Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: the Third of a Putative Five-Member Tungstoenzyme Family

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO₂, and H₂. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS⁻) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (K_m = 100 μM) and oxidizes a range of aldehydes. Formaldehyde (K_m = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C₅ semi- or dialdehyde, e.g., glutaric dialdehyde (K_m = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.     

Genome-wide profiling of sperm DNA methylation in relation to buffalo (Bubalus bubalis) bull fertility

The DNA methylation pattern in spermatozoa of buffalo bulls of different fertility status was investigated. Spermatozoa isolated DNA from two groups of buffalo bulls (n = 5), selected based on their artificial insemination–generated conception rate data followed by IVF efficiency, were studied for global methylation changes using a custom-designed 180 K buffalo (Bubalus bubalis) CpG island/promoter microarray. A total of 96 individual genes with another 55 genes covered under CpG islands were found differentially methylated in sperm of high-fertile and subfertile buffalo bulls. Important genes associated with biological processes, cellular components, and functions were identified to be differentially methylated in buffalo bulls with differential fertility status. The identified differentially methylated genes were found to be involved in germ cell development, spermatogenesis, capacitation, and embryonic development. The observations hint that methylation defects of sperm DNA may play a crucial role in determining the fertility of breeding bulls. This growing field of sperm epigenetics will be of great benefit in understanding the graded fertility conditions of breeding bulls in commercial livestock production system.     

Development of Satranidazole Mucoadhesive Gel for the Treatment of Periodontitis

The aim of the paper was to develop satranidazole-containing mucoadhesive gel for the treatment of periodontitis. Different mucoadhesive gels were prepared, using various gelling agents like sodium carboxymethylcellulose (SCMC), poloxamer 407, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and the mucoadhesive polymer carbopol 934P. The selected formulations (based on the mucoadhesive force) were studied for different mechanical properties, such as mucoadhesive strength, hardness, compressibility, adhesiveness, and cohesiveness through Texture Profile Analyzer. In vitro satranidazole release from the prepared formulations was also determined and compared with marketed preparation of metronidazole (Metrogyl® gel). The formulation SC30 (containing SCMC 3% w/v) showed maximum mucoadhesive strength (167.72 ± 3.76 g) and adhesiveness (−46.23 ± 0.34 N mm), with low hardness (9.81 ± 0.04 N) and compressibility (40.05 ± 0.48 N mm) and moderate cohesiveness (0.87 ± 0.01). SC30 formulation exhibited long-term release. Thus, SC30 gel was evaluated for its clinical effectiveness along with marketed metronidazole gel. At the end of the study (42 days of clinical studies), both formulations were found to significantly reduce the probing depth, plaque index, gingival index, calculus criteria, and bleeding index. However, the SC30 gel was more effective in reducing the above parameters than marketed metronidazole gel. This study confirmed the acceptability and effectiveness of satranidazole gel for treatment of periodontitis.Key words: mucoadhesive gel, periodontitis, satranidazole, texture profile analysis     

Anticancer activity of a broccoli derivative, sulforaphane, in barrett adenocarcinoma: potential use in chemoprevention and as adjuvant in chemotherapy

INTRODUCTION: The incidence of Barrett esophageal adenocarcinoma (BEAC) has been increasing at an alarming rate in western countries. In this study, we have evaluated the therapeutic potential of sulforaphane (SFN), an antioxidant derived from broccoli, in BEAC. METHODS: BEAC cells were treated with SFN, alone or in combination with chemotherapeutic, paclitaxel, or telomerase-inhibiting agents (MST-312, GRN163L), and live cell number determined at various time points. The effect on drug resistance/chemosensitivity was evaluated by rhodamine efflux assay. Apoptosis was detected by annexin V labeling and Western blot analysis of poly(ADP-ribose) polymerase cleavage. Effects on genes implicated in cell cycle and apoptosis were determined by Western blot analyses. To evaluate the efficacy in vivo, BEAC cells were injected subcutaneously in severe combined immunodeficient mice, and after the appearance of palpable tumors, mice were treated with SFN. RESULTS: SFN induced both time- and dose-dependent decline in cell survival, cell cycle arrest, and apoptosis. The treatment with SFN also suppressed the expression of multidrug resistance protein, reduced drug efflux, and increased anticancer activity of other antiproliferative agents including paclitaxel. A significant reduction in tumor volume was also observed by SFN in a subcutaneous tumor model of BEAC. Anticancer activity could be attributed to the induction of caspase 8 and p21 and down-regulation of hsp90, a molecular chaperon required for activity of several proliferation-associated proteins. CONCLUSIONS: These data indicate that a natural product with antioxidant properties from broccoli has great potential to be used in chemoprevention and treatment of BEAC.     

Murine aldo-keto reductase family 1 subfamily B: identification of AKR1B8 as an ortholog of human AKR1B10

Aldo-keto reductase family 1 member B10 (AKR1B10), over-expressed in multiple human cancers, might be implicated in cancer development and progression via detoxifying cytotoxic carbonyls and regulating fatty acid synthesis. In the present study, we investigated the ortholog of AKR1B10 in mice, an ideal modeling organism greatly contributing to human disease investigations. In the mouse, there are three aldo-keto reductase family 1 subfamily B (AKR1B) members, i.e., AKR1B3, AKR1B7, and AKR1B8. Among them, AKR1B8 has the highest similarity to human AKR1B10 in terms of amino acid sequence, computer-modeled structures, substrate spectra and specificity, and tissue distribution. More importantly, similar to human AKR1B10, mouse AKR1B8 associates with murine acetyl-CoA carboxylase-α and mediates fatty acid synthesis in colon cancer cells. Taken together, our data suggest that murine AKR1B8 is the ortholog of human AKR1B10.     

Identification of a red-emitting fluorescent ligand for in vitro visualization of human serotonin 5-HT(1A) receptors

The 5-HT(1A) receptor subtype is the most thoroughly studied serotonin receptor subtype. We report here the design, synthesis and characterization of two new fluorescent ligands for the 5-HT(1A) receptor. The new 1-arylpiperazine-based red-emitting fluorescent compound 6 displayed good binding affinity at the 5-HT(1A) receptor (K(i)=35 nM) and was able to label specifically the human 5-HT(1A) receptor stably expressed in CHO cells visualized using confocal laser scanning microscopy.     

Intra-Tumoral Heterogeneity in Metastatic Potential and Survival Signaling between Iso-Clonal HCT116 and HCT116b Human Colon Carcinoma Cell Lines

Background

Colorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS).

Materials and Methods

HCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days.

Results

Both HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death.

Conclusions

We demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer.