Identification and Characterization of a Periplasmic α-Carbonic Anhydrase (CA) in the Gametophytes of Saccharina japonica (Phaeophyceae).

Periplasmic or external carbonic anhydrases (CAs) have been well accepted as playing a crucial role in the acquisition of dissolved inorganic carbon; however, no cytological evidence or molecular information on these enzymes has been reported in seaweeds to date. In this study, the full-length cDNA sequence coding for a putative periplasmic Sjα-CA2 was cloned from the gametophytes of Saccharina japonica, an industrial brown seaweed. It was 1,728 bp in length and included a 263-bp 5'-untranslated region (UTR), a 577-bp 3'-UTR, and an 888-bp open reading frame encoding a protein precursor consisting of 295 amino acids. The mature protein, after removal of a predicted 28-residue signal peptide, was composed of 267 amino acids with a relative molecular weight of 29.27 kDa. Multisequence alignment and phylogenetic analysis indicated that it was a member of the α-CA family. Enzyme activity assays showed that the recombinant Sjα-CA2 in Escherichia coli possessed CO2 hydration and esterase activities, thus identifying this gene Sjα-CA2 in function. Immunogold electron microscopic observations with the prepared anti-Sjα-CA2 polyclonal antibody illustrated that Sjα-CA2 was located in periplasmic space of the kelp gametophyte cells. Quantitative real-time PCR results revealed that the transcription of Sjα-CA2 was induced by elevated HCO3- levels, but it was little changed while the kelp gametophytes were subjected to elevated CO2 concentrations. This study suggests that the periplasmic Sjα-CA2 might play a role in adapting to elevated environmental levels of HCO3- by dehydration of HCO3- to generate CO2 , which could be readily taken up by S. japonica gametophytes.

Evidence for postinitiation regulation of mRNA biogenesis in tuberculosis.

Mycobacterium tuberculosis infection alters macrophage gene expression and macrophage response to IFN-γ, a critical host defense cytokine. However, regulation of these changes is poorly understood. We report discordance of changes in nascent transcript and total nuclear RNA abundance for the transcription factors STAT1 and IRF1, together with lack of effect on their RNA half-lives, in human THP-1 cells infected with M. tuberculosis and stimulated with IFN-γ. The results indicate that negative postinitiation regulation of mRNA biogenesis limits the expression of these factors, which mediate host defense against M. tuberculosis through the cellular response to IFN-γ. Consistent with the results for STAT1 and IRF1, transcriptome analysis reveals downregulation of postinitiation mRNA biogenesis processes and pathways by infection, with and without IFN-γ stimulation. Clinical relevance for regulation of postinitiation mRNA biogenesis is demonstrated by studies of donor samples showing that postinitiation mRNA biogenesis pathways are repressed in latent tuberculosis infection compared with cured disease and in active tuberculosis compared with ongoing treatment or with latent tuberculosis. For active disease and latent infection donors from two populations (London, U.K., and The Gambia), each analyzed using a different platform, pathway-related gene expression differences were highly correlated, demonstrating substantial specificity in the effect. Collectively, the molecular and bioinformatic analyses point toward downregulation of postinitiation mRNA biogenesis pathways as a means by which M. tuberculosis infection limits expression of immunologically essential transcription factors. Thus, negative regulation of postinitiation mRNA biogenesis can constrain the macrophage response to infection and overall host defense against tuberculosis.

Lymphadenectomy influences the utilization of adjuvant radiation treatment for endometrial cancer.

OBJECTIVE: We analyzed the effect of lymphadenectomy on the use of adjuvant radiation treatment for women with stage I-II endometrial cancer. STUDY DESIGN: Women with stage I-II endometrioid adenocarcinomas treated between 1988 and 2006 and recorded in the Surveillance, Epidemiology, and End Results database were identified. The influence of lymphadenectomy (LND) on receipt of external beam radiation and brachytherapy stratified was examined. RESULTS: We identified 58,776 women including 26,043 who underwent LND (44.3%). Among women younger than 60 years of age with stage IA (grades 1, 2, and 3) tumors, LND had no impact on the use of radiation. Patients with stage IB (grade 2 or 3) and stage IC (grade 1 or 2) tumors who underwent lymph node dissection were less likely to undergo external beam radiation and more likely to receive vaginal brachytherapy (P < .05 for all). Furthermore, the extent of lymphadenectomy influenced the receipt of radiation. CONCLUSION: Women who undergo lymphadenectomy are less likely to receive whole pelvic radiotherapy.

