Biochemical Characteristics of Microbial Enzymes and Their Significance from Industrial Perspectives.

Microbes are ubiquitously distributed in nature and are a critical part of the holobiont fitness. They are perceived as the most potential biochemical reservoir of inordinately diverse and multi-functional enzymes. The robust nature of the microbial enzymes with thermostability, pH stability and multi-functionality make them potential candidates for the efficient biotechnological processes under diverse physio-chemical conditions. The need for sustainable solutions to various environmental challenges has further surged the demand for industrial enzymes. Fueled by the recent advent of recombinant DNA technology, genetic engineering, and high-throughput sequencing and omics techniques, numerous microbial enzymes have been developed and further exploited for various industrial and therapeutic applications. Most of the hydrolytic enzymes (protease being the dominant hydrolytic enzyme) have broad range of industrial uses such as food and feed processing, polymer synthesis, production of pharmaceuticals, manufactures of detergents, paper and textiles, and bio-fuel refinery. In this review article, after a short overview of microbial enzymes, an approach has been made to highlight and discuss their potential relevance in biotechnological applications and industrial bio-processes, significant biochemical characteristics of the microbial enzymes, and various tools that are revitalizing the novel enzymes discovery.

Pharmacological Properties of Chromobacterium violaceum Violacein at the Human Serotonin 2C Receptor.

The monoamine neurotransmitter serotonin (5-HT) plays a role in many physiological responses by interacting with various receptor subtypes. The 5-HT2C receptor subtype is a 7-transmembrane, G protein-coupled receptor (GPCR) that is involved in neuronal excitability, spatial learning, mood, and appetite. The microorganism Chromobacterium violaceum produces a purple pigment, violacein, which can be extracted and purified. Violacein has antibiotic, antileishmanial, antifungal and antitumoral properties in various cancer cell lines. Violacein is derived from the amino acid tryptophan as is 5-HT and therefore, the two have similar chemical structures. However, no one has reported the activity of violacein at 5-HT receptors. Therefore the Fentress lab decided to investigate whether or not violacein had an effect on 5-HT2C receptor trafficking. Human Embryonic Kidney (HEK) 293 cells expressing fluorescently-tagged 5-HT2C receptor were treated with 5-HT, violacein, water or vehicle and then cells were fixed and visualized with fluorescent microscopy. Violacein treatment did not cause receptor internalization. Recent studies suggest that the 5-HT2C receptor can activate the JAK/STAT pathway. To see if violacein can modulate this pathway, HEK 293 cells expressing 5-HT2C receptor were treated with either 5-HT, violacein, or pretreated with violacein followed by incubation with 5-HT. Phosphorylation states of JAK2 and STAT3 were examined by immunoblotting. Results determined that 5-HT2C receptor activation had no effect on JAK2 phosphorylation and that violacein blocked STAT3 phosphorylation. Primary radioligand binding determined that violacein has a low affinity for 5-HT2C receptor but has a higher affinity for adrenergic receptors. Future studies will examine G protein-coupling by measuring phosphoinositide hydrolysis and cAMP assay to investigate adrenergic pathways.

Potential anticancer effect of prostratin through SIK3 inhibition.

Prostratin, a phorbol ester natural plant compound, has been demonstrated to exert an anti-retroviral effect through activation of latent cluster of differentiation (CD)4+T lymphocytes and inhibition of viral entry into the cell through downregulation of chemokine receptor type 4 (CXCR4) expression. However, the potential effect of prostratin on cancer is yet to be defined. As CXCR4 is well known to induce cancer migration, it was hypothesized that prostratin induces an anti-cancer effect through inhibition of CXCR4 expression. The authors previously demonstrated that high stimulating conditions (sub-minimal IL-17, 0.1 ng/ml, synergized with high salt, Δ0.05 M NaCl) promote breast cancer cell proliferation and CXCR4 expression through upregulation of salt-inducible kinase (SIK)-3. The present study demonstrated that prostratin selectively exerted increased cytotoxicity (IC50 of 7 µM) when breast cancer cells were cultured in high stimulating conditions, compared with regular basal culture conditions (IC50 of 35 µM). Furthermore, the cytotoxic potential of prostratin was increased seven-fold in the four breast cancer cell lines (MCF-7, MDA-MB-231, BT-20 and AU-565) compared with the non-malignant MCF10A breast epithelial cell line. This suggested that prostratin specifically targets cancer cells over normal cells. Mechanistic studies revealed that prostratin inhibited CXCR4 expression in breast cancer cells through downregulation of SIK3 expression. Overall, the data suggest that prostratin is a novel drug target for the pro-oncogenic factor SIK3. These studies could form a basis for further research to evaluate the anticancer effect of prostratin in a combinatorial chemotherapeutic regimen.

Effects of precipitation changes on switchgrass photosynthesis, growth, and biomass: A mesocosm experiment.

