Influence of steam and dry heat pretreatment on fibre properties and cellulase degradation of cellulosic fibres

Cellulosic fabric samples of cotton, viscose, lyocell and modal were pretreated with steam and dry heat in the range of 100-190°C. The samples were then treated with a Trichoderma reesei cellulase preparation (total culture filtrate - TC), with mechanical agitation, at a high enzyme dosage of 75% by weight of fabric, for 8 hours. Generally, viscose proved to be easily degradable, followed by cotton and modal. The degree of hydrolysis was the least for Lyocell. Dry heat pretreatments exerted a lower influence on degradation rate than steam pretreatments which showed a distinct maximum at a steam temperature of 130°C. The hydrolysis rate varied strongly depending on treatment conditions and fibre type. Water retention values in treated substrates were changed by up to 20% of initial values.     

In vitro production of Indian citrus ringspot virus (ICRSV) free Kinnow plants employing thermotherapy coupled with shoot tip grafting

Indian citrus ringspot virus (ICRSV) is known to cause serious disease problem in Kinnow (Citrus nobilis Lour × C. deliciosa Tenora) plants. This work reports the elimination of ICRSV by using thermotherapy coupled with shoot tip grafting in vitro. Nodal segments from infected mother plants (indexed by indirect ELISA and RT-PCR) were treated both in water bath and moist hot air at different temperatures viz. 40, 45 and 50°C for 30, 60 and 120 min and cultured on MS medium containing 2-iP (1 mg/l) and malt extract (800 mg/l). Shoot tips were excised from the nodal sprouts and grafted on to rough lemon (C. jambhiri) rootstock under aseptic conditions. Water bath treatment was found to be more effective as compared to moist hot air treatment as maximum number of ICRSV free plants (36.84%) were obtained by grafting the tips (0.7 mm) taken from the nodal segments treated at 50°C in water bath for 2 h. In an alternate treatment regime, 1-year-old infected plants were kept at various temperatures viz.36, 38 and 40°C in a thermotherapy chamber. Maximum of 60% ICRSV free plants were obtained by grafting the tips (0.7 mm) from the plants placed at 40°C followed by the plants placed at 38°C (59.09%) and the least was observed in case of the plants placed at 36°C (40.74%). Only those plants/plantlets were considered virus free, which showed negative reaction both in Indirect ELISA and RT-PCR.     

Temporal and spatial expression analysis of gamma kafirin promoter from Sorghum (Sorghum bicolor L. moench) var. M 35-1

Different cis acting elements of gamma kafirin gene from Sorghum bicolor var. M 35-1 were amplified and cloned using different combination of the primers. The amplified promoter was replaced with CaMV35S promoter of vector pCMBIA-1304 and resultant vector contained beta-glucuronidase (gus) gene under the control of amplified gamma-kafirin promoter. The resulting fusants were then transformed in to different explants of sorghum via particle bombardment. The regulation of uid gene expression was analyzed to find out the minimum required 5' regulatory sequence and cis acting elements for the efficient expression. However no gus expression was detected in leaves of micropropagated plants, scutellum and calli at any stage of growth. The expression of gus, with pKaf gus-P4 gene construct, was detected in immature embryos and endosperm 20 days after pollination (DAP). The result suggest that at least three motifs (two GCN4 and one prolamin box) besides TATA and CATC boxes are required for the efficient expression of the kafirin gene of sorghum. The study shows that PCR based isolation of different motifs and regions can be used as an alternate to deletion analysis for observing the role of various motifs and their importance in the gene expression and regulation.     

Calcium ionophore and phorbol ester increase membrane binding of annexin a7 in alveolar type II cells

The fusion of lamellar body with plasma membrane, a distal obligatory step in exocytosis of lung surfactant, may be mediated by annexin a7 (anx a7; synexin). To understand the mechanism of anx a7 action, we tested the hypothesis that anx a7 binding to membranes would increase in order to facilitate membrane fusion during stimulation of lung surfactant secretion. Isolated rat alveolar type II cells were treated with established secretagogues of lung surfactant and the membrane and cytosol fractions were analyzed for in vitro binding of anx a7. In cells treated with calcium ionophore (A23187) or phorbol 12-myristate 13-acetate (PMA), anx a7 binding to the membrane fraction was increased by 120%, while that to the cytosol fraction was decreased by 40%, when compared with binding to corresponding fractions from control cells. Protein kinase inhibitors prevented the PMA effects on anx a7 binding. The lamellar body and plasma membrane fractions of A23187-treated cells also showed increased binding of anx a7. The lamellar bodies of A23187-treated cells showed lower K(m) for Ca(2+) and higher maximal binding of anx a7, when compared with those from control cells. Collectively, our findings suggest that these two agents modify membrane proteins to regulate anx a7 binding, which may facilitate increased membrane fusion activity during stimulation of surfactant secretion.     