Inhibition of aberrant androgen receptor induction of prostate specific antigen gene expression, cell proliferation and tumor growth by 17α-estradiol in prostate cancer.

PURPOSE: Androgen independent prostate cancer growth and metastasis are a major cause of prostate cancer death. Aberrant androgen receptor activation due to androgen receptor mutation is an important mechanism of androgen independence. We determined the effectiveness and mechanism of 17α-estradiol (Sigma®) in blocking aberrant androgen receptor activation due to androgen receptor mutation. MATERIALS AND METHODS: We used LNCaP and MDA Pca-2b prostatic tumor cells (ATCC®) containing a mutated androgen receptor and WT estrogen receptor ß to test 17α-estradiol inhibition of aberrant androgen receptor activation of prostate specific antigen gene expression and cell growth. Cotransfection analysis was used to further elucidate the mechanism of 17α-estradiol action. Xenograft animals with an LNCaP prostate tumor were prepared to study the in vivo effect of 17α-estradiol on tumor growth inhibition. RESULTS: In LNCaP cells 17α-estradiol produced a dose dependent inhibition of cyproterone acetate (Sigma) or dihydrotestosterone induced prostate specific antigen gene expression. In MDA Pca-2b cells 17α-estradiol inhibited cortisol (Sigma) induced prostate specific antigen expression and blocked dihydrotestosterone and cortisol induced cell proliferation in LNCaP and MDA Pca-2b cells, respectively. Cotransfection analysis showed that 17α-estradiol inhibition of aberrant androgen receptor activation of prostate specific antigen gene expression was medicated via estrogen receptors. In xenograft mice with LNCaP prostate cancer 17α-estradiol but not 17ß-estradiol (Sigma) significantly inhibited tumor growth, although each estrogen tended to decrease tumor growth. CONCLUSIONS: Results suggest that 17α-estradiol with less classic estrogenic activity is a potential therapeutic agent for androgen independent prostate cancer due to androgen receptor mutation.

NSC606985, a novel camptothecin analog, induces apoptosis and growth arrest in prostate tumor cells.

PURPOSE: Prostate cancer is a major cause of cancer mortality in American males. Once prostate cancer has metastasized, there is currently no curative therapy available. The development of effective agents is therefore a continuing effort to combat this disease. In the present study, the effects and potential mechanisms of NSC606985 (NSC), a water-soluble camptothecin analog, in prostate cancer cells were investigated. METHODS: Prostatic tumor cells, DU-145, LNCaP and PC-3, were used for the study. Cell proliferation, cell cycle, cell apoptosis and caspase 3/7 activity were determined in the presence or absence of NSC. The levels of Bax and Bak, and the release of cytochrome c from mitochondria were analyzed by Western blot. RESULTS: Treatment with NSC at nanomolar concentrations produced a time- and dose-dependent decrease in viable cell numbers of multiple prostate cancer cells. In DU-145 cells, NSC produced a time-and dose-dependent induction of cell apoptosis and cell cycle arrest as evidenced by cell morphological changes, increases in S-phase and sub-G1 cell fractions, an elevation of caspase 3/7 activity, DNA fragmentation and apoptotic cells. NSC increased the levels of apoptotic proteins, Bax and Bak, and induced a release of cytochrome c from mitochondria to cytosol in DU-145 cells. Co-administration of Z-VAD-FMK, a pan-caspase inhibitor, blocked NSC-induced caspase 3/7 activity and cell apoptosis without affecting NSC-induced cell cycle arrest. In contrast, co-administration of a PKCdelta inhibitor, rottlerin, had no significant effect on NSC induction of caspase activity, and slightly potentiated NSC-induced cell death. Furthermore, like camptothecin, a mutation of topoisomerase 1 that prevents the binding of camptothecin to the enzyme completely abolished the NSC effect in DU-145 cells. CONCLUSION: The data obtained suggest that NSC is able to decrease cell growth, induce cell apoptosis and cause growth arrest in prostatic tumor cells, which may involve an interaction with topoisomerase 1 and an activation of mitochondrial apoptotic pathway.

