Elucidation of Structure of Lipid A from the Marine Gram-Negative Bacterium Pseudoalteromonas haloplanktis ATCC 14393T

The chemical structure of lipid A, from the marine -proteobacterium Pseudoalteromonas haloplanktis 14393, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be -1,6-glucosaminobiose 1,4-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3 and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2, respectively.     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.     

New geographical distribution and host records of rust fungi from northern Thailand

Ten species of rust fungi (Crossopsora 2, Maravalia 1, Pileolaria 1, Puccinia 1, Ravenelia 1, Sphaerophragmium 1, Uredo 2, and Uromyces 1) are newly recorded together with six new host plants in Thailand.Contribution no. 194, Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

Y-chromosome Data and Tribal Affiliations of Allenopithecus and Miopithecus

The phylogeny of Old World monkeys has remained unresolved in part because of a lack of resolution in the Cercopithecinae. Competing morphological hypotheses have had Allen's swamp monkey (Allenopithecus nigroviridis) and the talapoins (Miopithecus spp.) as basal branches of either the tribe Cercopithecini or the tribe Papionini. Previous molecular analyses have not adequately addressed the issue. To better understand the evolutionary history of these primates, we sequenced and subjected to phylogenetic analysis 3.1 kb of 2 loci (TSPY and SRY) from the non-recombining portion of the Y-chromosome. Individuals from the genera Allenopithecus, Miopithecus, Erythrocebus, Chlorocebus, and Cercopithecus were surveyed and their sequences compared with those previously published for the Papionini and Colobinae. The results suggest Allenopithecus and Miopithecus are more closely related to the Cercopithecini than Papionini. Our data also support the hypothesis that within the Cercopithecini, Erythrocebus and Chlorocebus share a close evolutionary relationship, distinct from the other members of the tribe.     

Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L)

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.     

Validation of the <Emphasis Type="Italic">VRN-H2</Emphasis>/<Emphasis Type="Italic">VRN-H1</Emphasis> epistatic model in barley reveals that intron length variation in <Emphasis Type="Italic">VRN-H1</Emphasis> may account for a continuum of vernalization sensitivity

The epistatic interaction of alleles at the VRN-H1 and VRN-H2 loci determines vernalization sensitivity in barley. To validate the current molecular model for the two-locus epistasis, we crossed homozygous vernalization-insensitive plants harboring a predicted “winter type” allele at either VRN-H1 (Dicktoo) or VRN-H2 (Oregon Wolfe Barley Dominant), or at both VRN-H (Calicuchima-sib) loci and measured the flowering time of unvernalized F₂ progeny under long-day photoperiod. We assessed whether the spring growth habit of Calicuchima-sib is an exception to the two-locus epistatic model or contains novel “spring” alleles at VRN-H1 (HvBM5A) and/or VRN-H2 (ZCCT-H) by determining allele sequence variants at these loci and their effects relative to growth habit. We found that (a) progeny with predicted “winter type” alleles at both VRN-H1 and VRN-H2 alleles exhibited an extremely delayed flowering (i.e. vernalization-sensitive) phenotype in two out of the three F₂ populations, (b) sequence flanking the vernalization critical region of HvBM5A intron 1 likely influences degree of vernalization sensitivity, (c) a winter habit is retained when ZCCT-Ha has been deleted, and (d) the ZCCT-H genes have higher levels of allelic polymorphism than other winterhardiness regulatory genes. Our results validate the model explaining the epistatic interaction of VRN-H2 and VRN-H1 under long-day conditions, demonstrate recovery of vernalization-sensitive progeny from crosses of vernalization-insensitive genotypes, show that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity, and provide molecular markers that are accurate predictors of “winter vs spring type” alleles at the VRN-H loci.     

