Peptide: N- glycanase is expressed in prestalk cells and plays a role in the differentiation of prespore cells during development of Dictyostelium discoideum.

Peptide: N-glycanase (PNGase) enzyme is found throughout eukaryotes and plays an important role in the misfolded glycoprotein degradation pathway. This communication reports the expression patterns of the pngase transcript (as studied by the analysis of beta-galactosidase reporter driven by the putative pngase promoter) and protein (as studied by the analysis of beta-galactosidase reporter expressed under the putative pngase promoter as a fusion with the pngase ORF) during development and further elucidated the developmental defects of the cells lacking PNGase (png(-)). The results show that the DdPNGase is an essential protein expressed throughout development and beta-galactosidase activity was present in the anterior part of the slug. In structures derived from a null mutant for pngase, the prestalk A and AO patterning was expanded and covered a large section of the prespore region of the slugs. When developed as chimeras with wild type, the png(-) cells preferentially populate the prestalk/stalk region. When the mutants were mixed in higher ratios, they also tend to form the prespore/spore cells. The results emphasize that the DdPNGase has an essential role during development and the mutants have defects in a system that changes the physiological dynamics in the prespore cells. DdPNGase play a role in development both during aggregation and in the differentiation of prespore cells.

Identification and characterization of peptide: N-glycanase from Dictyostelium discoideum.

BACKGROUND: Peptide: N- glycanase (PNGase) enzyme cleaves oligosaccharides from the misfolded glycoproteins and prepares them for degradation. This enzyme plays a role in the endoplasmic reticulum associated degradation (ERAD) pathway in yeast and mice but its biological importance and role in multicellular development remain largely unknown. RESULTS: In this study, the PNGase from the cellular slime mold, Dictyostelium discoideum (DdPNGase) was identified based on the presence of a common TG (transglutaminase) core domain and its sequence homology with the known PNGases. The domain architecture and the sequence comparison validated the presence of probable functional domains in DdPNGase. The tertiary structure matched with the mouse PNGase. Here we show that DdPNGase is an essential protein, required for aggregation during multicellular development and a knockout strain of it results in small sized aggregates, all of which did not form fruiting bodies. The in situ hybridization and RT-PCR results show higher level of expression during the aggregate stage. The expression gets restricted to the prestalk region during later developmental stages. DdPNGase is a functional peptide:N-glycanase enzyme possessing deglycosylation activity, but does not possess any significant transamidation activity. CONCLUSIONS: We have identified and characterized a novel PNGase from D. discoideum and confirmed its deglycosylation activity. The results emphasize the importance of PNGase in aggregation during multicellular development of this organism.

A prospective randomized controlled study comparing intrathecal bupivacaine combined with fentanyl and sufentanil in abdominal and lower limb surgeries.

BACKGROUND: Hyperbaric bupivacaine along with either fentanyl or sufentanil as additive, has been widely used in spinal anesthesia. In the present study, we compared the analgesic effects of intrathecal fentanyl versus sufentanil combined with bupivacaine for surgical procedures over the abdomen and lower limbs. SETTINGS AND DESIGN: This was randomized controlled study conducted in a tertiary care hospital attached to a medical school. METHODS: Sixty American Society of Anesthesiologists I and II patients were randomized into three groups by sealed envelope technique. Group 1 was to receive bupivacaine with fentanyl; group 2 to receive bupivacaine with sufentanil and group 3 to receive bupivacaine with saline (control), intrathecally. The parameters checked were hemodynamic changes, onset and duration of sensory block, duration of analgesia and maximal sensory level achieved. STATISTICAL ANALYSIS: The data collected were analyzed using χ2 test and paired Student's t-test. RESULTS: The time taken for the onset of analgesia was longest in the control group followed by fentanyl group. The earliest onset of action of 9.35 ± 1.92 min was recorded in sufentanil group. Duration of sensory blockade and analgesia was longest for fentanyl group than the other groups. Adverse effects noted were more for sufentanil group but were self-limiting. CONCLUSION: Fentanyl with bupivacaine produced prolonged analgesia and delayed two-segment regression and demonstrated reduced incidence of complications as compared with intrathecal sufentanil. As the quality of analgesia was complete and comparable, fentanyl emerges as a better option for analgesia and it is much economical too when compared to sufentanil.

Dissecting the functional role of polyketide synthases in Dictyostelium discoideum: biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol.

Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds.