Relationship between NF-kappaB and trypsinogen activation in rat pancreas after supramaximal caerulein stimulation

Intra-acinar cell nuclear factor-kappaB (NF-kappaB) and trypsinogen activation are early events in secretagogue-induced acute pancreatitis. We have studied the relationship between NF-kappaB and trypsinogen activation in rat pancreas. CCK analogue caerulein induces early (within 15 min) parallel activation of both NF-kappaB and trypsinogen in pancreas in vivo as well as in pancreatic acini in vitro. However, NF-kappaB activation can be induced without trypsinogen activation by lipopolysaccharide in pancreas in vivo and by phorbol ester in pancreatic acini in vitro. Stimulation of acini with caerulein after 6 h of culture results in NF-kappaB but not trypsinogen activation. Protease inhibitors (AEBSF, TLCK, and E64d) inhibit both intracellular trypsin activity and NF-kappaB activation in caerulein stimulated acini. A chymotrypsin inhibitor (TPCK) inhibits NF-kappaB activation but not trypsin activity. The proteasome inhibitor MG-132 prevents caerulein-induced NF-kappaB activation but does not prevent trypsinogen activation. These findings indicate that although caerulein-induced NF-kappaB and trypsinogen activation are temporally closely related, they are independent events in pancreatic acinar cells. NF-kappaB activation per se is not required for the development of early acinar cell injury by supramaximal secretagogue stimulation.     

LEDGF binds to heat shock and stress-related element to activate the expression of stress-related genes

We have investigated the mechanism by which LEDGF protects cells against environmental stress. Our earlier report showed that a low level of LEDGF was present in the nucleus of most cell types and significant elevation of LEDGF level was induced by heat and oxidative stress. The cells overexpressing LEDGF-activated expression of heat shock proteins and enhanced survival of many cell types. Here we show that LEDGF binds to heat shock element (HSE) and stress-related regulatory element (STRE) to activate the expression of stress-related genes (Hsp27 and alphaB-crystallin). Apparently, HSE and STRE are present in promoters of many stress-related genes. Elevation of many stress-related proteins (STRPs) induced by LEDGF may protect cells against environmental stress. In yeast, it has been demonstrated that a single stress can activate the expression of multiple STRPs. This is known as "cross-protection," and now similar mechanism has been found in mammalian cells and LEDGF plays a vital role in it.     

C-terminal region amino acid substitutions contribute to catalytic differences between murine class alpha glutathione transferases mGSTA1-1 and mGSTA2-2 toward anti-diol epoxide isomers of benzo[c]phenanthrene

The molecular basis for catalytic differences between structurally closely related murine class alpha glutathione (GSH) transferases mGSTA1-1 and mGSTA2-2 in the GSH conjugation of anti-diol epoxide isomers of benzo[c]phenanthrene (anti-B[c]PDE) was investigated. GSH conjugation of both (-)- and (+)-enantiomers of anti-B[c]PDE was observed in the presence of mGSTA1-1 (60 and 40% GSH conjugation, respectively), whereas mGSTA2-2 exhibited a preference for the (-)-anti-isomer (>97%). In addition, the specific activity of mGSTA2-2 toward the (-)-anti-B[c]PDE isomer was relatively higher than that of mGSTA1-1. The amino acid sequences of mGSTA1-1 and mGSTA2-2 differ at 10 positions that are distributed in three sections. Section I contains amino acid residues in positions 65 and 95; section II contains residues in positions 157, 162, and 169, and section III contains residues in positions 207, 213, 218, 221, and 222. Enzyme activity measurements with chimeras of mGSTA1-1 and mGSTA2-2 revealed that amino acid substitutions in section III account for their differential enantioselectivity and catalytic activity toward anti-B[c]PDE. Site-directed mutagenesis of amino acid residues in section III of mGSTA2-2 with corresponding residues of mGSTA1-1 followed by activity measurements of the wild type and mutated enzymes indicates that leucine 207 and phenylalanine 221 may be critical for the high catalytic activity of mGSTA2-2 toward (-)-anti-B[c]PDE. Molecular modeling studies demonstrated that the active site of mGSTA1-1 accommodates both enantiomers of anti-B[c]PDE, whereas the (-)-anti-isomer interacts more favorably with active site residues in mGSTA2-2. The results of this study clearly indicate that amino acid substitutions in the C-terminal region contribute to catalytic differences between mGSTA1-1 and mGSTA2-2 with respect to anti-B[c]PDE.     

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.     

Activity of glutathione related enzymes and ovarian steroid hormones in different sizes of follicles from goat and sheep ovary of different reproductive stages

The investigations on enzymes related to glutathione like glutathione-S-transferase (GST) and glutathione peroxidase (GSH-Px) have been carried out mostly in human and rat ovaries, however the studies on these enzymes in ruminants are relatively absent. In the present study the changes in the activity of these enzymes, in different sizes of follicles from goat and sheep ovaries of different reproductive stages, were investigated. The results demonstrated that the activity of the enzyme GST increased with the increase in size of the follicles from small to large follicles of follicular phase ovary and from small to medium follicles of luteal phase ovary in both the species, thereafter it decreased in large follicles of luteal phase ovary. There was increasing pattern in the activity of GSH-Px in the follicular phase follicles and a decreasing pattern in the luteal phase follicles from both the species. Thus the changes in the activity of glutathione related enzymes namely GST and GSH-Px in different size follicles from both the species during different reproductive phases are evident from the results. It is reasonable, therefore, to assume that these enzymes may have functional role in the steroid hormone metabolism in ruminant ovary as reported in human ovary.     

Antiallergic/antiasthmatic effect of novel antiallergic hexapeptide-95/220 in various experimental models

The effects of newly synthesized antiallergic hexapeptide 95/220 was investigated on various allergic and asthmatic test models. This newly developed peptide was found to be more potent than clinically used drug disodium cromoglycate (DSCG). Hexapeptide 95/220 inhibited immediate hypersensitivity reactions such as passive cutaneous anaphylaxis (PCA) and mast cell degranulation in rats, antigen-induced bronchoconstriction in actively sensitized guinea pigs in dose dependent manner like DSCG. Antigen-induced contraction of guinea pig ileum was also markedly inhibited by this newly developed hexapeptide in the same fashion as ketotifen and DSCG did but at comparatively lower dose. Egg albumin-induced histamine release was also blocked by this hexapeptide from chopped lung tissues of sensitized guinea pigs. These results suggest that hexapeptide' 95/220 has potent inhibitory effect on immediate hypersensitivity reactions thereby inhibiting mediator release from mast cell. Moreover, this newly synthesized peptide is orally active and effective at lower doses as compared to standard drugs.     

In vitro regeneration of Hypericum patulum Thunb.--a medicinal plant

A protocol was developed for high frequency plant regeneration in H. patulum by shoot-tip culture. H. patulum plants were collected from a wild source growing at high altitude in the eastern Himalayas. Multiple buds were initiated from shoot-tips cultured on Murashige and Skoog's medium supplemented with BAP, kinetin. Addition of thiamin HCI, Ca-pantothenate and biotin enhanced multiple shoot formation. Upon transfer to phytohormone free liquid medium following a brief exposure to auxin, root formation occurred from the micro shoots . Rooted plants were hardened and transferred to soil. Regeneration potentiality was found to be constant throughout the year in long term cultures.     

Interconversion of free sugars in relation to activities of enzymes catalyzing synthesis and cleavage of sucrose in growing stem tissues of sorghum

Free sugar interconversion and activities of soluble acidic (pH 4.8) and neutral (pH 7.5) invertases, sucrose synthase (synthesis) and sucrose phosphate synthase were investigated in the growing nodes and internodes of sorghum (Sorghum vulgare). The results were substantiated with incorporation of 14C from supplied sucrose and hexoses into endogenous sugars of these stem tissues. With the advancement in plant growth, the content of total free sugars in apical nodes and internodes increased till 70 DAS (flowering stage) followed by a decline. In the corresponding basal tissues, the sugar build-up continued even beyond this stage of plant growth. Compared with basal stem tissues, the apical ones contained high activities of soluble invertases and a low proportion amongst free sugars of sucrose. The activities of sucrose-hydrolyzing enzymes were higher as compared with those of sucrose-synthesizing ones in both nodes and internodes and with the growth of plant, the activity of neutral invertase increased in these tissues. More 14C from supplied sucrose and hexoses appeared in extracted sugars from cut discs of apical nodes and internodes in comparison with their basal counterparts. 14C from supplied sucrose appeared in glucose, fructose and from supplied hexoses appeared in sucrose. The results suggest that in apical nodes and internodes, where a rapid cell division and cell expansion occur, sucrose is obligatorily inverted to meet the increased requirement of hexoses and there is a compartmentalized synthesis and cleavage of sucrose in the nodes and internodes of growing sorghum plant.     

Characterization of Lr46, a gene conferring partial resistance to wheat leaf rust

Components of resistance conferred by the Lr46 gene, reported as causing "slow rusting" resistance to leaf rust in wheat, were studied and compared with the effects of Lr34 and genes for quantitative resistance in cv. Akabozu. Lr34 is a gene that confers non-hypersensitive type of resistance. The effect of Lr46 resembles that of Lr34 and other wheats reported with partial resistance. At macroscopic level, Lr46 produced a longer latency period than observed on the susceptible recurrent parent Lalbahadur, and a reduction of the infection frequency not associated with hypersensitivity. Microscopically, Lr46 increased the percentage of early aborted infection units not associated with host cell necrosis and decreased the colony size. The effect of Lr46 is comparable to that of Lr34 in adult plant stage, but in seedling stage its effect is weaker than that of Lr34.