A locus controlling the activity of UDP-galactose:GM2(NeuGc) galactosyltransferase in mouse liver is linked to the H-2 complex

Genetic polymorphism in the expression of the G_M1(NeuGc) ganglioside has been shown in the liver of inbred strains of mice. Through analysis of the gangliosides of H-2 congenic and recombinant strains, this polymorphism was demonstrated to be controlled by a locus mapped left outside of the H-2 complex on chromosome 17, and the locus was assumed to control the level of the activity of G_M1(NeuGc) synthetase, UDP-galactose:G_M2(NeuGc) galactosyltransferase (E.C.2.4.1.62) [Hashimotoet al., J Biochem (1983) 94:2049-54].In the present study we analyzed the genetic linkage between the activity of the galactosyltransferase and the H-2 haplotype. For this purpose, we selected two inbred strains of mice, WHT/Ht and BALB/c, because they have different levels of the transferase activity and show different H-2 haplotypes; the specific activity of the transferase obtained with BALB/c was one-eighth of that with WHT/Ht, and BALB/c expressed the la.7 antigen as one of the products encoded in their H-2^d complex, whereas WHT/Ht did not. To analyze the linkage between these two phenotypes, WHT/Ht were mated with BALB/c to obtain the F₁ mice, and the female F₁ mice were then backcrossed to WHT/Ht. It was found that one half of the backcross generation expressed the la.7 antigen derived from BALB/c and had a significantly lower specific activity of the transferase than that of WHT/Ht, while the other half did not express the la.7 antigen but had the same specific activity of the transferase as that obtained with WHT/Ht.These results suggest that the locus controlling the level of the transferase activity in mouse liver is linked to the H-2 complex on chromosome 17.Abbreviations NeuGc N-glycolylneuraminic acid The ganglioside nomenclature is based on the system of Svennerholm, J Neurochem (1963) 10:613-23. The sialic acid species present is shown in parentheses after the ganglioside abbreviation.     

Molecular genetics of type 1 diabetes mellitus: Achievements and future trends

Type 1 diabetes mellitus (T1DM) is a widespread severe disease that results from autoimmune destruction of β cells in Langerhans islets of the pancreas. To date, several loci involved in T1DM have been reliably identified using various approaches: the MHC locus, VNTR within the 5′-nontranscibed region of the insulin gene (INS), CTLA4 (T-cell surface receptor), PTPN22, PTPN2 (T-cell tyrosine phosphatases), IL2 (interleukin 2, IL-2), IL2RA (IL-2 receptor α chain), KIAA0350 (unknown function), and IFIH1 (receptor for double-stranded DNA generated in virus infections). Functional analysis of their protein products confirmed the hypothesis that T1DM is underlain by deregulation of the mechanisms of immune tolerance and, on the other hand, a destructive immune response against the body’s own proteins after virus infection or some other immune stress. Thus the protein products of MHC, INS, PTPN22, and PTPN2 are involved in the intrathymic formation of the T-cell repertoire, responsible for immune defense of the body. On the other hand, nonspecific activation of T cells, which starts autoimmune destruction of pancreatic β cells, is most likely associated with the protein products of CTLA4, IL2, IL2RA, and, possibly, PTPN22 and PTPN2. Apart from the genes with unknown functions, the only exception is IFIH1, but its association with T1DM confirms that certain virus infections can activate autoreactive T cells and lead to T1DM.     

Structural Peculiarities of Na+,K+-ATPase Isozymes from the Calf Brain

Functionally active preparations of Na⁺,K⁺-ATPase isozymes from calf brain that contain catalytic subunits of three types (1, 2, and 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of their membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na⁺ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na⁺,K⁺-ATPase of the 11 type and minor amounts of isozymes of the 22(1) and the 31(2) type. The axolemma contains 21 and 31 isozymes. A carbohydrate analysis indicated that 11 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the 1 isoform. An enhanced sensitivity of the 3 catalytic subunit of Na⁺,K⁺-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR⁴⁹² Y⁴⁹³ was localized (residue numbering is that of the human 3 subunit). This sequence corresponds to one of the regions of the greatest variability in 1-, 2-, 3-, and 4-subunits, but at the same time, it is characteristic of the 3 isoforms of various species. The presence of the 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na⁺,K⁺-ATPase 31 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the 3 catalytic subunit was shown.     

