Fed-batch fermentation studies with Bacillus thuringiensis H-14 synthesising endotoxin.

Cell yield and toxicity of B. thuringiensis H-14 was improved markedly by adopting a simple fed-batch fermentation technique based on controlling glucose concentration. Maintenance of steady glucose concentration (0.3-0.5%) in the culture medium was achieved by the continuous addition of concentrated glucose solution. Addition of glucose at 3 g/hr/l of culture starting from 3rd hr till 16th hr of fermentation was found to yield cell densities of 80 g/l (wet weight) which represented a nearly 3-fold increase over the batch mode. A fivefold increase in toxicity was obtained by fed-batch fermentation. Cultivation of B. thuringiensis H-14 to high cell densities had no negative effect on sporulation and toxin synthesis. The rate of pH drop and dissolved oxygen level were within manageable limits.     

Multiple heme pocket subconformations of methemoglobin associated with distal histidine interactions

Electron paramagnetic resonance spectra of methemoglobin reveal that, in addition to the major tetragonal high-spin aqueous complex and the low-spin hydroxide complex, three other complexes associated with the interaction of the distal histidine are resolved. These are a rhombic high-spin and two classes of low-spin bis-histidine complexes. By freeze-quenching experiments it is shown that the rhombic high-spin and one of the low-spin bis-histidine complexes (B) are at equilibrium with the dominant species. Incubation in the 210-260 K temperature range shifts the total equilibrium toward a low-energy state with the distal histidine coordinated to the iron (complex C).     

Forage Crop <Emphasis Type="Italic">Lolium multiflorum</Emphasis> Assisted Synthesis of AgNPs and Their Bioactivities Against Poultry Pathogenic Bacteria in In Vitro

Italian ryegrass is one of main feed for livestock animals/birds. It has potential antioxidant metabolites that can improve their health and protect them against various infectious diseases. In this work, we studied synthesis of silver nanoparticles assisted by forage crop Lolium multiflorum as a green synthesis way. Potential antibacterial efficacy of these synthesized nanosized silver nanoparticles against poultry pathogenic bacteria was then studied. Aqueous extract of IRG was used as reducing agent for bio-reduction of silver salt to convert Ag⁺ to Ag⁰ metallic nano-silver. Size, shape, metallic composition, functional group, and crystalline nature of these synthesized silver nanoparticles were then characterized using UV–Vis spectrophotometer, FESEM, EDX, FT-IT, and XRD, respectively. In addition, antibacterial effects of these synthesized AgNPs against poultry pathogenic bacteria were evaluated by agar well diffusion method. UV–Vis spectra showed strong absorption peak of 440–450 nm with differ reaction time ranging from 30 min to 24 h. FESEM measurements revealed particles sizes of around 20–100 nm, majority of which were spherical in shape while a few were irregular. These biosynthesized silver nanoparticles using IRG extract exhibited strong antibacterial activities against poultry pathogenic microorganisms, including Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, and Bacillus subtilis. Overall results confirmed that IRG plant extract possessed potential bioactive compounds for converting silver ions into nanosized silver at room temperature without needing any external chemical for redox reaction. In addition, such synthesized AgNPs showed strong antibacterial activities against pathogenic bacteria responsible for infectious diseases in poultry.     

Bacterial antagonists and hexanal-induced systemic resistance of mango fruits against Lasiodiplodia theobromae causing stem-end rot

Induction of defense-related enzymes, such as phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), superoxide dismutase (SOD) and catalase (CAT) due to bacterial antagonists viz., Pseudomonas fluorescens (Pf1) and Bacillus subtilis (EPCO16) and plant-derived lipoxygenase volatile compound hexanal, were studied in mango fruits against Lasiodiplodia theobromae causing stem-end rot disease. The results showed increased induction of all the defense-related enzymes in mango fruits 3–5 days after dipping treatment with combination of bacterial antagonists and hexanal when compared to untreated control treatment and treatment with fungicide carbendazim in storage condition. The increased activity was observed up to 3 days after treatment and thereafter declined. Further, increased expression of specific isoforms of PO, PPO, SOD and CAT were also observed in the treatment effect of P. fluorescens (0.5%)?+?hexanal (0.02%) treated fruits against L. theobromae. From the results obtained, it is inferred that due to the enhancement of defense-related enzymes via the phenylpropanoid pathway and due to secretion of secondary metabolites that would play significant role in hindering the pathogen quiescence and further invasion in mango fruits and thereby prevent the fruit rot.     

