Bacillus pumilus BpCRI 6, a promising candidate for cellulase production under conditions of catabolite repression

Cellulose degrading organisms have been used for the conversion of cellulolytic materials into soluble sugars or solvents in several biotechnological and industrial applications. In this report, a mutant of Bacillus pumilus was obtained after chemical mutagenesis and screened for cellulase production. This mutant named BpCRI 6 was selected for its ability to produce cellulase under catabolite repression. Cellulase yield by BpCRI 6 was four times higher than that of the wild type under optimum growth conditions (pH 6.5, 25°C and Ca2+ 1mM). In shaking flask cultures, production of cellulase  by the wild type was completely repressed in the presence of 25 mM glucose, while BpCRI 6 strain still exhibited a residual cellulase production of 80 and 40% at 25 mM and 40 mM of glucose concentrations respectively. The mutant strain is stable and grows rapidly in liquid and solid media. Under conditions of catabolite repression (40 mM of glucose), the production of cellulase by this mutant is particularly significant when compared to Trichoderma reesei a well-known cellulase producer, which is under control of end-product inhibition. This is the first report of a successful catabolite repression insensitivity of cellulase production by a mutant of B. pumilus. (African Journal of Biotechnology: 2003 2(6): 140-146

Purification and characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens

Cellulases from the wild-type (WT) and two improved mutants (catabolite repression resistant mutant 4 and 24, abbreviated CRRmt4 and CRRmt24, respectively) of Pseudomonas fluorescens were purified to apparent homogeneity by ammonium sulphate precipitation, ion exchange chromatography on DEAE Sephadex A-50 and gel filtration on Sephadex G-100. Purification fold of about 5 was obtained for the WT and CRRmt24 while purification fold of about 7 was achieved for CRRmt4 by ammonium sulphate precipitation. Ion exchange chromatography gave purification fold of about 24, 22 and 25 for WT, CRRmt4 and CRRmt24, respectively. Gel filtration chromatography step yielded a homogeneous preparation with a specific activity of 6.8, 5.9 and 6.9 units/mg protein for the WT, CRRmt4and CRRmt24, respectively. The purified cellulase gave a single protein band on polyacrylamide gel electrophoresis. The molecular weights of the three cellulases were estimated to be 36, 26 and 36 kDa for the wild-type,CRRmt4 and CRRmt24, respectively. Km values of 3.6, 3.1, and 5.3 mg/ml were obtained for the wild-type, CRRmt4 and CRRmt24, respectively. The optimum pH value for the purified cellulases was 6.5 – 7.0 andthe enzymes were optimally active at temperature of 35°C. The activities of the purified cellulases were stimulated by low concentrations (10-30 mM) of Na+ and Mg++ while EDTA was found to inhibit enzyme activity at all concentrations

The molecular initiation and subsequent acquisition of disease resistance in plants

Interactions between disease resistance (R) genes in plants and their corresponding pathogen avirulence (Avr) genes are the key determinants of whether a plant is susceptible or resistance to a pathogen attack. Evidence has emerged that these gene-for-gene interactions in the perception of pathogenic invasions and development of acquired resistance in plants involve different molecular and hormonal transduction pathways, which are still poorly understood. It has become apparent that plants actively produce several phytohormones such as ethylene, jasmonate, salicylic acid, and reactive oxygen intermediates prior to upregulation of R genes. The physiological role of these molecules in plant resistance to pathogens is beginning to attract attention. The use of transgenic plants in recent attempts, including development of mutants with altered R genes, has provided new insights into the mechanisms involved in pathogen perception, signal transduction and subsequent resistance to disease in plants. This review tries to summarize current knowledge of pathogen-related genes in plants, and how they can be use to improve disease resistance in agronomically valuable plants. It also describes the molecular basis of defense mechanisms in plants under pathogen attack. (African Journal of Biotechnology: 2003 2(2): 26-32

A home made kit for plasmid DNA mini-preparation

Many methods have been used to isolate plasmid DNA, but some of them are time consuming especially when extracting a large number of samples. Here, we developed a rapid protocol for plasmid DNA extraction based on the alkaline lysis method of plasmid preparation (extraction at pH 8.0). Using this new method, a good plasmid preparation can be made in approximately one hour. The plasmids are suitable for any subsequent molecular applications in the laboratory. By applying the recommendations to avoid contaminations and to maximize the plasmid yield and quality during extraction, this protocol could be a valuable reference especially when analyzing a large number of samples. (African Journal of Biotechnology: 2003 2(4): 87

Regulatory mutations affecting the synthesis of cellulase in Pseudomonas fluorescens

Pseudomonas fluorescens, was cultured in basal medium containing carboxymethyl-cellulose (CMC) as inducer and glucose or glycerol as carbon and energy sources. Ethylmethanesulphonate (EMS) was used to mutagenize the wild-type organism to produce mutants. The isolated mutants were screened for the isolation of catabolite repression resistant mutants in the presence of 1% (w/v) glucose as carbon source. A total of fifty mutants were isolated. All the mutants produced cellulase in the presence of CMC as an inducer with specific activity of 0.057, 0.088 and 0.074 units/mg protein for the wild-type, catabolite repression resistant mutant4 (CRRmt4) and catabolite repression resistant mutant24 (CRRmt24), respectively. It was observed that addition of glucose or glycerol as carbon and energy sources to the culture medium resulted into considerable reduction in the cellulolytic activity. However, glycerol appeared to be a better carbon and energy source than glucose which inhibited enzyme expression in most of the strains used in this study. It was also observed that potent cellulase production occurred at the exponential growth phase of the organism. The isolated mutants were grouped into three classes based on their induction ratios namely; unimproved mutants, catabolite repression resistant mutants and mutants with highest induction ratio but sensitive to catabolite repression in the presence of high glucose concentration. The overall results obtained showed that cellulolytic activity in P. fluorescens was regulated by catabolite repression.Key words: Pseudomonas fluorescens, ethylmethanesulphonate, mutants, cellulose, catabolite repression, induction ratio.African Journal of Biotechnology Vol. 4 (8), pp. 838-84

A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration

Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK) and Src, and that this signalling pathway lies downstream of fascin-microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and may have implications for the design of therapies to target fascin in metastatic disease

Prognostic significance of fascin expression in advanced colorectal cancer: an immunohistochemical study of colorectal adenomas and adenocarcinomas

BACKGROUND: Fascin is an actin bundling protein with roles in the formation of cell protrusions and motility of mesenchymal and neuronal cells. Fascin is normally low or absent from epithelia, but is upregulated in several epithelial neoplasms where it may contribute to an invasive phenotype. Here, we report on the prevalence and potential clinical significance of fascin expression in relation to the progression of colorectal adenocarcinoma and to tumor cell proliferation as measured by Ki67 index. METHODS: Conventional tissue sections of 107 colorectal adenomas and 35 adenocarcinomas were analyzed by immunohistochemistry for fascin and Ki67 expression. Fascin expression and Ki67 proliferation index were also investigated by use of a tissue microarray containing cores from a further 158 colorectal adenocarcinomas and 15 adenomas linked to a CCF, IRB-approved database with a mean of 38 months of clinical follow-up. Survival analysis was carried out by the Kaplan-Meier and Cox regression methods. RESULTS: Fascin was not expressed by the normal colonic epithelium. In conventional sections, 16% of adenomas and 26% of adenocarcinomas showed fascin expression in greater than 10% of the tumor cells. In the clinically-annotated tumors, fascin immunoreactivity was more common in tumors located in the proximal colon (p = 0.009), but was not associated with age, gender, or TNM stage. Patients with stage III/IV adenocarcinomas (n = 62) with strong fascin immunoreactivity had a worse prognosis than patients with low or absent fascin, (3-year overall survival of 11% versus 43% for fascin-negative patients; p = 0.023). In adenomas, fascin and Ki67 tended to be inversely correlated at the cellular level; this trend was less apparent in adenocarcinomas. CONCLUSION: Fascin is upregulated in a proportion of adenomas, where its expression is often focal. Strong and diffuse expression was seen in a subset of advanced colorectal adenocarcinomas that correlated with shorter survival in stage III and IV patients. Fascin may have prognostic value as an early biomarker for more aggressive colorectal adenocarcinomas

Gamma-Secretase Represents a Therapeutic Target for the Treatment of Invasive Glioma Mediated by the p75 Neurotrophin Receptor

The multifunctional signaling protein p75 neurotrophin receptor (p75NTR) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here, we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75NTR is required for p75NTR-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75NTR or treatment of animals bearing p75NTR-positive intracranial tumors with clinically applicable γ-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75NTR was observed in p75NTR-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75NTR as a therapeutic target, suggesting that γ-secretase inhibitors may have direct clinical application for the treatment of malignant glioma

Experience of brentuximab vedotin in relapsed/refractory Hodgkin lymphoma and relapsed/refractory systemic anaplastic large-cell lymphoma in the Named Patient Program: Review of the literature

Brentuximab vedotin was made available via a Named Patient Program (NPP) to non-US/Canadian patients with relapsed/refractory (R/R) Hodgkin lymphoma (HL) or systemic anaplastic large-cell lymphoma (sALCL) until approval in their respective countries. We re-evaluated the efficacy and safety NPP data in a pooled analysis. Through a systematic literature review, 21 NPP publications were identified describing 14 cohorts (N=245). Among patients with a specified diagnosis, 207 had HL, 28 had ALCL, and one had CD30+ T-cell lymphoma (not specified). In cohorts reporting response, overall response and complete remission rates were 67\% and 26\%, respectively, in R/R HL, and 75\% and 74\%, respectively, in R/R ALCL. Incidences of grade 3/4 neurologic and hematologic toxicities were 6\% and 12\%, respectively; 5\% of patients discontinued because of toxicity. In real-world practice, response rates and tolerability to brentuximab vedotin are similar to those reported in the two pivotal phase 2 trials in these settings