A multicopy suppressor ofnin1-1 of the yeastSaccharomyces cerevisiae is a counterpart of theDrosophila melanogaster diphenol oxidase A2 gene,Dox-A2

NIN1 is an essential gene for growth of the yeastSaccharomyces cerevisiae and was recently found to encode a component of the regulatory subunit of the 26S proteasome. Thenin1-1 mutant is temperature sensitive and its main defect is in G₁/S progression and G₂/M progression at non-permissive temperatures. One of the two multicopy suppressors ofnin1-1, SUN2 (SUppressor of Nin1-1), was found to encode a protein of 523 amino acids whose sequence is similar to those ofDrosophila melanogaster diphenol oxidase A2 and the mouse mast-cell Tum^– transplantation antigen, P91A. The C-terminal half of Sun2p was found to be functional as Sun2p at 25° C, 30° C, and 34° C but not at 37° C. The open reading frame (ORF) of theDrosophila diphenol oxidase A2 gene (Dox-A2) was obtained from a lambda phage cDNA library using the polymerase chain reaction technique. TheDox-A2 ORF driven by theTDH3 promoter complemented the phenotype of a strain deleted forsun2. ThisDox-A2-dependent strain was temperature sensitive and accumulated dumb-bell-shaped cells, with an undivided nucleus at the isthmus, after temperature upshift. This morphology is similar to that ofnin1-1 cells kept at a restrictive temperature. These results suggest thatSUN2 is a functional counterpart ofDox-A2 and that these genes play a pivotal role in the cell cycle in each organism.     

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man₆₍₇₎GlcNAc₂-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 M, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man₉GlcNAc₂-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man₅GlcNAc₂-R and Man₆GlcNAc₂-R. Man₇GlcNAc₂-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).     

Biodegradation of disperse dye brown 3REL by microbial consortium of Galactomyces geotrichum MTCC 1360 and Bacillus sp. VUS

The consortium-GB (Galactomyces geotrichum MTCC 1360 and Bacillus sp. VUS) exhibited 100% decolorization ability with the dye Brown 3REL within 2 h at shaking condition with optima of pH 7 and at 50°C. However, G. geotrichum MTCC 1360 showed 39% decolorization within 24 h and Bacillus sp. VUS took 5 h for 100% decolorization, when incubated individually. Additional carbon and nitrogen sources like, starch, peptone, and urea were found to enhance decolorization. Induction in lignin peroxidase, tyrosinase, and riboflavin reductase was observed in consortium as that of individual organisms. GCMS identification showed different metabolites formed using consortium (2-(6,8-dichloro-quinazolin-4yloxy)-acetyl-urea and 2-(6,8-dichloro-quinazolin-4yloxy)-acetyl-formamide) and Bacillus sp. VUS (6,8-dichloro-4 methoxy-quinazoline) after 2 h of incubation with Brown 3REL. G. geotrichum MTCC 1360 showed minor modifications in structure of Brown 3REL. Phytotoxicity revealed non toxic nature of metabolites. This consortium-GB was also able to decolorize various industrial dyes.     

Cloning and morphological properties of Nicgl;CYCD3;1 gene in genetic tumors from interspecific hybrid of N. langsdorffii and N. glauca

Plant genetic tumors represent neoplastic growths, which arise spontaneously in hybrid plants without apparent external induction. To understand the molecular nature of unregulated cell proliferation, a cyclin D cDNA clone encoding a cyclin D of 1104bp was isolated from a genetic tumor and designated Nicgl;CYCD3;1 gene. DNA gel blot analysis suggested that there are two copies of Nicgl;CYCD3;1 in the genetic tumors. Northern analysis showed that this gene had the highest expression level in genetic tumor compared to Nicotiana glauca, N. langsdorffii and hybrid plants. Plant morphology of hybrid plant was an intermediate between N. glauca and N. langsdorffii and was altered in the genetic tumors. The cell cycle distribution in N. glauca was G0/G1, 90.59; S, 0.60; G2/M, 8.81; in N. langsdorffii it was G 0/G1, 86.22; S, 6.90; G2/M, 6.88; in hybrid plants it was G 0/G1, 96.40; S, 1.79; G2/M, 1.81; and in genetic tumors G 0/G1, 74.70; S, 2.35; G2/M, 22.94. These data provide new insights into the molecular mechanisms underlying genetic tumor formation from interspecific hybrid between N. langsdorffii and N. glauca.     

