was in the form of H(3)AsO(3). The results of the present study demonstrate an enhanced H(3)AsO(3) efflux system operating in mine site H. ericae as a mechanism for H(2)AsO(4)(-) resistance. The ecological significance of this mechanism of arsenate resistance is discussed.     

A study of histone-histone interactions by affinity chromatography

Homologous whole histone from calf thymus was adsorbed on Sepharose 4B columns with covalently coupled histone fractions H2a, H2b, H3 or H4 in 0.01 M phosphate buffer, pH 6.7–1 M NaCl. The adsorbed histones were eluted from the columns with 5 M urea in the same buffer. Electrophoretic analysis has shown that the different columns exhibit selective affinity to the histone fractions: the H2b column to histone H2b and H2a (with only weak affinity to histones H3 and H4), the H2a column to histones H2b and H3 (moderate affinity to histones H2a and H4), the H3 column to histones H3, H4, H2a (moderate affinity to histone H2b), and the H4 column to histone H3, H4 and H2b (weak affinity to histone H2a). Histone H1 displayed no fixation by either of the columns tested.     

Proton mobilities in water and in different stereoisomers of covalently linked gramicidin A channels

Proton conductivities in bulk solution (lambda(H)) and single-channel proton conductances (g(H)) in two different stereoisomers of the dioxolane-linked gramicidin A channel (the SS and RR dimers) were measured in a wide range of bulk proton concentrations ([H], 0.1-8000 mM). Proton mobilities (micro(H)) in water as well as in the SS and RR dimers were calculated from the conductivity data. In the concentration range of 0.1-2000 mM, a straight line with a slope of 0.75 describes the log (g(H))-log ([H]) relationship in the SS dimer. At [H] > 2000 mM, saturation is followed by a decline in g(H). The g(H)-[H] relationship in the SS dimer is qualitatively similar to the [H] dependence of lambda(H). However, the slope of the straight line in the log(lambda(H))-log([H]) plot is 0.96, indicating that the rate-limiting step for proton conduction through the SS dimer is not the diffusion of protons in bulk solution. The significant difference between the slopes of those linear relationships accounts for the faster decline of micro(H) as a function of [H] in the SS dimer in relation to bulk solution. In the high range of [H], saturation and decline of g(H) in the SS dimer can be accounted for by the significant decrease of micro(H) in bulk solution. At any given [H], g(H) in the RR dimer is significantly smaller than in the SS. Moreover, the g(H)-[H] relationship in the RR stereoisomer is qualitatively different from that in the SS. Between 1 and 50 mM [H], g(H) can be fitted with an adsorption isotherm, suggesting the presence of a proton-binding site inside the pore (pK(a) approximately 2), which limits proton exit from the channel. At 100 mM < [H] < 3000 mM, g(H) increases linearly with [H]. The distinctive shape of the g(H)-[H] relationship in the RR dimer suggests that the channel can be occupied simultaneously by more than one proton. At higher [H], the saturation and decline of g(H) in the RR dimer reflect the properties of micro(H) in bulk solution. In the entire range of [H], protons seem to cross the SS and RR channels via a Grotthuss-like mechanism. The rate-limiting step for proton transfer in the SS dimer is probably the membrane-channel/bulk solution interface. It is also proposed that the smaller g(H) in the RR dimer is the consequence of a different organization and dynamics of the H-bonded network of water molecules inside the pore of the channel, resulting in a slower proton transfer and multiple pore occupancy by protons.     

Association of nucleosome core particle DNA with different histone oligomers. Transfer of histones between DNA-(H2A,H2B) and DNA-(H3,H4) complexes

In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 M). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 M-NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.     

Comparative analysis of flagellin sequences from Escherichia coli strains possessing serologically distinct flagellar filaments with a shared complex surface pattern.

