Differential gene expression in CD3e- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation

 A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3e- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrβ, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function. Received: 3 June 1999 / Revised: 3 August 1999     

The expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase gene in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strains

The gene encoding for B. intermedius glutamyl endopeptidase (gseBi) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius glutamyl endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on endopeptidase expression. Activity of glutamyl endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius glutamyl endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

Prss16 is not required for T-cell development

PRSS16 is a serine protease expressed exclusively in cortical thymic epithelial cells (cTEC) of the thymus, suggesting that it plays a role in the processing of peptide antigens during the positive selection of T cells. Moreover, the human PRSS16 gene is encoded in a region near the class I major histocompatibility complex (MHC) that has been linked to type 1 diabetes mellitus susceptibility. The mouse orthologue Prss16 is conserved in genetic structure, sequence, and pattern of expression. To study the role of Prss16 in thymic development, we generated a deletion mutant of Prss16 and characterized T-lymphocyte populations and MHC class II expression on cortical thymic epithelial cells. Prss16-deficient mice develop normally, are fertile, and show normal thymic morphology, cellularity, and anatomy. The total numbers and frequencies of thymocytes and splenic T-cell populations did not differ from those of wild-type controls. Surface expression of MHC class II on cTEC was also similar in homozygous mutant and wild-type animals, and invariant chain degradation was not impaired by deletion of Prss16. These findings suggest that Prss16 is not required for quantitatively normal T-cell development.     

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from <Emphasis Type="Italic">Asclepias fruticosa</Emphasis> latex

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZα vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.     

Isolation and expression analysis of a poriferan <Emphasis Type="Italic">Antp</Emphasis>-class <Emphasis Type="Italic">Bar-/Bsh-</Emphasis>like homeobox gene

A novel non-Hox Antp-class gene (BarBsh-Hb) was isolated from the marine sponge Halichondria sp. This gene shares high sequence identity with eumetazoan genes from the Bsh and Bar gene families and can be distinguished from other non-Hox Antp-class genes by diagnostic residues. We also present an alignment of all known (full-length) poriferan non-Hox Antp-class genes. Maximum likelihood methods were employed to estimate phylogenetic relationships among non-Hox genes and BarBsh-Hb. We employed RT-PCR techniques to look at expression across different developmental stages (larval to rhagon). BarBsh-Hb product was present in newly released larvae, but expression was not detected 8–16 h post-release. Expression of BarBsh-Hb was detected in later-stage (>16 h post-release), free-swimming larvae until they settled and attached to the substratum, after which expression was down-regulated. In a separate set of experiments, low levels of expression were observed in normal adult tissue and disaggregated adult tissue, but BarBsh-Hb expression increased during tissue re-aggregation. These data increase the number of non-Hox homeobox genes identified in sponges and provide evidence of regulation of this non-Hox gene during sponge development. While the Bar and Bsh genes play important roles in the development of nervous tissue—especially visual systems—in metazoans, the specific role(s) BarBsh-Hb play(s) in sponge development is unclear and deserves greater attention.Edited by C. Desplan     

Characterization and Candidate Gene Analysis of the Yellow-Green Leaf Mutant <i>ygl16</i> in Rice (<i>Oryza sativa</i> L.)

Leaf color mutants are ideal materials for studying many plant physiological and metabolic processes such as photosynthesis, photomorphogenesis, hormone physiology and disease resistance. In this study, the genetically stable yellow-green leaf mutant ygl16 was identified from mutated “Xinong 1B”. Compared with the wild type, the pigment concentration and photosynthetic capacity of the ygl16 decreased significantly. The ultrastructural observation showed that the distribution of thylakoid lamellae was irregular in ygl16 chloroplasts, and the grana and matrix lamellae were blurred and loose in varied degrees, and the chloroplast structure was disordered, while the osmiophilic corpuscles increased. The results of the genetic analysis and mapping showed that the phenotype of ygl16 was controlled by a pair of recessive nuclear gene. The gene located in the 56Kb interval between RM25654 and R3 on the long arm of chromosome 10. The sequencing results showed that the 121st base of the first intron of the candidate gene OsPORB/FGL changed from A to T in the interval. qRT-PCR results showed that the expression of chlorophyll synthase-related genes in the mutant decreased.     

Evidence for a polyamine-mediated control of soluble nitrogen mobilization during post-clipping re-growth of white clover (Trifolium repens L.)