Penicillin acylases revisited: importance beyond their industrial utility

It is of great importance to study the physiological roles of enzymes in nature; however, in some cases, it is not easily apparent. Penicillin acylases are pharmaceutically important enzymes that cleave the acyl side chains of penicillins, thus paving the way for production of newer semi-synthetic antibiotics. They are classified according to the type of penicillin (G or V) that they preferentially hydrolyze. Penicillin acylases are also used in the resolution of racemic mixtures and peptide synthesis. However, it is rather unfortunate that the focus on the use of penicillin acylases for industrial applications has stolen the spotlight from the study of the importance of these enzymes in natural metabolism. The penicillin acylases, so far characterized from different organisms, show differences in their structural nature and substrate spectrum. These enzymes are also closely related to the bacterial signalling phenomenon, quorum sensing, as detailed in this review. This review details studies on biochemical and structural characteristics of recently discovered penicillin acylases. We also attempt to organize the available insights into the possible in vivo role of penicillin acylases and related enzymes and emphasize the need to refocus research efforts in this direction.     

Dysregulated cellular redox status during hyperammonemia causes mitochondrial dysfunction and senescence by inhibiting sirtuin-mediated deacetylation

Perturbed metabolism of ammonia, an endogenous cytotoxin, causes mitochondrial dysfunction, reduced NAD⁺/NADH (redox) ratio, and postmitotic senescence. Sirtuins are NAD⁺-dependent deacetylases that delay senescence. In multiomics analyses, NAD metabolism and sirtuin pathways are enriched during hyperammonemia. Consistently, NAD⁺-dependent Sirtuin3 (Sirt3) expression and deacetylase activity were decreased, and protein acetylation was increased in human and murine skeletal muscle/myotubes. Global acetylomics and subcellular fractions from myotubes showed hyperammonemia-induced hyperacetylation of cellular signaling and mitochondrial proteins. We dissected the mechanisms and consequences of hyperammonemia-induced NAD metabolism by complementary genetic and chemical approaches. Hyperammonemia inhibited electron transport chain components, specifically complex I that oxidizes NADH to NAD⁺, that resulted in lower redox ratio. Ammonia also caused mitochondrial oxidative dysfunction, lower mitochondrial NAD⁺-sensor Sirt3, protein hyperacetylation, and postmitotic senescence. Mitochondrial-targeted Lactobacillus brevis NADH oxidase (MitoLbNOX), but not NAD+ precursor nicotinamide riboside, reversed ammonia-induced oxidative dysfunction, electron transport chain supercomplex disassembly, lower ATP and NAD⁺ content, protein hyperacetylation, Sirt3 dysfunction and postmitotic senescence in myotubes. Even though Sirt3 overexpression reversed ammonia-induced hyperacetylation, lower redox status or mitochondrial oxidative dysfunction were not reversed. These data show that acetylation is a consequence of, but is not the mechanism of, lower redox status or oxidative dysfunction during hyperammonemia. Targeting NADH oxidation is a potential approach to reverse and potentially prevent ammonia-induced postmitotic senescence in skeletal muscle. Since dysregulated ammonia metabolism occurs with aging, and NAD⁺ biosynthesis is reduced in sarcopenia, our studies provide a biochemical basis for cellular senescence and have relevance in multiple tissues.     

Pyridine nucleotides in lung and liver of hypoxic rats

Adult male albino rats of Wistar strain in the weight range of 170–200 g were exposed to acute hypobaric hypoxia of 25,000 ft at 32°C for 90, 180, 270 and 360 min. respectively. The extramitochondrial dehydrogenases generating NADPH and the mitochondrial dehydrogenases generating NADH were studied in lung and liver. The oxidized and the reduced forms of these two nucleotides were determined at 90 min. and 360 min. of hypoxia. Hypoxia caused an initial decline in the activities of all the dehydrogenases studied. The activities of mitochondrial and pentose phosphate dehydrogenases of liver were high at 360 min. of hypoxia whereas the activities of extramitochondrial dehydrogenases of lung were generally low at all levels of hypoxic exposure. The NADPH levels of hypoxic rat lung were correspondingly low. However, the activities of liver mitochondrial dehydrogenases were elevated at 360 min. of hypoxia in spite of NADH accumulation in hypoxic rat liver. A decreased NAD/NADH ratio was observed in lung at 90 min. and 360 min. of hypoxia.