Determination of the plasmid size and location of d-endotoxin genes of Bacillus thuringiensis by pulse field gel electrophoresis

The genes encoding the d-endotoxins of Bacillus thuringiensis are located on plasmids ranging in size from 45 to 1000 kb. Plasmid size and variety are diagnostic features for characterizing subspecies of this aerobic spore-forming crystalliferous entomopathogen. Two of 25 B. thuringiensis isolates obtained from Middle Tennessee were characterized into subspecies on the basis of size, number, and varieties of plasmids they host using pulse field gel electrophoresis (PFGE). By using specific pulse angle (120°), field strength (5 V/cm), pulse time (26 h), and agarose concentration (1.2%) these DNA molecules were separated from other genomic DNA. The purified DNA product the agarose gel was tested for homology to 49 d-endotoxin gene using PCR.. The PFGE data for 5 of the 25 isolates collected in Tennessee showed distinct banding patterns. Two of the isolates had only 1 band whereas the others had more than 4 ranging from 45 to 1000 kb in size. Isolate 6 and 10 both yielded one 49 kb fragment that contained the cry1A gene.African Journal of Biotechnology Vol. 4 (7), pp. 580-585, 200

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

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 \u3c 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 \u3c 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 (\u3e9.1 fold, p \u3c 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

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

L-asparaginase II Production by Escherichia coli

Growth of Escherichia coli A-l under aerobic conditions in an enriched medium with a total amount of 0.2 per cent glucose was biphasic and asparaginase II activity was detected after depletion of ammonia from the growth medium in the second phase of growth. Glucose was exhausted two hours before ammonia and three hours before asparaginase II activity was detected. The concentration of 3',5'-cyclic adenosine monophosphate was found to fluctuate when the dissolved oxygen in the medium reached a low level, when glucose and ammonia were exhausted, and when the cells entered the second stationary phase of growth. Culture tube studies of the growth of E_j_ coli A-l in three per cent nutrient broth with varied concentrations of ammonium chloride and potassium nitrate gave lower specific activity of asparaginase II when this was compared to that seen in three per cent nutrient broth alone. The addition of glucose to the same medium before asparaginase II activity was detected resulted in the production of acid by E. coli A-l with cessation of growth; however, addition after L-asparaginase synthesis had started did not affect the specific activity of the enzyme. The addition of ammonium chloride suppressed L-asparaginase synthesis, but addition after enzyme synthesis started had no affect. These findings suggest that asparaginase II is produced by E. coli A-l in response to low concentrations of ammonia and that exogenously supplied nitrogen compounds may play a major role in the regulation of this enzyme. It is suggested that E. coli A-l produced L-asparaginase in order to obtain ammonia for the synthesis of glutamine from glutamate. The synthesis of glutamine from glutamate is the first step of a highly branched pathway which ultimately leads to the synthesis of many of the important macromolecules of the cell

DISTRIBUTION OF CRAYFISH SPECIES IN SELECT NORTH DAKOTA STREAMS

Crayfish have an integral role in aquatic ecosystems, serving as herbivores, predators, detritivores, and prey for fishes and other aquatic and terrestrial animals (Momot 1995, Taylor et al. 1996, Martin 1997). Many crayfish populations are currently declining as a result of habitat alteration (Taylor et al. 1996) and invasive predators (e.g., rusty crayfish Orconectes rusticus [Girard; Lodge et al. 2000]). Knowledge of crayfish distribution and biology in North America and specifically North Dakota is sparse or lacking (Taylor et al. 1996) and baseline information is necessary to identify effects of anthropogenic alterations. Early inventories documented 3 crayfish species in North Dakota: calico crayfish Orconectes immunis (Hagen), virile crayfish, O. virilis (Hagen), and devil crayfish, Cambarus diogenes Girard (Harris 1903, Crocker and Barr 1968, Hobbs 1989, Taylor et al. 1996); however an extensive statewide crayfish survey with site specific information has not been conducted. Additionally, the rusty crayfish is present in the surrounding states of Minnesota and South Dakota (Olden et al. 2006) and in southern Manitoba (Phillips et al. 2009). This invasive species has been known to displace native crayfishes (Hill and Lodge 1999) and cause severe ecological alterations that can affect fishes. The rusty crayfish can limit macrophyte growth which can remove habitat and alter nutrient cycles (Hill and Lodge 1999, Byron and Wilson 2001). Our objectives were to document presence and distribution of native and non-native crayfish species in central and western North Dakota

Distribution of buffalograss polyploid variation in the southern Great Plains

Buffalograss [Buchloë dactyloides (Nutt.) Engelm.] is indigenous to the short-grass prairies of North America and is a polyploid series of diploid, tetraploid, and hexaploid individuals. It has a base chromosome number of x = 10. The distribution pattern of these ploidy levels is not well-defined, especially in the southern Great Plains. We predicted the ploidy levels of 273 buffalograsses from the southern Great Plains of North America using flow cytometry to measure cellular DNA content. The buffalograss accessions were grouped into four distinct ploidy level groups. Very few diploid accessions were collected (2.6% of the collection), and all were found in northwest Texas and eastern New Mexico. Tetraploid accessions (23% of the collection) were found exclusively in the western regions of the southern Great Plains. Hexaploids were the most prevalent ploidy level, representing 73% of the collection and found throughout the collection area. Pentaploid accessions were also found in field sites (1.8% of the collection). No clear pattern of adaptation for ploidy levels is apparent from these data. In other collections, cold hardiness appears associated with higher ploidy levels, but this pattern is not apparent in the southern Great Plains

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

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 TGFβ) 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