Expression of a novel gene, gluP, is essential for normal Bacillus subtilis cell division and contributes to glucose export

BACKGROUND: The Bacillus subtilis glucokinase operon was predicted to be comprised of the genes, yqgP (now named gluP), yqgQ, and glcK. We have previously established a role for glcK in glucose metabolism. In the absence of enzymes that phosphorylate glucose, such as GlcK and/or enzyme II(Glc), accumulated cytoplasmic glucose can be transported out of the cell. Genes within the glucokinase operon were not previously known to play a role in glucose transport. Here we describe the expression of gluP and its function in glucose transport. RESULTS: We found that transcription of the glucokinase operon was regulated, putatively, by two promoters: σ(A )and σ(H). Putative σ(A )and σ(H)-recognition sites were located upstream of and within gluP, respectively. Transcriptional glucokinase operon – lacZ fusions and Northern blotting were used to analyze the expression of gluP. GluP was predicted to be an integral membrane protein. Moreover, the prediction of GluP structure revealed interesting signatures: a rhomboid domain and two tetracopeptide repeat (TPR) motifs. Microscopic analysis showed that GluP minus cells were unable to divide completely, resulting in a filamentous phenotype. The cells were grown in either rich or minimal medium. We found GluP may be involved in glucose transport. [(14)C]-glucose uptake by the GluP minus strain was slightly less than in the wild type. On the other hand, trehalose-derived glucose in the growth medium of the GluP minus strain was detected in very low amounts. Experimental controls comprised of single or multiple genes mutations within the glucose transporting phosphotransferase system. CONCLUSIONS: gluP seems to be regulated only by a putative σ(A)-dependent promoter. The glucose uptake and export assays suggest that GluP is important for glucose export and may act as an exporter. This also supports the role of the glucokinase operon in glucose utilization

Bacillus subtilis GlcK activity requires cysteines within a motif that discriminates microbial glucokinases into two lineages

BACKGROUND: Bacillus subtilis glucokinase (GlcK) (GenBank NP_390365) is an ATP-dependent kinase that phosphorylates glucose to glucose 6-phosphate. The GlcK protein has very low sequence identity (13.7%) to the Escherichia coli glucokinase (Glk) (GenBank P46880) and some other glucokinases (EC 2.7.1.2), yet glucose is merely its substrate. Our lab has previously isolated and characterized the glcK gene. RESULTS: Microbial glucokinases can be grouped into two different lineages. One of the lineages contains three conserved cysteine (C) residues in a CXCGX(2)GCXE motif. This motif is also present in the B. subtilis GlcK. The GlcK protein occurs in both monomer and homodimer. Each GlcK monomer has six cysteines. All cysteine residues have been mutated, one-by-one, into alanine (A). The in vivo GlcK enzymatic activity was assayed by functional complementation in E. coli UE26 (ptsG ptsM glk). Mutation of the three motif-specific residues led to an inactive enzyme. The other mutated forms retained, or in one case (GlcK(C321A)) even gained, activity. The fluorescence spectra of the GlcK(C321A )showed a red shift and enhanced fluorescence intensity compare to the wild type's. CONCLUSIONS: Our results emphasize the necessity of cysteines within the CXCGX(2)GCXE motif for GlcK activity. On the other hand, the C321A mutation led to higher GlcK(C321A )enzymatic activity with respect to the wild type's, suggesting more adequate glucose phosphorylation

Molecular cloning, genomic characterization and over-expression of a novel gene, XRRA1, identified from human colorectal cancer cell HCT116(Clone2_XRR )and macaque testis

BACKGROUND: As part of our investigation into the genetic basis of tumor cell radioresponse, we have isolated several clones with a wide range of responses to X-radiation (XR) from an unirradiated human colorectal tumor cell line, HCT116. Using human cDNA microarrays, we recently identified a novel gene that was down-regulated by two-fold in an XR-resistant cell clone, HCT116(Clone2_XRR). We have named this gene as X-ray radiation resistance associated 1 (XRRA1) (GenBank BK000541). Here, we present the first report on the molecular cloning, genomic characterization and over-expression of the XRRA1 gene. RESULTS: We found that XRRA1 was expressed predominantly in testis of both human and macaque. cDNA microarray analysis showed three-fold higher expression of XRRA1 in macaque testis relative to other tissues. We further cloned the macaque XRRA1 cDNA (GenBank AB072776) and a human XRRA1 splice variant from HCT116(Clone2_XRR )(GenBank AY163836). In silico analysis revealed the full-length human XRRA1, mouse, rat and bovine Xrra1 cDNAs. The XRRA1 gene comprises 11 exons and spans 64 kb on chromosome 11q13.3. Human and macaque cDNAs share 96% homology. Human XRRA1 cDNA is 1987 nt long and encodes a protein of 559 aa. XRRA1 protein is highly conserved in human, macaque, mouse, rat, pig, and bovine. GFP-XRRA1 fusion protein was detected in both the nucleus and cytoplasm of HCT116 clones and COS-7 cells. Interestingly, we found evidence that COS-7 cells which over-expressed XRRA1 lacked Ku86 (Ku80, XRCC5), a non-homologous end joining (NHEJ) DNA repair molecule, in the nucleus. RT-PCR analysis showed differential expression of XRRA1 after XR in HCT116 clones manifesting significantly different XR responses. Further, we found that XRRA1 was expressed in most tumor cell types. Surprisingly, mouse Xrra1 was detected in mouse embryonic stem cells R1. CONCLUSIONS: Both XRRA1 cDNA and protein are highly conserved among mammals, suggesting that XRRA1 may have similar functions. Our results also suggest that the genetic modulation of XRRA1 may affect the XR responses of HCT116 clones and that XRRA1 may have a role in the response of human tumor and normal cells to XR. XRRA1 might be correlated with cancer development and might also be an early expressed gene

