Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line <Emphasis Type="Italic">in vitro</Emphasis>

Induction of p53 gene expression in cancer cells can lead to both cell cycle arrest and apoptosis. To clarify whether the level of p53 expression determines the apoptotic response of hepatocellullar carcinoma (HCC) cells, we assessed the effect of various levels of expression of p53 gene on a p53-deficient HCC cell line, Hep3B, utilizing a doxycycline (Dox)-regulated inducible p53 expression system. Our results showed that apoptosis was induced in HCC cells with high levels of p53 expression. However, lower level of p53 expression induced only cell cycle arrest but not apoptosis. Bax expression was up-regulated following high levels of p53 expression, while bcl-2 expression was not altered by the level of p53 expression. Moreover, p21 expression was observed in both high and low expression of p53. These results suggest the level of p53 expression could determine if the HCC cells would go into cell cycle arrest or apoptosis. Bax may participate, at least in part, in inducing p53-dependent apoptosis and the induction of p21 alone was able to cause cell cycle arrest but not apoptosis.     

Diversity of Archaea in marine sediments from Skan Bay, Alaska, including cultivated methanogens, and description of Methanogenium boonei sp. nov

Methanogenesis in cold marine sediments is a globally important process leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. We employed a multidisciplinary approach that combined culture-dependent and -independent analyses with geochemical measurements in the sediments of Skan Bay, Alaska (53 degrees N, 167 degrees W), to investigate methanogenesis there. Cultivation-independent analyses of the archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present at Skan Bay and that methanogens constituted a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO(2)-reducing methanogens: AK-3, AK7, and AK-8. The phylogenetic positions and physiological characteristics of these strains are described. We propose a new species, Methanogenium boonei, with strain AK-7 as the type strain.     

Distinct NKT cell subsets are induced by different Chlamydia species leading to differential adaptive immunity and host resistance to the infections

We investigated the role of NKT cells in immunity to Chlamydia pneumoniae and Chlamydia muridarum infections using a combination of knockout mice and specific cellular activation approaches. The NKT-deficient mice showed exacerbated susceptibility to C. pneumoniae infection, but more resistance to C. muridarum infection. Activation of NKT reduced C. pneumoniae in vivo growth, but enhanced C. muridarum infection. Cellular analysis of invariant NKT cells revealed distinct cytokine patterns following C. pneumoniae and C. muridarum infections, i.e., predominant IFN-gamma in the former, while predominant IL-4 in the latter. The cytokine patterns of CD4(+) and CD8(+) T cells matched those of NKT cells. Our data provide in vivo evidence for a functionally diverse role of NKT cells in immune response to two intracellular bacterial pathogens. These results suggest that distinct NKT subsets are induced by even biologically closely related pathogens, thus leading to differential adaptive immune response and infection outcomes.     

Synthesis and identification of novel oxa-steroids as progesterone receptor antagonists

A novel series of oxa-steroids 6 derived from (8S, 13S, 14R)-7-oxa-estra-4,9-diene-3,17-dione 1 have been synthesized and identified as potent and selective progesterone receptor antagonists. These novel oxa-steroids showed similar potency to mifepristone. Preliminary SAR study resulted in the most potent 17-phenylethynyl oxa-steroid 6i wih an IC(50) of 1.4nM. In contrast to the equipotent mifepristone toward the progesterone receptor (PR) and glucocorticoid receptor (GR), compound 6i had over 200-fold selectivity for PR over GR.     

Synthesis of 3-(1H-benzimidazol-2-yl)-5-isoquinolin-4-ylpyrazolo[1,2-b]pyridine, a potent cyclin dependent kinase 1 (CDK1) inhibitor

The novel compound 3-(1H-benzimidazol-2-yl)-5-isoquinolin-4-ylpyrazolo[1,2-b]pyridine was discovered to be a potent CDK1 inhibitor. Described here is the chemistry for its synthesis, including Pd(II) catalyzed Stille coupling reaction and sulfur(0) induced benzimidazole formation. Its effects on VEGFR-2 kinase activity and tumour cell proliferation are also described.     