Escherichia coli morphotype E flagellar filaments have a characteristic surface pattern of short-pitch loops when examined by electron microscopy. Seven of the 50 known E. coli H (flagellar antigen) serotypes (H1, H7, H12, H23, H45, H49, and H51) produce morphotype E filaments. Polymerase chain reaction was used to amplify flagellin structural (fliC) genes from E. coli strains producing morphotype E flagellar filaments and from strains with flagellar filaments representing other morphotypes. A single DNA fragment was obtained from each strain, and the size of the amplified DNA correlated with the molecular mass of the corresponding flagellin protein. This finding and hybridization data suggest that these bacteria are monophasic. fliC genes from three E. coli serotypes (H1, H7, and H12) possessing morphotype E flagellar filaments were sequenced in order to assess the contribution of conserved flagellin primary sequence to the characteristic filament architecture. The H1 and H12 fliC sequences were identical in length (1,788 bp), while the H7 fliC sequence was shorter (1,755 bp). The deduced molecular masses of the FliC proteins were 60,857 Da (H1), 59,722 Da (H7), and 60,978 Da (H12). The H1, H7, and H12 flagellins demonstrated 98 to 99% identity over the amino-terminal region (190 amino acid residues) and 89% (H7) to 99% (H1 and H12) identity in the carboxy-terminal region (100 amino acid residues). The complete primary amino acid sequences for H1 and H12 flagellins differed by only 10 amino acids, accounting for previously reported serological cross-reactions. However, the central region of H7 flagellin had only 38% identity with H1 and H12 flagellins.The characteristic morphology of morphotype E flagellar filaments is therefore not dependent on a highly conserved primary sequence within the exposed central region. Comparison of morphotype E E. coli flagellins with those from E. coli K-12, Serratia marcescens, and several Salmonella serovars supported the established concept of highly conserved terminal regions flanking a variable central region.     

Study on genetic variations of PPARα gene and its effects on thermal tolerance in Chinese Holstein

Peroxisome proliferator-activated receptor alpha (PPARα) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. In this research, polymerase chain reaction (PCR) technique was used to amplify 766 and 589 bp fragments of intron 3 and 7 of PPARα gene in Chinese Holstein (n = 771). Sequencing results showed that three novel single nucleotide polymorphisms (SNPs) were identified at position 44087 (G/A), 65550 (G/A), and 65676(G/A) in the PPARα gene. PCR–restriction fragment length polymorphism technology was used to genotype the three SNPs. Association analysis showed that cows with H1H8 (P < 0.05), H2H8 (P < 0.01), H5H7 (P < 0.05), H5H8 (P < 0.05), and H8H8 (P < 0.05) haplotype combinations had lower potassium content in erythrocytes than those with H2H6 haplotype combination. Cows with H1H8, and H8H8 haplotype combinations had lower decrease rate of milk yield than those with H2H6 and H6H8 haplotype combinations (P < 0.05). Cows with H2H8 and H8H8 haplotype combinations had lower rectal temperature than those with H5H8 and H7H7 haplotype combinations (P < 0.05). In conclusion, H8H8 haplotype combination may be advantageous for heat resistance traits in Chinese Holstein cattle.     

黄河内蒙古段表层沉积物细菌多样性及群落结构类型

为掌握黄河内蒙古段表层沉积物微生物多样性、群落结构类型及其影响因素,采用高通量测序技术分析了6个采样点表层沉积物中的微生物多样性及群落结构。研究结果表明,黄河内蒙古段沉积物细菌丰度大小排序为乌拉特前旗(H3)老牛湾(H6)临河(H2)包头(H4)托县(H5)乌海(H1),微生物多样性排序为H6 H2 H5 H4 H3 H1,乌海沉积物中细菌丰度和微生物多样性都是最低的一个采样点。黄河内蒙古段表层沉积物中三大优势菌群分别为变形菌门(Proteobacteria,相对丰度32.39%)、绿弯菌门(Chloroflexi,13.25%)和拟杆菌门(Bacteroidetes,12.16%)。细菌群落丰度与环境因子之间的冗余分析结果显示,沉积物中总有机碳(TOC)、离子交换容量(CEC)和总磷(TP)、总氮(TN)等环境因子对黄河内蒙古段沉积物细菌群落分布影响较大,负相关系数分别为82.5%、80.1%、85.5%和85.2%;微生物多样性与环境理化因子相关性分析结果表明,黄河沉积物微生物多样性格局与对氮磷等营养物质的损耗有直接关系。     

Identification of EPEC and non-EPEC serotypes in the EPEC O serogroups by PCR-RFLP analysis of the fliC gene

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC) was performed in 233 strains of enteropathogenic Escherichia coli (EPEC) O serogroups for determining their flagellar antigen (H) status. The serological detection of flagellin is the basis for the H-codes typing system in E. coli. Thus, it is impossible to serotype nonmotile bacteria (i.e. to assign H-codes). Twenty-eight fliC restriction patterns were obtained for motile (H2, H4, H6, H7, H8, H9, H10, H11, H12, H18, H21, H27, H32, H34, H35, H40 and H51) and nonmotile serotypes (H(-)). Each motile serotype was characterized by one or two fliC specific restriction patterns. The only exception was serogroup O128ab, where a common restriction pattern was found for serotypes O128ab:H2 and O128ab:H35, even after digestion with RsaI, AluI and Sau3AI endonucleases. These two serotypes were, however, discriminated by single strand conformation polymorphism (SSCP) analysis of RsaI restriction fragments. Nonmotile strains showed fliC restriction patterns identical to some known H serotypes. The PCR-RFLP analysis of fliC gene proved to be a useful method for identifying the H variants in motile and nonmotile EPEC O serogroups.     

Immunohistochemical distribution of the histone H1(0)/H5 variant in various tissues of adult Xenopus laevis

The cellular distribution of the histone H1(0)/H5 variant has been examined immunohistochemically in various tissues of adult Xenopus laevis, using monoclonal antibodies against this variant that was isolated from erythrocyte nuclei. The H1(0)/H5 variant appears not to be erythrocyte-specific and appears to be present in all cell types of liver, stomach, and skin. In contrast, in oocyte nuclei the H1(0)/H5 variant cannot be detected, whereas they do contain H1; the nuclei of spermatogenic cells contain the H1(0)/H5 variant, but probably less than the somatic cells. In Xenopus no H1(0) variant distinct from H5 seems to occur and the H1(0)/H5 variant apparently may perform a functional role related to mammalian H1(0).     

A phylogenetic review of the genus Hexabathynella Schminke, 1972 (Crustacea, Malacostraca, Bathynellacea): with a description of four new species

The genus Hexabathynella (Crustacea, Malacostraca, Bathynellacea) is revised in the sense of the phylogenetic systematics and four new species are described from South Africa ( H. monoaethetasca sp. nov. and H. africana sp. nov. ) and America ( H. schrieveri sp. nov. and H. virginiae sp. nov. ). A comparative analysis of all observable outer structures distributed in 18 known and four new species resulted in a re-evaluation of 18 characters and character states. The phylogenetic analysis using the program PAUP yielded one most-parsimonious tree, which suggests the grouping of ( H. decora (( H. halophila  +  H. aotearoae ) + ( H. pauliani ( H. monoaethetasca  + H. africana )))) + ( H. knoepffleri ( H. nicoleiana ( H. hebrica , H. tenera , H. longiappendiculata , H. breviappendiculata , H. nestica ) +  H. virginiae (( H. minuta ( H. valdecasasi + H. otayana )) + ( H. hessleri + H. muliebris ))) +  H. paranaensis ( H. szidati + H. schrieveri )). The tree is 57 steps long and has a consistency index of 0.6140, a retention index of 0.7982 and a rescaled consistency index of 0.4901. The result does not agree with the previous analyses on the genus. In terms of sampling and coding, the characters used in the previous study are critically assessed.  © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 147 , 71–96.     

The hydrogenase cytochrome b heme ligands of Azotobacter vinelandii are required for full H(2) oxidation capability

The hydrogenase in Azotobacter vinelandii, like other membrane-bound [NiFe] hydrogenases, consists of a catalytic heterodimer and an integral membrane cytochrome b. The histidines ligating the hemes in this cytochrome b were identified by H(2) oxidation properties of altered proteins produced by site-directed mutagenesis. Four fully conserved and four partially conserved histidines in HoxZ were substituted with alanine or tyrosine. The roles of these histidines in HoxZ heme binding and hydrogenase were characterized by O(2)-dependent H(2) oxidation and H(2)-dependent methylene blue reduction in vivo. Mutants H33A/Y (H33 replaced by A or Y), H74A/Y, H194A, H208A/Y, and H194,208A lost O(2)-dependent H(2) oxidation activity, H194Y and H136A had partial activity, and H97Y,H98A and H191A had full activity. These results suggest that the fully conserved histidines 33, 74, 194, and 208 are ligands to the hemes, tyrosine can serve as an alternate ligand in position 194, and H136 plays a role in H(2) oxidation. In mutant H194A/Y, imidazole (Imd) rescued H(2) oxidation activity in intact cells, which suggests that Imd acts as an exogenous ligand. The heterodimer activity, quantitatively determined as H(2)-dependent methylene blue reduction, indicated that the heterodimers of all mutants were catalytically active. H33A/Y had wild-type levels of methylene blue reduction, but the other HoxZ ligand mutants had significantly less than wild-type levels. Imd reconstituted full methylene blue reduction activity in mutants H194A/Y and H208A/Y and partial activity in H194,208A. These results indicate that structural and functional integrity of HoxZ is required for physiologically relevant H(2) oxidation, and structural integrity of HoxZ is necessary for full heterodimer-catalyzed H(2) oxidation.     

