A three-dimensional homology model of the O-acetylserine sulfhydrylase-B from Salmonella typhimurium

O-acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants. The overall pathway involves the substitution of the beta-acetoxy group of O-acetyl-L-serine with inorganic bisulfide. Two isozymes are present in S. typhimurium, the A- and B-isozymes, expressed under aerobic and anaerobic conditions, respectively. No crystal structure is presently available for the B-isozyme. Kinetic data indicate the catalytic mechanism of OASS-B is ping-pong, as found for the A-isozyme, but kinetic parameters and substrate specificity differ. In order to estimate whether structural differences may be responsible for the kinetic differences, a homology model was built using the structure of OASS-A as the template for the OASS-B model. The beta-subunit of tryptophan synthase and cystathionine beta-synthase were used for comparison. Differences between the OASS-A structure and the homology model for OASS-B are discussed.     

Geminin is bound to chromatin in G2/M phase to promote proper cytokinesis

Previous studies suggested that geminin plays a vital role in both origin assembly and DNA re-replication during S-phase; however, no data to support a role for geminin in G2/M cells have been described. Here it is shown that in G2/M-phase, geminin participates in the promotion of proper cytokinesis. This claim can be supported through a series of observations. First, geminin in G2/M is loaded onto chromatin after it is tyrosine phosphorylated. It is unlike S-phase geminin that resides in the nuclear soluble fraction, where it is exclusively S/T phosphorylated. Secondly, on chromatin, geminin gets S/T phosphorylated in late G1; this modification causes the release of geminin from the chromatin. Cyclins bind and phosphorylate geminin in a sequential, cell cycle-dependent manner. These modifications correlated well with geminin departure from the chromatin. This suggests that cyclin functions to either release geminin from chromatin or at least keep it at bay until late S-phase. Thirdly, depletion of geminin from a diploid mammary epithelial cell line (HME) causes cells to arrest in late G2/M-phase. Massive serine-10 phosphorylated histone H3 staining and survivin localization to mid-body were observed; this suggests that they could be arrested in either mitosis or at cytokinesis. Finally, while in the absence of geminin, cyclin B1, chk1 and cdc7 are all over expressed. This paper will demonstrate that only cdc7 is important in maintaining the cytokinesis arrest in the absence of geminin. Only double depletion of geminin and cdc7 induce apoptosis. Our results taken together show, for the first time, that phosphorylation-induction activates oscillation of geminin between both nuclear soluble and chromatin compartments. Chromatin-bound geminin species functions to initiate or maintain proper cytokineses. In the absence of geminin, cells arrest in cytokinesis; this defines a novel checkpoint, monitored by cdc7, rather than cyclin B1 or chk1.     

Effects of sarcolectin (SCL) on human peripheral blood mononuclear cells

Sarcolectin (SCL) is a tissue growth factor found in various human or animal tissues, functioning in balance with interferons (IFNs) that can inhibit growth and affect cell differentiation. Like somatotropin, SCL is found in the pituitary gland. In humans, the SCL gene is located on chromosome 12 (q12-q13) and expressed as a 55 kDa protein consisting of 469 amino-acids. After a single activation of peripheral blood mononuclear cells (PBMC) obtained from more than 30 individuals, highly significant cell proliferation was found to peak after 7 days in culture. The presence of adherent cells was necessary for cell proliferation. SCL induced over-expression of alpha-IL-2 receptor (CD25) leading to proliferation of CD3+/CD4+/CD45RO+ T cells. Thus in PBMC, SCL induced CD4+ T cell growth and expression of inflammatory cytokine genes, including TNF-alpha, IL-1beta, IL-6 and IL-8. IFNs are also produced following activation as a feedback response which is maintained for about 20 days.     

BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells

The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ERalpha signaling. However, many ERalpha-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ERalpha signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ERalpha-negative cells. We previously noticed that both ERalpha-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ERalpha-negative cell lines even exceeded its over-expression level in ERalpha-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ERalpha-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.     