Mitochondrial superoxide production in skeletal muscle fibers of the rat and decreased fiber excitability

Mammalian skeletal muscles generate marked amounts of superoxide (O₂·^–) at 37°C, but it is not well understood which is the main source of O₂·^– production in the muscle fibers and how this interferes with muscle function. To answer these questions, O₂·^– production and twitch force responses were measured at 37°C in mechanically skinned muscle fibers of rat extensor digitorum longus (EDL) muscle. In mechanically skinned fibers, the sarcolemma is removed avoiding potential sources of O₂·^– production that are not intrinsically part of the muscle fibers, such as nerve terminals, blood cells, capillaries and other blood vessels in the whole muscle. O₂·^– production was also measured in split single EDL muscle fibers, where part of the sarcolemma remained attached, and small bundles of intact isolated EDL muscle fibers at rest, in the presence and absence of modifiers of mitochondrial function. The results lead to the conclusion that mitochondrial production of O₂·^– accounts for most of the O₂·^– measured intracellularly or extracellularly in skeletal muscle fibers at rest and at 37°C. Muscle fiber excitability at 37°C was greatly improved in the presence of a membrane permeant O₂·^– dismutase mimetic (Tempol), demonstrating a direct link between O₂·^– production in the mitochondria and muscle fiber performance. This implicates mitochondrial O₂·^– production in the down-regulation of skeletal muscle function, thus providing a feedback pathway for communication between mitochondria and plasma membranes that is not directly related to the main function of mitochondria as the power plant of the mammalian muscle cell. excitation-contraction coupling; mechanically skinned fiber; physiological temperature     

Identifying the predator complex of <Emphasis Type="Italic">Homalodisca vitripennis</Emphasis> (Hemiptera: Cicadellidae): a comparative study of the efficacy of an ELISA and PCR gut content assay

A growing number of ecologists are using molecular gut content assays to qualitatively measure predation. The two most popular gut content assays are immunoassays employing pest-specific monoclonal antibodies (mAb) and polymerase chain reaction (PCR) assays employing pest-specific DNA. Here, we present results from the first study to simultaneously use both methods to identify predators of the glassy winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). A total of 1,229 arthropod predators, representing 30 taxa, were collected from urban landscapes in central California and assayed first by means of enzyme-linked immunosorbent assay (ELISA) using a GWSS egg-specific mAb and then by PCR using a GWSS-specific DNA marker that amplifies a 197-base pair fragment of its cytochrome oxidase gene (subunit I). The gut content analyses revealed that GWSS remains were present in 15.5% of the predators examined, with 18% of the spiders and 11% of the insect predators testing positive. Common spider predators included members of the Salticidae, Clubionidae, Anyphaenidae, Miturgidae, and Corinnidae families. Common insect predators included lacewings (Neuroptera: Chrysopidae), praying mantis (Mantodea: Mantidae), ants (Hymenoptera: Formicidae), assassin bugs (Hemiptera: Reduviidae), and damsel bugs (Hemiptera: Nabidae). Comparison of the two assays indicated that they were not equally effective at detecting GWSS remains in predator guts. The advantages of combining the attributes of both types of assays to more precisely assess field predation and the pros and cons of each assay for mass-screening predators are discussed.     

SelSA,selenium analogs of SAHA as potent histone deacetylase inhibitors

Cancer treatment and therapy has moved from conventional chemotherapeutics to more mechanism-based targeted approach. Disturbances in the balance of histone acetyltransferase (HAT) and deacetylase (HDAC) leads to a change in cell morphology, cell cycle, differentiation, and carcinogenesis. In particular, HDAC plays an important role in carcinogenesis and therefore it has been a target for cancer therapy. Structurally diverse group of HDAC inhibitors are known. The broadest class of HDAC inhibitor belongs to hydroxamic acid derivatives that have been shown to inhibit both class I and II HDACs. Suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA), which chelate the zinc ions, fall into this group. In particular, SAHA, second generation HDAC inhibitor, is in several cancer clinical trials including solid tumors and hematological malignancy, advanced refractory leukemia, metastatic head and neck cancers, and advanced cancers. To our knowledge, selenium-containing HDAC inhibitors are not reported in the literature. In order to find novel HDAC inhibitors, two selenium based-compounds modeled after SAHA were synthesized. We have compared two selenium-containing compounds; namely, SelSA-1 and SelSA-2 for their inhibitory HDAC activities against SAHA. Both, SelSA-1 and SelSA-2 were potent HDAC inhibitors; SelSA-2 having IC50 values of 8.9 nM whereas SAHA showed HDAC IC₅₀ values of 196 nM. These results provided novel selenium-containing potent HDAC inhibitors.