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein: A POTENTIAL MECHANISM PROMOTING HUMAN CANCER*

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70–80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of “gene lesions responsible for human inherited disease” were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2–4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA)³ is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11–14), and ATR (15)) or generation of dominant negative inhibitors (e.g. CHEK2 (16) and VWOX (17)). In breast cancer, aberrantly spliced forms of progesterone and estrogen receptors are found (reviewed in Ref. 3). Intronic mutations inactivate p53 through aberrant splicing and intron retention (18). Somatic mutations with the potential to alter splicing are frequent in some cancers (19–25). Single nucleotide polymorphisms in the cyclin D1 proto-oncogene predispose to aberrant splicing and the cyclin D1b intronic splice variant (26–29). Cyclin D1b confers anchorage independence, is tumorogenic in vivo, and is detectable in human tumors (30), but as yet no clinical studies have confirmed an impact on outcome. On the other hand, aberrant splicing of HAS1 shows an association between aberrant splice variants and malignancy, suggesting that such variants may be potential therapeutic targets and diagnostic indicators (19, 31–33). Increased HA expression has been associated with malignant progression of multiple tumor types, including breast, prostate, colon, glioma, mesothelioma, and multiple myeloma (34). The three mammalian HA synthase (HAS) isoenzymes synthesize HA and are integral transmembrane proteins with a probable porelike structural assembly (35–39). Although in humans, the three HAS genes are located on different chromosomes (hCh19, hCh8, and hCh16, respectively) (40), they share a high degree of sequence homology (41, 42). HAS isoenzymes synthesize a different size range of HA molecules, which exhibit different functions (43, 44). HASs contribute to a variety of cancers (45–55). Overexpression of HASs promotes growth and/or metastatic development in fibrosarcoma, prostate, and mammary carcinoma, and the removal of the HA matrix from a migratory cell membrane inhibits cell movement (45, 53). HAS2 confers anchorage independence (56). Our work has shown aberrant HAS1 splicing in multiple myeloma (5) and Waldenstrom''s macroglobulinemia (6). HAS1 is overexpressed in colon (57), ovarian (58), endometrial (59), mesothelioma (60), and bladder cancers (61). A HAS1 splice variant is detected in bladder cancer (61).Here, we characterize molecular and biochemical characteristics of HAS1 variants (HAS1-Vs) (5), generated by aberrant splicing. Using transient transfectants and tagged HAS1 family constructs, we show that HAS1-Vs differ in cellular localization, de novo HA localization, and turnover kinetics, as compared with HAS1-FL, and dominantly influence HAS1-FL when co-expressed. HAS1-Vs proteins form intra- and intermolecular associations among themselves and with HAS1-FL, including covalent interactions and multimer formation. HAS1-Vc supports vigorous cellular transformation of NIH3T3 cells in vitro, and HAS1-Vc-transformed NIH3T3 cells are tumorogenic in vivo.     

The tumour suppressor C/EBPδ inhibits FBXW7 expression and promotes mammary tumour metastasis

Inflammation and hypoxia are known to promote the metastatic progression of tumours. The CCAAT/enhancer-binding protein-δ (C/EBPδ, CEBPD) is an inflammatory response gene and candidate tumour suppressor, but its physiological role in tumourigenesis in vivo is unknown. Here, we demonstrate a tumour suppressor function of C/EBPδ using transgenic mice overexpressing the Neu/Her2/ERBB2 proto-oncogene in the mammary gland. Unexpectedly, this study also revealed that C/EBPδ is necessary for efficient tumour metastasis. We show that C/EBPδ is induced by hypoxia in tumours in vivo and in breast tumour cells in vitro, and that C/EBPδ-deficient cells exhibit reduced glycolytic metabolism and cell viability under hypoxia. C/EBPδ supports CXCR4 expression. On the other hand, C/EBPδ directly inhibits expression of the tumour suppressor F-box and WD repeat-domain containing 7 gene (FBXW7, FBW7, AGO, Cdc4), encoding an F-box protein that promotes degradation of the mammalian target of rapamycin (mTOR). Consequently, C/EBPδ enhances mTOR/AKT/S6K1 signalling and augments translation and activity of hypoxia-inducible factor-1α (HIF-1α), which is necessary for hypoxia adaptation. This work provides new insight into the mechanisms by which metastasis-promoting signals are induced specifically under hypoxia.