17alpha-estradiol inhibits LAPC-4 prostatic tumor cell proliferation in cell cultures and tumor growth in xenograft animals.

BACKGROUND: Blockade of androgen activity is a major effective therapy for advanced prostate cancer. Estrogen analogs have been used for prostate cancer therapy for years presumably by inhibiting testosterone biosyntheses, but with considerable adverse events due to their classic estrogenic activity. With the discovery of the estrogen receptor (ER) beta and its presence in prostate tumor cells, evaluation of estrogen analogs with less classic estrogenic activity in prostate cancer therapy is emerging. METHODS: The effects of 17alpha-estradiol (alphaE2), a stereo-isomer of 17beta-estradiol (betaE2), on dihydrotestosterone (DHT)-induced cell growth and gene expressions were examined in androgen-dependent LAPC-4 prostatic tumor cells and in LAPC-4 xenograft animals, and compared to those of betaE2. RESULTS: Both alphaE2 and betaE2 attenuated DHT induction of PSA gene expression, cell proliferation, and cell growth in cultured LAPC-4 cells. The inhibition of cell proliferation was associated with a blockade of DHT-induced cyclin A and cyclin D1 expression by alphaE2 and betaE2. In LAPC-4 xenograft mice, alphaE2 significantly inhibited tumor growth without altering the plasma testosterone level, while betaE2 failed to inhibit tumor growth even though it significantly inhibited PSA gene expression. CONCLUSION: alphaE2 is an effective agent for inhibition of DHT-induced PSA, cyclin A, cyclin D1 gene expression, and cell proliferation in LAPC-4 cells, and tumor growth in LAPC-4 xenograft mice.

IFN-alpha beta secreted during infection is necessary but not sufficient for negative feedback regulation of IFN-alpha beta signaling by Mycobacterium tuberculosis.

IFN-alphabeta functions in the transition from innate to adaptive immunity and may impinge on the interaction of Mycobacterium tuberculosis with its host. Infection by M. tuberculosis causes IFN-alphabeta secretion and down-regulation of IFN-alphabeta signaling in human APC and the human monocytic cell line THP-1, which provides a model for these studies. Neutralization of secreted IFN-alphabeta prevents inhibition of IFN-alpha signaling during infection, but several lines of evidence distinguish inhibition due to infection from a negative feedback response to only IFN-alphabeta. First, greater inhibition of IFN-alpha-stimulated STAT-1 tyrosine phosphorylation occurs 3 days postinfection than 1 or 3 days after IFN-alphabeta pretreatment. Second, LPS also induces IFN-alphabeta secretion and causes IFN-alphabeta-dependent down-regulation of IFN-alpha signaling, yet the inhibition differs from that caused by infection. Third, IFN-alpha signaling is inhibited when cells are grown in conditioned medium collected from infected cells 1 day postinfection, but not if it is collected 3 days postinfection. Because IFN-alphabeta is stable, the results with conditioned medium suggest the involvement of an additional, labile substance during infection. Further characterizing signaling for effects of infection, we found that cell surface IFN-alphabeta receptor is not reduced by infection, but that infection increases association of protein tyrosine phosphatase 1c with the receptor and with tyrosine kinase 2. Concomitantly, IFN-alpha stimulation of tyrosine kinase 2 tyrosine phosphorylation and kinase activity decreases in infected cells. Moreover, infection reduces the abundance of JAK-1 and tyrosine-phosphorylated JAK-1. Thus, the distinctive down-regulation of IFN-alpha signaling by M. tuberculosis occurs together with a previously undescribed combination of inhibitory intracellular events.

Posttranscriptional inhibition of gene expression by Mycobacterium tuberculosis offsets transcriptional synergism with IFN-gamma and posttranscriptional up-regulation by IFN-gamma.

