Adenylyl cyclase G is activated by an intramolecular osmosensor

Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor. The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation. To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGDeltacat) and was driven by a UV-inducible promoter. After UV induction of ACGDeltacat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced. Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGDeltacat. However, high osmolality did not alter the dimer/monomer ratio. This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality. To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium. This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes.     

Lyn- and ERK-mediated vs. Ca2+-mediated neutrophil O2- responses with thermal injury

We evaluated thedependency of neutrophil O production on PTK-Lyn andMAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn wasassessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessedby Western blot analysis. O production was measuredby isoluminol-enhanced luminometry. Imaging technique was employed tomeasure neutrophil [Ca²⁺]_i in individualcells. Thermal injury caused marked upregulation of Lyn and ERK1/2accompanying enhanced neutrophil O production.Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478)before burn injury caused complete inhibition of the respective kinaseactivation. Both AG556 and AG1478 produced an ~66% inhibition inO production. Treatment with diltiazem (DZ) producedan ~37% inhibition of O production withoutaffecting Lyn or ERK1/2 activation with burn injury. Ca²⁺mobilization was upregulated with burn injury but not affected bytreatment of burn rats with AG556. Unlike the partial inhibition ofburn-induced O production by AG556, AG1478, or DZ,platelet-activating factor antagonist (PAFa) treatment of burn ratsproduced near complete inhibition of O production.PAFa treatment also blocked activation of Lyn. The findings suggestthat the near complete inhibition of O production byPAFa was a result of blockade of PTK as well as Ca²⁺signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result ofpotentiation of Ca²⁺-linked and -independent signalingtriggered by inflammatory agents such as PAF.

     

Gut-associated lymphoid T cell suppression enhances bacterial translocation in alcohol and burn injury

The mechanism of alcohol-mediated increased infection in burn patients remains unknown. With the use of a rat model of acute alcohol and burn injury, the present study ascertained whether acute alcohol exposure before thermal injury enhances gut bacterial translocation. On day 2 postinjury, we found a severalfold increase in gut bacterial translocation in rats receiving both alcohol and burn injury compared with the animals receiving either injury alone. Whereas there were no demonstrable changes in intestinal morphology in any group of animals, a significant increase in intestinal permeability was observed in ethanol- and burn-injured rats compared with the rats receiving either injury alone. We further examined the role of intestinal immune defense by determining the gut-associated lymphoid (Peyer's patches and mesenteric lymph nodes) T cell effector responses 2 days after alcohol and burn injury. Although there was a decrease in the proliferation and interferon-gamma by gut lymphoid T cells after burn injury alone; the suppression was maximum in the group of rats receiving both alcohol and burn injuries. Furthermore, the depletion of CD3(+) cells in healthy rats resulted in bacterial accumulation in mesenteric lymph nodes; such CD3(+) cell depletion in alcohol- and burn-injured rats furthered the spread of bacteria to spleen and circulation. In conclusion, our data suggest that the increased intestinal permeability and a suppression of intestinal immune defense in rats receiving alcohol and burn injury may cause an increase in bacterial translocation and their spread to extraintestinal sites.     

Development of cloning vectors and transformation methods for<Emphasis Type="Italic"> Amycolatopsis</Emphasis>

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA-rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains. Electronic Publication     

Casein hydrolysis in PEG-salt two phase system by Eudragit-chymotrypsin bioconjugate

A bioconjugate of -chymotrypsin and Eudragit S-100 was used in an aqueous two-phase system (polyethylene glycol/phosphate) for casein hydrolysis. More product was obtained by replacing the lower salt phase with a fresh one during the reaction. The bioconjugate could be reused six times for casein hydrolysis.     

Gene dosage and selective expression modify phenotype in a Drosophila model of human mitochondrial disease

Human mitochondrial disease manifests with a wide range of clinical phenotypes of varying severity. To create a model for these disorders, we have manipulated the Drosophila gene technical knockout, encoding mitoribosomal protein S12. Various permutations of endogenous and transgenic alleles create a range of phenotypes, varying from larval developmental arrest through to mild neurological defects in the adult, and also mimic threshold effects associated with human mtDNA disease. Nuclear genetic background influences mutant phenotype by a compensatory mechanism affecting mitochondrial RNA levels. Selective expression of the wild-type allele indicates critical times and cell-types in development, in which mitochondrial protein synthesis deficiency leads to specific phenotypic outcomes.     

One step purification of peanut phospholipase D by precipitation with alginate

A simple titrimetric assay with soybean lecithin has been used for screening phospholipase D activity from some plant sources, viz. peanut, wheat germ, cabbage and carrot. The enzyme from peanut has been purified by binding to alginate which is a water soluble polymer. The purification consisted of co-precipitation of enzyme with alginate upon addition of 0.06 M Ca⁺⁺. The enzyme was eluted from the polymer using 0.2 M sodium chloride. The activity recovery was 61% with 34 fold purification.     

Molecular analysis of autosomal dominant hereditary ataxias in the Indian population: high frequency of SCA2 and evidence for a common founder mutation

Expansion of CTG/CAG trinucleotide repeats has been shown to cause a number of autosomal dominant cerebellar ataxias (ADCA) such as SCA1, SCA2, SCA3/ MJD, SCA6, SCA7, SCA8 and DRPLA. There is a wide variation in the clinical phenotype and prevalence of these ataxias in different populations. An analysis of ataxias in 42 Indian families indicates that SCA2 is the most frequent amongst all the ADCAs we have studied. In the SCA2 families, together with an intergenerational increase in repeat size, a horizontal increase with the birth order of the offspring was also observed, indicating an important role for parental age in repeat instability. This was strengthened by the detection of a pair of dizygotic twins with expanded alleles showing the same repeat number. Haplotype analysis indicates the presence of a common founder chromosome for the expanded allele in the Indian population. Polymorphism of CAG repeats in 135 normal individuals at the SCA loci studied showed similarity to the Caucasian population but was significantly different from the Japanese population.