A putative contribution of polyamines to the control of peptidase activity expression during re-growth was studied in source organs (roots and stolons) of defoliated white clover (Trifolium repens L.). Endopeptidase activity increased in roots during the first 6 days following complete defoliation, while exopeptidase expression seemed to be restricted to the early hours of re-growth. These changes correlated with an immediate 80% decline in the content of total free polyamines, mainly represented by the diamine cadaverine. The inhibitory capacities of cadaverine and spermine were tested on enzyme activity in vitro in order to elucidate whether the endogenous polyamine level was associated with the cut-induced endopeptidase expression. Cadaverine seemed to inhibit endopeptidase activity of stolons but not root endopeptidase activity. These data support the view that polyamines may play a role in the regulation of peptidase expression in source organs of white clover during post-clipping re-growth. The existence of different endopeptidase isoforms in roots and stolons is discussed in relation to the molecular mechanisms by which polyamines may regulate their activities.Abbreviations AP aminopeptidase - Cad cadaverine - CP carboxypeptidase - EP endopeptidase - PA(s) polyamine(s) - Spm spermine     

Validation of reference genes for real-time quantitative PCR studies in gene expression levels of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang

Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.     

Distinct Spatiotemporal Expression of Serine Proteases Prss23 and Prss35 in Periimplantation Mouse Uterus and Dispensable Function of Prss35 in Fertility

PRSS23 and PRSS35 are homologous proteases originally identified in mouse ovaries. In the periimplantation mouse uterus, Prss23 was highly expressed in the preimplantation gestation day 3.5 (D3.5) uterine luminal epithelium (LE). It disappeared from the postimplantation LE and reappeared in the stromal compartment next to the myometrium on D6.5. It was undetectable in the embryo from D4.5 to D6.5 but highly expressed in the embryo on D7.5. Prss35 became detectable in the uterine stromal compartment surrounding the embryo on D4.5 and shifted towards the mesometrial side of the stromal compartment next to the embryo from D5.5 to D7.5. In the ovariectomized uterus, Prss23 was moderately and Prss35 was dramatically downregulated by progesterone and 17β-estradiol. Based on the expression of Prss35 in granulosa cells and corpus luteum of the ovary and the early pregnant uterus, we hypothesized that PRSS35 might play a role in female reproduction, especially in oocyte development, ovulation, implantation, and decidualization. This hypothesis was tested in Prss35^(−/−) mice, which proved otherwise. Between wild type (WT) and Prss35^(−/−) mice, superovulation of immature females produced comparable numbers of cumulus-oocyte complexes; there were comparable numbers of implantation sites detected on D4.5 and D7.5; there were no obvious differences in the expression of implantation and decidualization marker genes in D4.5 or D7.5 uteri. Comparable mRNA expression levels of a few known protease-related genes in the WT and Prss35^(−/−) D4.5 uteri indicated no compensatory upregulation. Comparable litter sizes from WT × WT and Prss35 ^(−/−)× Prss35 ^(−/−) crosses suggested that Prss35 gene was unessential for fertility and embryo development. Prss35 gene has been linked to cleft lip/palate in humans. However, no obvious such defects were observed in Prss35^(−/−) mice. This study demonstrates the distinct expression of Prss23 and Prss35 in the periimplantation uterus and the dispensable role of Prss35 in fertility and embryo development.     

Expression of Apoptotic and Antioxidant Enzyme Genes in Sheep Oocytes and In Vitro Produced Embryos

The present study was to find out the expression pattern and relative expression level of apoptotic (Bcl2, Bax, Casp3, and PCNA) and antioxidant enzyme [(GPx, Cu/Zn-SOD (SOD1) and Mn-SOD (SOD2)] genes in sheep oocytes and developing embryos produced in vitro by conventional RT-PCR and real time qPCR, respectively. Different developmental stages of embryos were produced in vitro from oocytes collected from local slaughter house ovaries. RT-PCR amplicons showed expression of Bcl2 and PCNA in all stages except at morula. In contrast Bax and Casp3 were expressed in all stages. GPx and SOD1 were expressed in all stages but SOD2 was not expressed in 8–16 cells, although expressed in the remaining stages. The qPCR analysis reflected that Bcl2 expression was significantly (P < 0.05) downregulated in morula and maximum upregulated expression was observed in in vitro matured oocytes. Higher upregulated expression (P < 0.05) of Bax was in morula and downregulated expression was at 2-4 cells. Casp3 was significantly upregulated at 8–16 cells and downregulated in in vitro matured oocyte. PCNA expression was highest at blastocyst and least expression was at morula. GPx was expressed significantly highest in matured oocytes and least expression was at zygote. SOD1 was expressed significantly highest at 8–16 cells and least expression was at zygote. Expression of SOD2 was least among all the antioxidant enzymes but significantly higher expression of SOD2 was in immature oocyte; however, least expression was at 8–16 cells. It can be concluded from the study that the sheep embryos produced in vitro are highly sensitive to culture condition, which alters the expression level of apoptotic and antioxidant enzyme genes.     

