Substitution of one calcium-binding amino acid strengthens substrate binding in a thermophilic alginate lyase.

Ligand binding is sensitive to temperatures since noncovalent bonds between the binding site and ligand could be broken by heat. How metal ion-binding amino acids in alginate lyase evolve to achieve tight substrate binding in a hostile environment remains unknown. An endolytic alginate lyase AlgAT0 specifically cleaved the M-G glycosidic bond and released disaccharides as the main end product. Four conserved calcium-binding sites were predicted and the supplement of Ca2+ led to enhanced substrate binding and protein stability. Among the four conserved calcium-binding sites, one substitution of aspartate for glutamate in AlgAT0 was proved to stimulate Ca2+ affinity. This study suggested that substrate affinity of polysaccharide lyases could be improved by tight binding to Ca2+ via one amino acid substitution.

Direct bioconversion of brown algae into ethanol by thermophilic bacterium Defluviitalea phaphyphila.

BACKGROUND: Brown algae are promising feedstocks for biofuel production with inherent advantages of no structural lignin, high growth rate, and no competition for land and fresh water. However, it is difficult for one microorganism to convert all components of brown algae with different oxidoreduction potentials to ethanol. Defluviitalea phaphyphila Alg1 is the first characterized thermophilic bacterium capable of direct utilization of brown algae. RESULTS: Defluviitalea phaphyphila Alg1 can simultaneously utilize mannitol, glucose, and alginate to produce ethanol, and high ethanol yields of 0.47 g/g-mannitol, 0.44 g/g-glucose, and 0.3 g/g-alginate were obtained. A rational redox balance system under obligate anaerobic condition in fermenting brown algae was revealed in D. phaphyphila Alg1 through genome and redox analysis. The excess reducing equivalents produced from mannitol metabolism were equilibrated by oxidizing forces from alginate assimilation. Furthermore, D. phaphyphila Alg1 can directly utilize unpretreated kelp powder, and 10 g/L of ethanol was accumulated within 72 h with an ethanol yield of 0.25 g/g-kelp. Microscopic observation further demonstrated the deconstruction process of brown algae cell by D. phaphyphila Alg1. CONCLUSIONS: The integrated biomass deconstruction system of D. phaphyphila Alg1, as well as its high ethanol yield, provided us an excellent alternative for brown algae bioconversion at elevated temperature.

Characterization of a thermostable endo-1,3(4)-ß-glucanase from Caldicellulosiruptor sp. strain F32 and its application for yeast lysis.

ß-1,3-Glucans, important structural components of cell wall or nutritional components of the endosperm, are extensively found in bacteria, fungi, yeast, algae, and plants. The structural complexity of ß-1,3-glucans implies that the enzymatic depolymerization of polysaccharides needs combined activities of distinct enzymes. In this study, Lam16A-GH, the catalytic module of a putative glycoside hydrolase (GH) family 16 laminarinase/lichenase from thermophilic bacterium Caldicellulosiruptor sp. F32, was purified and characterized through heterologous expression in Escherichia coli. Lam16A-GH can hydrolyze both ß-1,3-glucan (laminarin) and ß-1,3-1,4-glucan (barley ß-glucan) revealed by analysis of the products of polysaccharide degradation using thin-layer chromatography (TLC). The time required for the loss of 50 % of its activity is 45 h under the optimal condition of 75 °C and pH 6.5. Oligosaccharides degradation assay indicated that Lam16A-GH can catalyze endo-hydrolysis of the ß-1,4 glycosidic linkage adjacent to a 3-O-substituted glucosyl residue in the mixed linked ß-glucans, as well as the ß-1,3 linkage. The survival rate of Saccharomyces cerevisiae cells depends on the addition of Lam16A-GH, and the cytoplasm protein was released from the apparently deconstructed yeast cells. These results indicate that the bi-functional thermostable Lam16A-GH exhibits unique enzymatic properties and potential for yeast lysis.

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae.

Brown algae are one of the largest groups of oceanic primary producers for CO2 removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2(T). Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-d-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing D-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD(+), and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats.

Brassicibacter thermophilus sp. nov., a thermophilic bacterium isolated from coastal sediment.

A novel thermophilic, obligately anaerobic bacterium, strain Cel2f(T), was isolated from a cellulolytic community enriched from coastal marine sediment. Cells were Gram-stain-negative, non-motile, non-spore-forming and rod-shaped. Optimal growth temperature and pH of strain Cel2f(T) were 55 °C and pH 7.0, respectively. NaCl was essential for the growth of strain Cel2f(T) and the strain showed enhanced growth in the presence of sea salt; the optimum sea salt concentration for growth was 7% (w/v). Thiosulfate, sulfate and sulfite were potential electron acceptors. The major fatty acids of strain Cel2f(T) were iso-C15 : 0, C16 : 0, and C18 : 0. Polar lipid analysis indicated the presence of phosphatidylethanolamine and phosphatidylglycerol. Strain Cel2f(T) contained menaquinone MK-7 as the isoprenoid quinone, and the DNA G+C content was 31.3 mol%. Phylogenetic analysis revealed that the nearest relative of strain Cel2f(T) was Brassicibacter mesophilus BM(T) with 93.8% 16S rRNA gene sequence similarity. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, strain Cel2f(T) represents a novel species of genus Brassicibacter, for which the name Brassicibacter thermophilus sp. nov. is proposed. The type strain is Cel2f(T) ( = JCM 30480(T) = CGMCC 1.5200(T)).