Systematics, biogeography, and evolution of Hemidactylus geckos (Reptilia: Gekkonidae) elucidated using mitochondrial DNA sequences

With more than 80 species inhabiting all warm continental land masses and hundreds of intervening continental and oceanic islands, Hemidactylus geckos are one of the most species-rich and widely distributed of all reptile genera. They consequently represent an excellent model for biogeographic, ecological, and evolutionary studies. A molecular phylogeny for Hemidactylus is presented here, based on 702 bp of mtDNA (303 bp cytochrome b and 399 bp 12S rRNA) from 166 individuals of 30 species of Hemidactylus plus Briba brasiliana, Cosymbotus platyurus, and several outgroups. The phylogeny indicates that Hemidactylus may have initially undergone rapid radiation, and long-distance dispersal is more extensive than in any other reptilian genus. In the last 15 My, African lineages have naturally crossed the Atlantic Ocean at least twice. They also colonized the Gulf of Guinea, Cape Verde and Socotra islands, again sometimes on more than one occasion. Many extensive range extensions have occurred much more recently, sometimes with devastating consequences for other geckos. These colonizations are likely to be largely anthropogenic, involving the 'weedy' commensal species, H. brookii s. lat, H. mabouia, H. turcicus, H. garnotii, and H. frenatus. These species collectively have colonized the Mediterranean region, tropical Africa, much of the Americas and hundreds of islands in the Pacific, Indian, and Atlantic oceans. Five well-supported clades are discernable in Hemidactylus, with the African H. fasciatus unallocated. 1. Tropical Asian clade: (Cosymbotus platyurus (H. bowringii, H. karenorum, H. garnotii)) (H. flaviviridis (Asian H. brookii, H. frenatus)). 2. African H. angulatus and Caribbean H. haitianus. 3. Arid clade, of NE Africa, SW Asia, etc.: (H. modestus (H. citernii, H. foudai)) (H. pumilio (H. granti, H. dracaenacolus) (H. persicus, H. macropholis, H. robustus, H. turcicus (H. oxyrhinus (H. homoeolepis, H. forbesii))). 4. H. mabouia clade (H. yerburii, H. mabouia). 5. African-Atlantic clade: H. platycephalus ((H. agrius, H. palaichthus) (H. longicephalus, H. greeffi, H. bouvieri, Briba brasiliana))). Cosymbotus and Briba are synonymized with Hemidactylus, and African populations of H. brookii separated as H. angulatus, with which H. haitianus may be conspecific. Some comparatively well-sampled widespread species show high genetic variability (10-15% divergence) and need revision, including Cosymbotus platyurus, H. bowringii, Asian H. brookii, H. frenatus, H. angulatus, and H. macropholis. In contrast, most populations of H. mabouia and H. turcicus are very uniform (1-2% divergence). Plasticity of some of the morphological features of Hemidactylus is confirmed, although retention of primitive morphologies also occurs.     

DNA stable pentaploid H1 (ES) cells obtained from an octaploid cell induced from tetraploid cells polyploidized using demecolcine

Pentaploid H1 (ES) cells (5H1 cells) were accidentally obtained through one‐cell cloning of octaploid H1 (ES) cells (8H1 cells) that were established from tetraploid H1 (ES) cells (4H1 cells) polyploidized using demecolcine. The number of chromosomes of 5H1 cells was 100, unlike the 40 of diploid H1 (ES) cells (2H1 cells), 80 of 4H1, and 160 of 8H1 cells. The durations of G₁, S, and G₂/M phases of 5H1 cells were 3, 7, and 6 h, respectively, almost the same as those of 2H1, 4H1, and 8H1 cells. The cell volume of 5H1 cells was half of that of 8H1 cells, suggesting that 5H1 cells were created through abnormal cell divisions of 8H1 cells. The morphology of growing 5H1 cells was a spherical cluster similar to that of 2H1 cells and differing from the flagstone‐like shape of 4H1 and 8H1 cells. Pentaploid solid tumors were formed from 5H1 cells after interperitoneal injection into the mouse abdomen, and they contained endodermal, mesodermal, and ectodermal cells as well as undifferentiated cells, suggesting both that the DNA content of 5H1 cells was retained during tumor formation and that the 5H1 cells were pluripotent. The DNA content of 5H1 cells was stable in long‐term culturing as 2H1 cells, meaning that 5H1 and 2H1 cells shared similarities in DNA structure. The excellent stability of the DNA content of 5H1 cells was explained using a hypothesis for the DNA structure of polyploid cells because the pairing of homologous chromosomes in 5H1 cells is spatially forbidden. J. Cell. Physiol. 223: 369–375, 2010. © 2010 Wiley‐Liss, Inc.