Hepatic chemoprotective enzyme responses to 2-substituted selenazolidine-4(R)-carboxylic acids

In epidemiology and human supplementation studies, as well as many animal models, selenium has shown antitumorigenic activity. The mechanism of action, however, has not been satisfactorily resolved. Selenium supplementation affects many enzymes in addition to those where selenocysteine is an essential component. Such enzymes include cytoprotective detoxifying enzymes, and the regulation of these enzymes by a set of 2-substituted selenazolidine-4(R)-carboxylic acids (SCAs) has been investigated. Following seven consecutive daily doses of these prodrugs of L-selenocysteine, changes in hepatic enzyme activities and/or mRNA levels of glutathione transferase (GST), microsomal epoxide hydrolase (mEH), NAD(P)H-quinone oxidoreductase (NQO), UDP-glucuronosyltransferase (UGT), glutathione peroxidase (GPx), and thioredoxin reductase (TR) have been observed. Among the enzymes examined, UGTs and GPx were found to be the least affected. Among the compounds, 2-oxoSCA produced the most changes and 2-phenylSCA produced the least, none. For no two compounds was the pattern of changes identical, and for a single compound, few changes were reproduced in common by the two routes of administration investigated. In general, more changes were elicited following intraperitoneal (i.p.) administration than with the intragastric (i.g.) route. This dominance was typified by 2-butylSCA and 2-cyclohexylSCA where enzyme activity elevations (TR and mEH with both, NQO with 2-butylSCA) were seen only with the i.p. route. With 2-oxoSCA, however, GST, TR, and NQO activities were found to be elevated independent of route. Only with GST (both routes) and TR (i.p. route), elevations in mRNAs accompanied the 2-oxoSCA elicited elevations of activities at the time of sacrifice. For some enzymes, most notably mEH with compounds administered i.p., elevations in mRNAs were not manifest as increased enzyme activity. Thus, although constituting a closely related series of compounds, each 2-substituted SCA produced its own unique pattern of changes, and for most members, changes were predominant following i.p. administration.     

Impact of DNA repair genes polymorphism (XPD and XRCC1) on the risk of breast cancer in Egyptian female patients

The genes involved in DNA repair system play a crucial role in the protection against mutations. It has been hypothesized that functional deficiencies in highly conserved DNA repair processes resulting from polymorphic variation may increase genetic susceptibility to breast cancer (BC). The aim of the present study was to evaluate the association of genetic polymorphisms in 2 DNA repair genes, XPD (Asp312Asn) and XRCC1 (A399G), with BC susceptibility. We further investigated the potential combined effect of these DNA repair variants on BC risk. Both XPD (xeroderma pigmentosum group D) and XRCC1 (X-ray repair cross-complementing group 1) polymorphisms were characterized in 100 BC Egyptian females and 100 healthy women who had no history of any malignancy by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method and PCR with confronting two-pair primers (PCR-CTPP), using DNA from peripheral blood in a case control study. Our results revealed that the frequencies of AA genotype of XPD codon 312 polymorphism were significantly higher in the BC patients than in the normal individuals (P ≤ 0.003), and did not observe any association between the XRCC1 Arg399Gln polymorphism and risk of developing BC. Also, no association between both XPD Asp312Asn and XRCC1 A399G polymorphisms and the clinical characteristics of disease. Finally, the combination of AA(XPD) + AG(XRCC1) were significantly associated with BC risk. Our results suggested that, XPD gene is an important candidate gene for susceptibility to BC. Also, gene–gene interaction between XPD(AA) + XRCC1(AG) polymorphism may be associated with increased risk of BC in Egyptian women.     

A marine-derived Streptomyces sp. MS449 produces high yield of actinomycin X2 and actinomycin D with potent anti-tuberculosis activity

In the course of our screening program for anti-Mycobacterium bovis bacillus Calmette-Guérin (BCG) and anti-Mycobacterium tuberculosis H37Rv (MTB H37Rv) agents from our marine natural product library, a newly isolated actinomycete strain, designated as MS449, was picked out for further investigation. The strain MS449, isolated from a sediment sample collected from South China Sea, produced actinomycin X(2) and actinomycin D in substantial quantities, which showed strong inhibition of BCG and MTB H37Rv. The structures of actinomycins were elucidated by nuclear magnetic resonance and mass spectrometric analysis. The strain MS449 was taxonomically characterized on the basis of morphological and phenotypic characteristics, genotypic data, and phylogenetic analysis. The 16S rRNA gene sequence of the strain was determined and a database search indicated that the strain was closely associated with the type strain of Streptomyces avermitilis (99.7?% 16S rRNA gene similarity). S. avermitilis has not been previously reported to produce actinomycins. The marine-derived strain of Streptomyces sp. MS449 produced notably higher quantities of actinomycin X(2) (1.92?mg/ml) and actinomycin D (1.77?mg/ml) than previously reported actinomycins producing strains. Thus, MS449 was considered of great potential as a new industrial producing strain of actinomycin X(2) and actinomycin D.