Host defense against Mycobacterium tuberculosis requires the cytokine IFN-gamma and IFN regulatory factor 1 (IRF-1), a transcription factor that is induced to high levels by IFN-gamma. Therefore, we chose to study regulation of IRF-1 expression as a model for effects of M. tuberculosis on response to IFN-gamma. We found that IRF-1 mRNA abundance increased far more than transcription rate in human monocytic THP-1 cells stimulated by IFN-gamma, but less than transcription rate in cells infected by M. tuberculosis. IFN-gamma stimulation of infected cells caused a synergistic increase in IRF-1 transcription, yet IRF-1 mRNA abundance was similar in uninfected and infected cells stimulated by IFN-gamma, as was the IRF-1 protein level. Comparable infection by Mycobacterium bovis bacillus Calmette-Guérin failed to induce IRF-1 expression and had no effect on the response to IFN-gamma. We also examined the kinetics of transcription, the mRNA t(1/2), and the distribution of IRF-1 transcripts among total nuclear RNA, poly(A) nuclear RNA, and poly(A) cytoplasmic RNA pools in cells that were infected by M. tuberculosis and/or stimulated by IFN-gamma. Our data suggest that infection by M. tuberculosis inhibits RNA export from the nucleus. Moreover, the results indicate that regulated entry of nascent transcripts into the pool of total nuclear RNA affects IRF-1 expression and that this process is stimulated by IFN-gamma and inhibited by M. tuberculosis. The ability of infection by M. tuberculosis to limit the increase in IRF-1 mRNA expression that typically follows transcriptional synergism may contribute to the pathogenicity of M. tuberculosis.

Inhibition of response to alpha interferon by Mycobacterium tuberculosis.

We previously reported that infection by Mycobacterium tuberculosis, the causative agent of tuberculosis, leads to secretion of alpha/beta interferon (IFN-alpha/beta). While IFN-alpha/beta ordinarily stimulates formation of signal transducer and stimulator of transcription-1 (STAT-1) homodimers and IFN-stimulated gene factor-3 (ISGF-3), only ISGF-3 is found in infected human monocytes and macrophages. We have now investigated the basis for this unusual profile of transcription factor activation and its consequences for regulation of transcription, as well as the impact of infection on response to IFN-alpha. After infection, IFN-alpha stimulation of STAT-1 homodimers is inhibited in monocytes and macrophages, while stimulation of ISGF-3 increases in monocytes but tends to decline in macrophages. Effects of infection on the abundance of ISGF-3 subunits, STAT-1, STAT-2, and interferon regulatory factor 9, and on tyrosine phosphorylation of STAT-1 and STAT-2 explain the observed changes in DNA-binding activity, which correlate with increased or inhibited transcription of genes regulated by ISGF-3 and STAT-1. Infection by Mycobacterium bovis BCG does not inhibit IFN-alpha-stimulated tyrosine phosphorylation of STAT-1, formation of homodimers, or transcription of genes regulated by STAT-1 homodimers, suggesting that inhibition of the response to IFN-alpha/beta by M. tuberculosis is an aspect of pathogenicity. Thus, this well-known feature of infection by pathogenic viruses may also be a strategy employed by pathogenic bacteria.

Host defense responses to infection by Mycobacterium tuberculosis. Induction of IRF-1 and a serine protease inhibitor.

Alveolar macrophages and newly recruited monocytes are targets of infection by Mycobacterium tuberculosis. Therefore, we examined the expression of interferon regulatory factor 1 (IRF-1), which plays an important role in host defense against M. tuberculosis, in undifferentiated and differentiated cells. Infection induced IRF-1 in both. IRF-1 from undifferentiated, uninfected monocytic cell lines was modified during extraction to produce specific species that were apparently smaller than intact IRF-1. After infection by M. tuberculosis or differentiation, intact IRF-1 was recovered. Subcellular fractions were assayed for the ability to modify IRF-1 or inhibit its modification. A serine protease on the cytoplasmic surface of an organelle or vesicle in the "lysosomal/mitochondrial" fraction from undifferentiated cells was responsible for the modification of IRF-1. Thus, the simplest explanation of the modification is cleavage of IRF-1 by the serine protease. Recovery of intact IRF-1 correlated with induction of a serine protease inhibitor that was able to significantly reduce the modification of IRF-1. The inhibitor was present in the cytoplasm of M. tuberculosis-infected or -differentiated cells. It is likely that induction of both IRF-1 and the serine protease inhibitor in response to infection by M. tuberculosis represent host defense mechanisms.