Synthesis and evaluation of 18F-labeled carbonic anhydrase IX inhibitors for imaging with positron emission tomography

Two carbonic anhydrase IX (CA IX) inhibitors were radiolabeled with (18)F, and evaluated for imaging CA IX expression. Despite good affinity for CA IX and excellent plasma stability, uptake of both tracers in CA IX-expressing HT-29 tumor xenografts in mice was low. (18)F-FEC accumulated predominately in the liver and nasal cavity, whereas a significant amount of (18)F-U-104 was retained in blood. Due to minimal uptake in HT-29 tumors compared to other organs/tissues, these two tracers are not suitable for use for CA IX-targeted imaging

Protease IgA1 Ureaplasma urealyticum bukan enzim tapi logam dan zimografik

Abstrak                 Ureaplasma urealyticum merupakan organisme komensal terkecil pada traktus urogenital manusia. Mikroba itu berperan menting dan berasosiasi dengan beragam penyakit ibu dan bayi baru lahir. Salah satu faktor penyebab terjadinya penyakit trsebut ialah protease IgA1. Protease IgA1 mampu memecah antibodi mukosa terdepan manusia, IgA1. Enzim tersebut dikelompokkan menjadi beberapa tipe berdasarkan hambatan aktivitasnya oleh berbagai senyawa inhibitor. Penelitian ini menguji inhibisi aktivitas protease IgA1 ureaplasma terhadap inhibitor protease logam, EDTA, dan inhibitor protease serin, PMSF. Ternyata EDTA dengan konsentrasi 0,1, 1,0 dan 10,0mM, dan PMSF konsentrasi 0,1, 0,5 dan 1,0 mM tidak mampu menghambat aktifitas pemecahan IgA1 oleh protein total sel uraplasma. Selanjutnya, pengukuran aktivitas proteolisis protease IgA1 dilakukan menggunakan teknik zomografi dengan substrat kasein dan kontrol positif Igase N. Gonorrhoeae. Namun hasil zomografi menggunakan SDS-PAGE untuk menguji hal di atas tiak memperlihatkan pemecahan fregmen kasein 29 kDa seperti yang diharapkan. Sehingga dapat disimpulkan protease IgA1 ureaplasma bukanlah tipe protease logam dan tidak memiliki sifat zimogram. Kata kunci : proteaseIgA1, protease logam, protease serin, inhibitor protease, kasein, zimogram. Abstract                 Ureaplasma urealyticum is one of the smallest inhabitants of human urogenital tract. The microbe plays important role in diseases involving mother and her newborn. One of the pathogenic factor is IgA1 protease. The enzyme cleaves human first line mucolas antibody. IgA1. Based on its inhibition, the enzyme is grouped into several categories. Weinhibited the enzyme activity using inhibitors such as EDTA and PMSF againt metallo- and serine- protease activities. Respectively. Neither EDTA at 0.1, 1.0 and 10.0 mM nor PMSF at 0,1, 0,5 and 1.0 mM was able to inhibit the IgA1 cleavage by total ureaplasma protein extract. Subsequently, using casein as enzymatic substrate and Igase purified from N. Gonorrhoeae as positive control, we tested proteolytic activity of the ureaplasma IgA1 protease. Zymography of the assay on an SDS-PAGE resulted that none of 29 kDa casein fregment was cleaved by the enzyme the result suggest that ureaplasma IgA1 protease is neither metallo- nor a zymographic- type protease Keyword : metallo-protease, serine-protease, IgA1 protease, IgA1, Igease, protease inhibitor, casein, zymogra

Transcriptomics of diapause in an isogenic self-fertilizing vertebrate.

BackgroundMany vertebrate species have the ability to undergo weeks or even months of diapause (a temporary arrest of development during early ontogeny). Identification of diapause genes has been challenging due in part to the genetic heterogeneity of most vertebrate animals.ResultsHere we take the advantage of the mangrove rivulus fish (Kryptolebias marmoratus or Kmar)-the only vertebrate that is extremely inbred due to consistent self-fertilization-to generate isogenic lineages for transcriptomic dissection. Because the Kmar genome is not publicly available, we built de novo genomic (642 Mb) and transcriptomic assemblies to serve as references for global genetic profiling of diapause in Kmar, via RNA-Seq. Transcripts unique to diapause in Kmar proved to constitute only a miniscule fraction (0.1 %) of the total pool of transcribed products. Most genes displayed lower expression in diapause than in post-diapause. However, some genes (notably dusp27, klhl38 and sqstm1) were significantly up-regulated during diapause, whereas others (col9a1, dspp and fmnl1) were substantially down-regulated, compared to both pre-diapause and post-diapause.ConclusionKmar offers a strong model for understanding patterns of gene expression during diapause. Our study highlights the importance of using a combination of genome and transcriptome assemblies as references for NGS-based RNA-Seq analyses. As for all identified diapause genes, in future studies it will be critical to link various levels of RNA expression with the functional roles of the coded products

Complete mitochondrial genome of a self-fertilizing fish Kryptolebias marmoratus (Cyprinodontiformes, Rivulidae) from Florida.

The complete mitochondrial genome was sequenced in a mangrove rivulus Kryptolebias marmoratus from western Florida using next-generation sequencing. The 17 329 bp-long genome was identical in length and 99.8% similar to a previously published genome of this species from a specimen of unknown geographic origin. Gene arrangement in K. marmoratus is similar to other cyprinodontiform fishes, except for the presence of a second copy of the control region inserted upstream of the nad1 gene