Climate changes, including chronic changes in precipitation amounts, will influence plant physiology and growth. However, such precipitation effects on switchgrass, a major bioenergy crop, have not been well investigated. We conducted a two-year precipitation simulation experiment using large pots (95 L) in an environmentally controlled greenhouse in Nashville, TN. Five precipitation treatments (ambient precipitation, and -50%, -33%, +33%, and +50% of ambient) were applied in a randomized complete block design with lowland "Alamo" switchgrass plants one year after they were established from tillers. The growing season progression of leaf physiology, tiller number, height, and aboveground biomass were determined each growing season. Precipitation treatments significantly affected leaf physiology, growth, and aboveground biomass. The photosynthetic rates in the wet (+50% and +33%) treatments were significantly enhanced by 15.9% and 8.1%, respectively, than the ambient treatment. Both leaf biomass and plant height were largely increased, resulting in dramatically increases in aboveground biomass by 56.5% and 49.6% in the +50% and +33% treatments, respectively. Compared to the ambient treatment, the drought (-33% and -50%) treatments did not influence leaf physiology, but the -50% treatment significantly reduced leaf biomass by 37.8%, plant height by 16.3%, and aboveground biomass by 38.9%. This study demonstrated that while switchgrass in general is a drought tolerant grass, severe drought significantly reduces Alamo's growth and biomass, and that high precipitation stimulates its photosynthesis and growth.

Violacein induces p44/42 mitogen-activated protein kinase-mediated solid tumor cell death and inhibits tumor cell migration.

Microbial secondary metabolites have emerged as alternative novel drugs for the treatment of human cancers. Violacein, a purple pigment produced by Chromobacterium violaceum, was investigated in the present study for its anti-tumor properties in tumor cell lines. Clinically applicable concentrations of violacein were demonstrated to inhibit the proliferative capacity of tumor cell lines according to a crystal violet proliferation assay. The underlying mechanism was the promotion of apoptotic cell death, as indicated by poly(ADP ribose) polymerase cleavage and p44/42 mitogen-activated protein kinase signaling determined by western blot analysis. Collectively, this provided mechanistic evidence that violacein elicits extracellular-signal regulated kinase-induced apoptosis via the intrinsic pathway. The anti-malignant properties of violacein in the present study were further demonstrated by its inhibitory effects on brain tumor cell migration, specifically glioblastomas, one of the most invasive and therapeutically resistant neoplasms in the clinic. Additionally, solid tumors examined in the present study displayed differential cellular responses and sensitivities to violacein as observed by morphologically induced cellular changes that contributed to its anti-migratory properties. In conclusion, violacein is a novel natural product with the potential to kill several types of human tumor cell lines, as well as prevent disease recurrence by antagonizing cellular processes that contribute to metastatic invasion.

Synergistic effect of pro-inflammatory TNFα and IL-17 in periostin mediated collagen deposition: potential role in liver fibrosis.

BACKGROUND: The pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-17, have been implicated in the pathogenesis of liver fibrosis. In this study, we investigated the role of TNFα and IL-17 toward induction of profibrotic factor, periostin. METHODS: HepG2 cells were cultured and treated with inflammatory cytokines, TNFα and IL-17. Computational promoter sequence analysis of the periostin promoter was performed to define the putative binding sites for transcription factors. Transcription factors were analyzed by Western blot and Chromatin Immunoprecipitation. Periostin and transcription factor expression analysis was performed by RT-PCR, Western blot, and fluorescence microscopy. Type I collagen expression from fibroblast cultures was analyzed by Western blot and Sircol soluble collagen assay. RESULTS: Activation of HepG2 Cells with TNFα and IL-17 enhanced the expression of periostin (3.5 and 4.4 fold, respectively p<0.05) compared to untreated cells. However, combined treatment with both TNFα and IL-17 at similar concentration demonstrated a 13.3 fold increase in periostin (p<0.01), thus suggesting a synergistic role of these cytokines. Periostin promoter analysis and specific siRNA knock-down revealed that TNFα induces periostin through cJun, while IL-17 induced periostin via STAT-3 signaling mechanisms. Treatment of the supernatant from the cytokine activated HepG2 cells on fibroblast cultures induced enhanced expression of type I collagen (>9.1 fold, p<0.01), indicative of a direct fibrogenic effect of TNFα and IL-17. CONCLUSION: TNFα and IL-17 induced fibrogenesis through cJun and STAT-3 mediated expression of profibrotic biomarker, periostin. Therefore, periostin might serve as a novel biomarker in early diagnosis of liver fibrosis.

Draft Genome Sequence of New Bacillus cereus Strain tsu1.

This paper reports the draft genome sequence of new Bacillus cereus strain tsu1, isolated on an agar-cellulose plate. The draft genome sequence is 5.81 Mb, revealing 5,673 coding sequences. It contains genes for cellulose-degradation and biosynthesis pathways of polyhydroxybutyrate (PHB) and 8 rRNA genes (5S, 16S, and 23S).

