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

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

Transcriptomic profiling of radiation-resistant or -sensitive human colorectal cancer cells : Acute effects of a single X-radiation dose

We previously isolated several clones that were closely-related genetically from a human colorectal tumor (HCT116) cell line. These clones displayed significantly different X-radiation response phenotypes. In this paper, we investigated how a single dose of X-radiation modulated the transcriptomic profiles of either the radiation-resistant (HCT116Clone2_XRR) or the radiation-sensitive (HCT116CloneK_XRS) clone when each was compared to a reference clone, HCT116Clone10_control. The latter represented a control clone that displayed a similar X-radiation response as the parental HCT116 cells. Pooled RNAs were obtained from HCT116Clone2_XRR, HCT116CloneK_XRS or HCT116Clone10_control cells either before or at 10 min, 6 or 24 h after treatment with 4-Gy X-radiation. Transcriptomic profiles were assessed by cDNA microarrays. At least three independent experiments were carried out for each time point and statistical analysis was performed by paired t-test (p<0.05). From 19,200 genes/ESTs examined, we identified only 120 genes/ESTs that were differentially expressed at any one of these four time points. Interestingly, different patterns of gene modulation were observed between the radiation-sensitive and radiation-resistant clones. However, the fold changes of gene modulation were generally small (2-3 fold). Surprisingly, only 12.7% of 79 genes involved in DNA damage sensor/repair and cell cycle and between 2.6 and 9.2% of 76 genes involved in apoptosis, were significantly modulated in these early time points following irradiation. By comparison, up to 10% of 40 known housekeeping genes were differentially expressed. Thus in our experimental model, we were able to detect the up-regulation or down-regulation of mostly novel genes and/or pathways in the acute period (up to 24 h) following a single dose of 4-Gy X-radiationNRC publication: Ye

Synthesis and evaluation of 18

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