Protective action of glutamate antibodies on increased expression of genes of programmed death of rat brain cells induced by injection of a β-amyloid fragment (25–35)

Glutamate antibodies intranasally administered to Wistar rats at a dose of 300 μg/kg reduced the elevated levels of expression of Aifml, Casp3, and Parp1 genes in the prefrontal cortex and Aifml and Casp3 genes in the hippocampus on the third day after administration of the β-amyloid fragment Aβ_25–35 into the Meynert nuclei of the brain. Changes in Aifm1, Bax, Casp3, and Parp1 gene expression were not found in the hypothalamus, and changes in Bax gene expression were not found in the brain structures studied. The discovered features of gene expression in the prefrontal cortex and hippocampus are considered in terms of development of various cell-death programs, which are modulated by glutamate antibodies.     

The impact of Caspase-1 deletion on apoptosis and acute kidney injury in a murine transplant model

BackgroundCaspase-1 knockout mice (Casp1KO) are protected from Acute Kidney Injury (AKI) after warm ischemia/reperfusion injury in non-transplant models. Since Caspase-1 plays a central role as an inflammatory response initiator, we hypothesized that Casp1KO mice would be protected from AKI following transplant.MethodsRenal tubular cells (RTECs) were subjected to cold storage and rewarming (CS/REW). C57Bl/6 J wild type or Casp1KO kidneys were subjected to CI for 30 min and then transplanted into wild type recipients (CI + Txp). The recipients underwent bilateral native nephrectomy at the time of transplant. Serum creatinine (sCr) was measured 24 h after native nephrectomy to assess transplant function.ResultsWe found that RTECs subjected to CS/REW had significantly increased expression of the Caspase-1 and inflammasome protein NLRP1. Wild type kidneys subjected to CI + Txp into wild type recipients also demonstrated significantly increased Caspase-1 and NLRP1 protein expression compared to kidneys transplanted from Casp1KO donors into wild type recipients. Caspase-1 deletion results in significantly decreased RTEC apoptosis in transplanted Casp1KO vs WT kidneys. Surprisingly, however, renal function, ATN scores including brush border injury, cast formation and tubular simplification were similar in both groups and not significantly different.ConclusionsOur data suggest that other triggers of inflammation and programmed necrosis may need to be inhibited in addition to attenuating Caspase-1 to fully prevent AKI after kidney transplant. Importantly, requirements may be distinct for AKI induced by transplantation as opposed to other transient models such as the clamp model of AKI.     

Survival associated pathway identification with group <Emphasis Type="Italic">L</Emphasis><Subscript><Emphasis Type="Italic">p</Emphasis></Subscript> penalized global AUC maximization

It has been demonstrated that genes in a cell do not act independently. They interact with one another to complete certain biological processes or to implement certain molecular functions. How to incorporate biological pathways or functional groups into the model and identify survival associated gene pathways is still a challenging problem. In this paper, we propose a novel iterative gradient based method for survival analysis with group L _p penalized global AUC summary maximization. Unlike LASSO, L _p (p < 1) (with its special implementation entitled adaptive LASSO) is asymptotic unbiased and has oracle properties [1]. We first extend L _p for individual gene identification to group L _p penalty for pathway selection, and then develop a novel iterative gradient algorithm for penalized global AUC summary maximization (IGGAUCS). This method incorporates the genetic pathways into global AUC summary maximization and identifies survival associated pathways instead of individual genes. The tuning parameters are determined using 10-fold cross validation with training data only. The prediction performance is evaluated using test data. We apply the proposed method to survival outcome analysis with gene expression profile and identify multiple pathways simultaneously. Experimental results with simulation and gene expression data demonstrate that the proposed procedures can be used for identifying important biological pathways that are related to survival phenotype and for building a parsimonious model for predicting the survival times.     