Peroxidase from Bacillus sp. VUS and its role in the decolorization of textile dyes

Peroxidase was purified by an ion exchange chromatography followed by gel filtration chromatography from dye degrading Bacillus sp. strain VUS. The optimum pH and temperature of the enzyme activity was 3.0 and 65°C, respectively. This enzyme showed more activity with n-propanol than other substrates tested viz. xylidine, 3-(3,4-dihydroxy phenyl) Lalanine (L-DOPA), hydroxyquinone, ethanol, indole, and veratrole. Km value of the enzyme was 0.076 mM towards n-propanol under standard assay conditions. Peroxidase was more active in presence of the metal ions like Li²⁺, Co²⁺, K²⁺, Zn²⁺, and Cu²⁺ where as it showed less activity in the presence of Ca²⁺ and Mn²⁺. Inhibitors like ethylenediamine tetraacetic acid (EDTA), glutamine, and phenylalanine inhibited the enzyme partially, while sodium azide (NaN₃) completely. The crude as well as the purified peroxidase was able to decolourize different industrial dyes. This enzyme decolourized various textile dyes and enhanced percent decolourization in the presence of redox mediators. Aniline was the most effective redox mediator than other mediators tested. Gas chromatography-Mass spectrometry (GC-MS) confirmed the formation of 7-Acetylamino-4-hydroxy-naphthalene-2-sulphonic acid as the final product of Reactive Orange 16 indicating asymmetric cleavage of the dye.     

Molecular and Structural Characterization of Barley Vernalization Genes

Vernalization, the requirement of a period of low temperature to induce transition from the vegetative to reproductive state, is an evolutionarily and economically important trait in the Triticeae. The genetic basis of vernalization in cultivated barley (Hordeum vulgare subsp. vulgare) can be defined using the two-locus VRN-H1/VRN-H2 model. We analyzed the allelic characteristics of HvBM5A, the candidate gene for VRN-H1, from ten cultivated barley accessions and one wild progenitor accession (subsp. spontaneum), representing the three barley growth habits – winter, facultative, and spring. We present multiple lines of evidence, including sequence, linkage map location, and expression, that support HvBM5A being VRN-H1. While the predicted polypeptides from different growth habits are identical, spring accessions contain a deletion in the first intron of HvBM5A that may be important for regulation. While spring HvBM5A alleles are typified by the intron-localized deletion, in some cases, the promoter may also determine the allele type. The presence/absence of the tightly linked ZCCT-H gene family members on chromosome 4H perfectly correlates with growth habit and we conclude that one of the three ZCCT-H genes is VRN-H2. The VRN-H2 locus is present in winter genotypes and deleted from the facultative and spring genotypes analyzed in this study, suggesting the facultative growth habit (cold tolerant, vernalization unresponsive) is a result of deletion of the VRN-H2 locus and presence of a winter HvBM5A allele. All reported barley vernalization QTLs can be explained by the two-locus VRN-H1/VRN-H2 model based on the presence/absence of VRN-H2 and a winter vs. spring HvBM5A allele. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

荧光转基因斑马鱼Tg(ttn.2:EGFP)的构建

为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因斑马鱼品系,本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体,并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎,通过荧光检测、遗传杂交筛选和分子鉴定等方法,成功建立了能稳定遗传的Tg(ttn.2:EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明,绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点,结果表明:No.33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上,No.34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg(ttn.2:EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外,绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。     

The purification of the three trimannosyl-N-acetylchitobiose pentasaccharides from the urine of swainsonine-intoxicated sheep

A simple method for the preparative resolution of three Man₃GlcNAc₂ isomers called Ia, Ib and II has been designed. It consists mainly of the use of concanavalin A-Sepharose which allowed the total purification of Man₃GlcNAc₂-Ia, and then of anion-exchange resin in borate buffer-gradient to separate the Ib and II isomers. The purity of each oligosaccharide was checked by two HPLC methods. The use of these oligosaccharides for different analytical and biosynthetic purposes is discussed, and the unexpected resistance of one of the Man₃GlcNAc₂ alditols to the action of endo--N-acetylglucosaminidase H is noted.