Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

Screening of phytotoxicity of Alternaria macrospora MKP1 against Parthenium hysterophorus L.

Parthenium hysterophorus L. an exotic, pernicious weed is considered as one of the most troublesome weeds for agricultural sector by virtue of its high ecological amplitude and adaptability. Microbes and their by-products are now proved to be a worthy alternative to toxic chemicals used for weed management. Alternaria macrospora MKPI was isolated from the parthenium leaves infected with leaf blight and found pathogenic to the weed. The herbicidal potential of cell free culture filtrate of A. macrospora MKP1 has been tested against parthenium by employing detached leaf bioassay and seed germination bioassay and a significant damage was exhibited by the cultural filtrate of pathogen to the parthenium leaves and seeds.     

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and β-glucosidase activities of 1,270, 146, 34, and 15 U mL^?1, respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k _cat/K _m?=?1,440 mg?mL^?1?s^?1) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.     

Genetic and Epigenetic Variation across Genes Involved in Energy Metabolism and Mitochondria of Chinese Hamster Ovary Cell Lines

The increasingdemandfor biopharmaceutical products drives the search for efficient cell factories that are able to sustainably support rapid growth, high productivity, and product quality. As these depend on energy generation, here the genomic variation in nuclear genes associated with mitochondria and energy metabolism and the mitochondrial genome of 14 cell lines is investigated. The variants called enable reliable tracing of lineages. Unique sequence variations are observed in cell lines adapted to grow in protein‐free media, enriched in signaling pathways or mitogen‐activated protein kinase 3. High‐producing cell lines bear unique mutations in nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND2 and ND4) and in peroxisomal acyl‐CoA synthetase (ACSL4), involved in lipid metabolism. As phenotypes are determined not only by functional mutations, but also by the exquisite regulation of expression patterns, it is not surprising that ≈50% of the genes investigated here are found to be differentially methylated and thus epigenetically controlled, enabling a clear distinction of high producers, and cells adapted to a minimal, glutamine (Gln)‐free medium. Similar pathways are enriched as those identified by genome variation. This strengthens the hypothesis that these phenomena act together to define cell behavior.     

Methylation: a regulator of HIV-1 replication?

Pakistan, the second most populous Muslim nation in the world, has started to finally experience and confront the HIV/AIDS epidemic. The country had been relatively safe from any indigenous HIV cases for around two decades, with most of the infections being attributable to deported HIV positive migrants from the Gulf States. However, the virus finally seems to have found a home-base, as evidenced by the recent HIV outbreaks among the injection drug user community. Extremely high-risk behavior has also been documented among Hijras (sex workers) and long-distance truck drivers. The weak government response coupled with the extremely distressing social demographics of this South-Asian republic also helps to compound the problem. The time is ripe now to prepare in advance, to take the appropriate measures to curtail further spread of the disease. If this opportunity is not utilized right now, little if at all could be done later.