Violacein inhibits matrix metalloproteinase mediated CXCR4 expression: potential anti-tumor effect in cancer invasion and metastasis.

Matrix metalloproteinases (MMP-2 and -9) play an important role in the tumor metastasis through cleavage of proinflammatory cytokines. Violacein a small molecule produced by Chromobacterium violaceum and has been implicated with anti-cancer effects. In this study we investigated the molecular basis of violacein mediated downregulation of CXCL12/CXCR4, chemokine-receptor ligand interaction. Zymography analysis demonstrated that violacein significantly inhibited the cytokine (TNFα and TGFß) mediated MMP-2 activation in MCF-7 breast cancer cell line. MMP-2 plays a critical role in the secretion of inflammatory chemokine, CXCL12, involved in cell migration and cancer metastasis. ELISA analysis demonstrated that violacein inhibited the secretion of CXCL12 from the activated MCF-7 cells. Further, we show that MMP-2/-9 act synergistically at two distinct steps towards the membrane expression of the tumor metastasis chemokine receptor, CXCR4. Violacein efficiently downregulated the CXCR4 membrane expression through MMP-9 inhibition. Taken together, these studies demonstrate a unique anti-tumor mechanism of action of violacein through reduction of CXCL12/CXCR4 interaction. These studies could offer a novel venue for violacein in cancer therapy.

Genetics and smoking behavior.

Accumulating data support the role of genetic factors in smoking initiation, progression to tobacco dependence, and smoking persistence. This review summarizes current research on the heritability of tobacco use phenotypes and genetic association studies of smoking-related behaviors. Although progress has been made in genetics research on smoking behavior, many studies have methodological limitations, including insufficient samples for detecting gene-gene and gene-environment interactions and use of less refined phenotypes. Pharmacogenetic investigations also are identifying variants in drug-metabolizing enzymes, receptors, and transporters that modify therapeutic response to smoking cessation medications; however, the field is relatively new, and most findings in this area have yet to be replicated. As this research advances, it will be important to study and address practical, economic, ethical, and social barriers to the translation of genetics research on tobacco use to clinical practice.

The clinical effectiveness of healing touch.

OBJECTIVES: (1) to determine the clinical effectiveness of Healing Touch (HT) on variables assumed to be related to health enhancement; (2) to determine whether practitioner training level moderates treatment effectiveness. DESIGN: Mixed-method repeated measures design with quasi-experimental and naturalistic approaches, paired with nomothetic and idiographic analyses. SETTING/LOCATION: Practitioner's offices or client's home. SUBJECTS: Twenty-two (22) clients who had never experienced HT. INTERVENTIONS: Three treatment conditions: no treatment (NT), HT only (standard HT care), and HT+ (Standard HT care plus music plus guided imagery). OUTCOME MEASURES: Secretory immunoglobulin A (sIgA) concentrations in saliva, self-reports of stress levels, client perceptions of health enhancement, and qualitative questionnaires about individual effects. RESULTS: Clients of practitioners with more training experienced statistically significant positive sIgA change over the HT treatment series, while clients of practitioners with less experience did not. Clients reported a statistically significant reduction of stress level after both HT conditions. Perceived enhancement of health was reported by 13 of 22 clients (59%). Themes of relaxation, connection, and enhanced awareness were identified in the qualitative analysis of the HT experience. Pain relief was reported by 6 of 11 clients (55%) experiencing pain. CONCLUSIONS: The data support the clinical effectiveness of HT in health enhancement, specifically for raising sIgA concentrations, lowering stress perceptions and relieving pain. The evidence indicates that positive responses were not exclusively as a result of placebo, that is, client beliefs, expectations, and behaviors regarding HT.

Properties of Cholesterol Dissimilation by Rhodococcus equi.

The identification of serum cholesterol as a risk factor in the development of coronary heart disease has resulted in attempts to reduce dietary cholesterol intake. As a result, the removal of cholesterol from food products has become a challenging research objective. We report here the loss of 40% of the cholesterol found in egg yolk preparations following treatment with sonicated extracts of Rhodococcus equi . Extracts of R. equi 21107 and R. equi 33706 removed 3.33% and 3.09% of the cholesterol from egg yolk per min per mg of crude enzyme protein, respectively. Incubation of fresh cream with R. equi 33706 extracts resulted in only a 2.4% reduction in cholesterol content (rate of reduction: 0.35% cholesterol per min per mg of protein). Cholesterol degradation by R. equi 33706 had an optimum temperature of 40°C and an optimum pH of 8.0, and there was no apparent requirement for divalent metal ions. Approximately 44% of enzyme activity was lost after a 60 min exposure at 60°C. Thin layer chromatographic analysis of cholesterol degradation products revealed only a few steroid-like compounds, primarily 4-cholesten-3-one and 1,4-cholestadiene-3-one.