Dysgonomonas macrotermitis sp. nov., isolated from the hindgut of a fungus-growing termite.

A Gram-stain-negative, facultatively anaerobic, non-motile and coccoid- to short-rod-shaped bacterium, designated strain Dys-CH1(T), was isolated from the hindgut of a fungus-growing termite Macrotermes barneyi. The optimal pH and cultivation temperature of strain Dys-CH1(T) were pH 7.2-7.6 and 35-37 °C, respectively. Sequence analysis of 16S rRNA gene showed that Dys-CH1(T) shared 94.6 % and 90.9 % similarity with Dysgonomonas capnocytophagoides JCM 16697(T) and Dysgonomonas gadei CCUG 42882(T), respectively. Strain Dys-CH1(T) was found to be different from other species of the genus Dysgonomonas with validly published names with respect to taxonomically important traits, including habitat, biochemical tests, DNA G+C content, bile resistance, fatty-acid composition and susceptibility to antimicrobial agents. On the basis of these characteristics, strain Dys-CH1(T) represents a novel species of the genus Dysgonomonas for which the name Dysgonomonas macrotermitis sp. nov. is proposed. The type strain is Dys-CH1(T) ( = JCM 19375(T) = DSM 27370(T)).

Down-regulation of CD74 inhibits growth and invasion in clear cell renal cell carcinoma through HIF-1α pathway.

OBJECTIVES: To investigate the expression and function of CD74 in normal renal tissue and clear cell-renal cell carcinoma (ccRCC), as well as related renal tubule epithelial lines. We also analyzed the association between clinicopathological characteristics of ccRCCs and the expression levels of CD74. METHODS: Immunostaining of CD74 was performed in 107 patients' renal tissue and cell lines. We evaluated the association between clinicopathological characteristics of ccRCC and CD74 levels using image analysis. CD74 expression levels were also analyzed by Western blot. Lentivirus-mediated CD74 knockdown inhibited the growth and invasion, of ccRCC cell lines 786-O in vitro and in vivo. Cell proliferation, apoptosis, and invasion as well as HIF-1α pathway-related proteins, were estimated by Western blot. All experiments were repeated at least 3 times. RESULTS: Immunostaining and image analysis showed strong immunoreactions of CD74 in all patients' ccRCC tissue and malignant cell lines, while CD74 expression levels were associated with tumor grade (P = 0.013). Western blot indicated that ccRCC tissue and malignant cell lines expressed higher levels of CD74 and hypoxia inducible factor 1α (HIF-1α) than adjacent normal renal tissue and normal cell HK-2. Vitro and vivo tests demonstrated that lentivirus-mediated CD74 knockdown inhibited the proliferation of ccRCC cell lines, induced G1/S arrest and apoptosis, and inhibited invasion. Inhibition of CD74 resulted in down-regulation of HIF-1α pathway proteins. CONCLUSIONS: CD74 was overexpressed in human ccRCCs and associated with tumor grade, and inhibition of CD74 produced ccRCC proliferation arrest, induced apoptosis, and inhibited invasion, which impinged on HIF-1α pathway-related proteins. It might represent a potential therapeutic target for ccRCC.

Small interfering RNA targeting HMGN5 induces apoptosis via modulation of a mitochondrial pathway and Bcl-2 family proteins in prostate cancer cells.

We investigated the importance of HMGN5, a nuclear protein that binds to nucleosomes, unfolds chromatin, and affects transcription, in the LNCaP prostate cancer cell line. We also examined the molecular mechanisms that promote apoptosis of LNCaP cells after infection with small interfering RNA (siRNA) targeting HMGN5 (siRNA-HMGN5). The androgen-dependent LNCaP human prostate cancer cells were infected with siRNA-HMGN5. Apoptosis was detected using the Annexin V-PE/7-AAD double staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. Mitochondrial membrane potential was measured by JC-1 staining. HMGN5 and GAPDH mRNA expression were determined using real-time PCR. Bcl-2 and other apoptosis-related protein levels were determined by Western blot analysis. Caspase activity was measured by cleavage of the caspase substrate. Infection with siRNA targeting HMGN5 efficiently and specifically reduced the HMGN5 expression in LNCaP cells. The downregulation of HMGN5 induced remarkable apoptosis of LNCaP cells and resulted in the reduction of mitochondrial membrane potential. The induction of cell apoptosis was accompanied by the upregulation of Bax, the Bax/Bcl-2 ratio and the activation of caspase3. The HMGN5-targeted siRNA was effective in downregulating the expression of HMGN5 in androgen-dependent prostate cancer cells and inducing cell apoptosis via the regulation of a caspase-related mitochondrial pathway and Bcl-2 family proteins. This study suggests that HMGN5 may be a potential molecular target with therapeutic relevance for the treatment of prostate cancer.

