Isolation and characterization of plasma membrane fromAcanthamoeba culbertsoni

A rapid method for preparation of plasma membrane fromAcanthamoeba culbertsoni involving toluene treatment followed by lithium bromide extraction is described. In the plasma membrane preparation, 5′-nucleotidase, Na⁺ + K⁺ -ATPase, Mg²⁺ -ATPase and glucose-6-phosphatase activities were enriched. The membrane preparation was free from nucleic acid, cytochrome P-450 and cytochrome b5. Amino acid (¹⁴C-Ieucine) was not incorporated in the plasma membrane in 2 min. Succinic dehydrogenase was not detectable in the plasma membrane preparation. The molar ratio of cholesterol and phospholipids was 0.95 which is characteristics for plasma membranes. Under electronmicroscopy the preparation was homogenous without any other component of the cell. Plasma membrane proteins and glycoproteins were separated on acrylamide gel electrophoresis     

Localization of GABA-like immunoreactivity in the ectostriatum of domestic chicks: GABA immunocytochemistry combined with Golgi impregnation

Brain Cell Biology - The distribution of GABA-like immunoreactivity (GABA-LI) in the ectostriatal core (Ec) of domestic chicks (one to two days old) was investigated using (1) preembedding GABA...     

Characterisation of a 5-bp deletion in exon 4 of the factor VIII gene: concordance with slipped-mispairing at DNA replication

In an attempt to characterize disease producing mutations in the factor VIII gene we screened exons 4, 7, 8, 11, 12 and 16 by PCR-SSCP (polymerase chain reactionsingle strand conformation polymorphism), in 12 randomly selected haemophilia A patients. These exons were chosen because they have been reported to harbour a disproportionately high number of mutations relative to their size. Using this strategy we detected a frame-shifting 5-bp deletion (TACCT, involving nucleotides 519–523), which is predicted to result in a severely truncated factor VIII polypeptide, terminating approximately midway through the conserved A1 domain and resulting in the observed severe phenotype. We also showed that the sequence in the vicinity of the observed deletion is concordant with the modified slipped-mispairing at DNA replication model of Krawczak and Cooper.     

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

Thermoluminescence studies of zinc borate (ZnB2O4) phosphor doped with rare earths for dosimetric aspects

A series of ZnB₂O₄ phosphors doped with different concentrations of Eu and Dy (0.05 0.1, 0.2, 0.5, 1.0 mol%) and co-doped with Ce (1, 2, 5, 7, 10 mol%) respectively was prepared via the solid-state reaction technique and the thermoluminescence (TL) behaviour of gamma ray-irradiated samples was studied. The synthesized samples were irradiated with γ-rays for the dose range 0.03–1.20 kGy. The TL intensity variations with dose, dopant concentration, and the effect of co-doping were studied. The TL response curves for ZnB₂O₄:Eu³⁺ and ZnB₂O₄:Dy³⁺, ZnB₂O₄:Eu³,Ce³⁺ and ZnB₂O₄:Dy³⁺,Ce³⁺ phosphor were observed. It was revealed that ZnB₂O₄:Eu³⁺ showed a linear TL behaviour for the dose 0.03–1.20 kGy and ZnB₂O₄:Dy³⁺ showed linearity for the gamma dose range 0.03–0.10 kGy. Furthermore, fading for all the samples was observed to be less than 10% for a storage period of 30 days. In addition to this, the trapping parameters, especially activation energies were evaluated using the Ilich method and the initial rise method. The activation energy values obtained from both methods were in complete agreement with each other.     

Production, Purification, and Characterization of Recombinant Human Hemoglobin Rainier Expressed inSaccharomyces cerevisiae

Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the β145 tyrosine is substituted with cysteine. The α and β^Rainierglobin cDNAs were cloned in a high copy number vector and expressed inSaccharomyces cerevisiaeunder the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual α and β^Rainierglobin chains. Coexpression of both α and β^RainiercDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the α and β chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A₀. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.     

A novel cyclomaltodextrin glucanotransferase from Bacillus firmus that degrades raw starch

Summary An alkalophilic Bacillus firmus secreting the enzyme cyclomaltodextrin glucanotransferase was isolated from soil. The enzyme attacked raw starch to produce cyclodextrins. Maximum cyclodextrins were produced from tapioca starch followed by potato and corn starch. About 49 % of tapioca starch (at 10 and 50 g/l) was converted to cyclodextrins. The main reaction products were and -cyclodextrins with 40 % and 8 % yield respectively. On prolonged incubation small amount of -cyclodextrin was also produced. The ratio of cyclodextrins was dependent on the initial substrate concentration as well as reaction time.NCL communication number 6203     

Citric acid fermentation by the mutant strain of theAspergillus niger resistant to manganese ions inhibition

Summary A new mutant strain,Aspergillus niger GS-III, showing resistance to manganese ions inhibition of citric acid fermentation on a sugarcane molasses containing medium was induced fromAspergillus niger KCU 520, a high citric acid-yielding strain. In submerged, surface or continuous cultures in the presence of manganese ions concentration upto 1.5 ppm the mutant strain yielded citric acid about 90 KgM^–3 . The citric acid yield was comparable to that obtained with the parental strain KCU 520 in the absence of manganese ions, but it was atleast 3-fold higher than that obtained by the latter in the presence of manganese ions. The mutant strain immobilized in calcium alginate beads was used in combination with surface-stabilized cultures for about 36-days in a continuous flow horizontal fermenter without any apparent loss in citric acid productivity. These results indicate that the manganese-resistant mutant is stable and may be used in the presence of sufficient manganese ions concentration (1.5 ppm) in the fermentation medium. This capability of the mutant strainA. niger GS-III has been correlated with greatly reduced levels (about one-thirds) of the NADP⁺ -isocitric dehydrogenase, one of the control points for citric acid accumulation.