Effect of the regulation system of metabolic nitrogen exchange on biosynthesis of serine proteinases from <Emphasis Type="Italic">Bacillus intermedius</Emphasis>

The regulatory link between biosynthesis of Bacillus intermedius subtilisin-like serine proteinase and nitrogen metabolism in B. intermedius cells was determined. The level of the enzyme biosynthesis by the recombinant strain of Bacillus subtilis in the medium containing ammonium ions was three- to fivefold less than that in the medium with poorly utilized sodium nitrate. Accumulation of glutamyl endopeptidase in a culture liquid of this microorganism did not depend on the source of nitrogen present in the medium. During cultivation in the rich medium, the productivity of subtilisin-like proteinase in the recombinant B. subtilis strain carrying a mutation in the NrgB sensor protein was demonstrated to increase threefold compared to that of the control strain. In the minimal culture medium, mutation in the nrgB gene abolished the effect of a nitrogen source on the level of the subtilisin-like proteinase gene expression. At the same time, this mutation did not affect glutamyl endopeptidase biosynthesis. Thus, expression of the gene coding for subtilisin-like proteinase from B.intermedius is suggested to be positively regulated by the regulatory system of nitrogen metabolism.     

The HyPRP gene <Emphasis Type="Italic">EARLI1</Emphasis> has an auxiliary role for germinability and early seedling development under low temperature and salt stress conditions in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The effect of the hybrid proline-rich protein (HyPRP) gene EARLI1 on the rate of germination (germinability) of Arabidopsis seeds and seedling growth under low temperature and salt stress conditions was investigated. EARLI1 was induced during germination in embryonic tissues, and was strongly expressed in certain parts of young seedlings. Comparisons of control, overexpressing (OX), and knockout (KO) lines indicated that higher than wild type levels of EARLI1 improved germinability, root elongation, and reduction of sodium accumulation in leaves under salt stress, as well as germinability under low-temperature stress. Abscisic acid (ABA) contents were relatively low after prolonged salt stress, suggesting that EARLI1 has an ABA-independent effect on germinability under these conditions. Overexpression of EARLI1 during germination enhanced the sensitivity of seeds to exogenously applied ABA, suggesting that EARLI1 has an ABA-dependent negative effect on seed germinability under high ABA stress conditions. Well-known stress response marker genes such as COR15a, KIN1, P5SC1, and RD29 were unaffected whereas P5SC2, RD22, or RAB18 were only slightly affected in OX and KO plants. The pleiotropic effects of EARLI1 during stress and an absence of strong regulatory effects on stress marker genes suggest that this HyPRP gene has an auxiliary role for various stress protection responses in Arabidopsis.     

Anammox Bacterial Diversity in Various Aquatic Ecosystems Based on the Detection of Hydrazine Oxidase Genes (<Emphasis Type="Italic">hzoA</Emphasis>/<Emphasis Type="Italic">hzoB</Emphasis>)

Anammox bacteria belonging to the phylum Planctomycetes are responsible for N removal through NH₄⁺ oxidation coupled with NO₂⁻ reduction. Microbial diversity and ecology of anammox bacteria have not yet been fully revealed due to limitations of 16S rRNA analysis. The hydrazine oxidase gene in cluster 1 (hereafter hzoA/hzoB) was suggested as a proper genetic marker due to its high expression and ubiquitous presence in anammox bacteria. We conducted a comparative analysis of 16S rRNA and hzoA/hzoB genes to reveal anammox bacterial diversity and distribution in various aquatic environments. Phylogenetic analyses of 16S rRNA and hzoA/hzoB genes showed the dominance of Scalindua organisms in marine ecosystems, but there was no congruence of 16S rRNA and hzoA/hzoB gene phylogenies among the freshwater anammox bacteria associated with Brocadia sp., Jettenia sp., and Anammoxoglobus sp. Higher diversity of anammox bacteria was revealed based on hzoA/hzoB genes than 16S rRNA genes in the examined environments. Multiple regression analysis showed that salinity had significant influence on differential distribution and diversity of anammox bacteria in different ecosystems. Thus, molecular detection and resulting phylogeny of the hzoA/hzoB gene generated a better understanding of anammox bacterial diversity and their ecological distribution in various aquatic ecosystems.