Hyperthermia synergizes with tissue factor knockdown to suppress the growth and hepatic metastasis of colorectal cancer in orthotopic tumor model.

BACKGROUND: Tissue factor (TF) is a significant risk factor for tumor growth and hepatic metastasis in patients with colorectal cancer (CRC). This study aimed to investigate whether hyperthermia has synergistic anti-tumor effects with TF knockdown in suppressing CRC progression and metastasis in vitro and in vivo. METHODS: Human colorectal cancer LOVO cells were treated by hyperthermia at 44°C for 2 hr or/and TF siRNA. Then the cells were subjected to colony formation assay. Apoptosis was analyzed by flow cytometry, confocal microscopy, and transmission electron microscopy. The cell migration and invasion abilities were analyzed by wound healing and matrigel assay. In addition, orthotopic nude mice model of CRC was established. RESULTS: Hyperthermia synergized with TF knockdown to reduce colony formation ability, induce apoptosis, and suppress the migration and invasion of LOVO cells in vitro. Moreover, hyperthermia in combination with TF depletion inhibited the growth and hepatic metastasis of CRC in orthotopic nude mice model. Mechanistically, the synergistic effects were at least partly mediated by inducing JNK mediated apoptosis and suppressing matrix metalloproteinases (MMPs) mediated invasion. CONCLUSIONS: Hyperthermia in combination with TF-targeted therapy could be a potential approach for CRC treatment.

Knockdown of the nucleosome binding protein 1 inhibits the growth and invasion of clear cell renal cell carcinoma cells in vitro and in vivo.

BACKGROUND: The nucleosome binding protein 1 (HMGN5/NSBP1) is a member of the HMGN protein family and is highly expressed in several kinds of cancer. Nevertheless, the role of NSBP1 in clear cell renal cell carcinoma (ccRCC) remains unclear. This study aimed to confirm the oncogenic role of NSBP1 in ccRCC using in vitro and in vivo models and explore the mechanism by which NSBP1 contributes to ccRCC tumorigenesis. METHODS: NSBP1 expression was detected in renal tissues from 152 ccRCC patients by immunohistochemistry, and examined in ccRCC cell lines by RT-PCR and Western blot analysis. ccRCC cells were transfected by NSBP1 RNAi and cell viability, apoptosis and invasion were detected by cell vitality test, flow cytometry and transwell assay in vitro. Xenograft in nude mice was also employed to examine the tumorigenesis of ccRCC cells depleted of NSBP1. RESULTS: Immunohistostaining showed strong immunoreactivity of NSBP1 in all ccRCC tissues and NSBP1 expression level was associated with tumor grade (p = 0.04). NSBP1 expression at mRNA and protein levels was high in ccRCC cell lines. Knockdown of NSBP1 induced cell cycle arrest and apoptosis, and inhibited invasion in 786-O cells. Western blot analysis demonstrated increased expression of Bax and decreased expression of Bcl-2, CyclinB1, VEGF, VEGFR-2, MMP-2, MMP-9, c-fos and c-jun in 786-O cells depleted of NSBP1. In vivo study further showed that knockdown of NSBP1 affected the tumorigenesis of ccRCC cells in nude mice. CONCLUSIONS: NSBP1 plays oncogenic role in ccRCCs by promoting cell proliferation and invasion, and could be exploited as a target for ccRCC treatment.

[Clinical study of diethylstilbestrol in hormone refractory prostate cancer].

OBJECTIVE: To summarize the clinical outcome and adverse events of estrogen therapy for hormone refractory prostate cancer. METHODS: A total of 32 patients with hormone refractory prostate cancer received diethylstilbestrol (DES) 2 mg daily at our institute. The data of PSA (prostate-specific antigen) change, time to progression, overall survival rate, disease-specific survival rate and adverse events were collected and analyzed. RESULTS: The data of 29 patients were complete. The mean duration of DES dosing was 8.6 ± 0.9 months. Among them, 8 (27.5%) patients achieved a PSA response with a 50% decrement of PSA or more. Seven (24.1%) patients had a stable level of PSA (50% < PSA < 125%) while 14 of 29 (48.3%) maintained a PSA progression with a 25% increment of PSA or more. The overall median time to progression was 4 (1 - 12) months. And the median time to progression was 6 (5-12) months in the PSA response group. The overall survival rate was 48.3% and disease-specific survival rate 55.2%. The main adverse events were gynecomastia (10/29, 34.5%) and deep vein thrombosis (3/29, 10.3%). CONCLUSION: When used for the treatment of hormone refractory prostate cancer, diethylstilbestrol at a daily dose of 2 mg can achieve a PSA response in 27.5% patients and a PSA stability in 24.1% patients. And the median time to progression is 4 months. Estrogen is efficacious for the patients with hormone refractory prostate cancer.