Detergent dissection of membrane proteins of Entamoeba histolytica and its effect on lymphokine release in in vitro.

Fifty-two amoebic liver abscess cases were assessed for the release of lymphokines (LMIF) using detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200) obtained with sodium deoxycholate treatment. Lymphokines release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was tested by leukocyte migration inhibition test on blood samples from amoebic liver abscess cases. A significant increase was noted in the release of LMIF and 100% positivity was observed with DDMP compared to whole amoebic extract with a positivity of 73%. The difference between means of the above two with regards to release of LMIF was found to be highly significant (P less than 0.005). This shows the patients had high degree of leukocyte sensitization to surface antigens of E. histolytica compared to the whole amoebic lysate. These findings suggest that the antigens shed might have important role as a potent antigen in elicitation of CMI response in amoebic liver abscess cases.     

Time course studies on the functional evaluation of experimental chronic myocardial infarction in rats

In vivo models of myocardial infarction induced by coronary artery ligation (CAL) in rats usually suffer from high early mortality and a low rate of induction. This study investigated the time course initiation of chronic myocardial infarction (CMI) in albino rats and the possibility of reducing early mortality rate due to myocardial infarction by modification of the surgical technique. CAL was carried out by passing the suture through the epicardial layer around the midway of the left anterior descending coronary artery including a small area of the myocardium to avoid mechanical damage to the heart geometry. In addition, the role of endothelin-1 (ET-1) in rat heart with congestive heart failure was critically assessed. Time course initiation experiments were designed by sacrificing the animals at different time intervals and by carrying out physiological, biochemical, histopathological, electron microscopical and immunohistochemical studies. Specific markers of myocardial injury, viz. cardiac troponin-T (cTnT), high sensitivity C-reactive protein, lactate dehydrogenase and fibrinogen were measured at different time points. Serum marker enzymes and activities of lysosomal hydrolases were found to be elevated on the eighth day post-ligation. Histopathological studies demonstrated focal areas showing fibrovascular tissue containing fibroblasts, collagenous ground substance and numerous small capillaries replacing cardiac muscle fibers. Transmission electron micrographs exhibited mitochondrial changes of well-developed irreversible cardiac injury, viz. swelling, disorganization of cristae, appearance of mitochondrial amorphous matrix densities, significant distortion of muscle fibers and distinct disruption of the intercalated discs. Immunoblotting studies confirmed the presence of alpha 2-macroglobulin which supported the inflammatory response. The severity of the CMI was inferred by the measurement of the level of ET-1 in plasma and left ventricle which was significantly higher in the CMI rats than in the sham-operated rats. Immunohistochemical studies at different time intervals showed that there was a significant immunoexpression of ET-1 on the eighth day post-ligation. This study conclusively showed that ligation of left anterior descending artery minimized mortality and ET-1 was expressed during CMI.     

Misfolded proteins and human diseases

Though protein folding is a regular phenomenon inside the cellular milieu, slight differences in the folding pattern of proteins may lead to disease-causing forms. Many diseases have been identified that are caused by these misfolded macromolecules and a considerable amount of focus is still directed towards better understanding of the processes that lead to these misfolded structures. Further progress in the field of how soluble proteins begin to misfold and how resultant oligomers begin dysfunction offers exciting prospects for specific molecular intervention.     

AhpF can be dissected into two functional units: tandem repeats of two thioredoxin-like folds in the N-terminus mediate electron transfer from the thioredoxin reductase-like C-terminus to AhpC

AhpF, the flavin-containing component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the NADH-dependent reduction of an active-site disulfide bond in the other component, AhpC, which in turn reduces hydroperoxide substrates. The amino acid sequence of the C-terminus of AhpF is 35% identical to that of thioredoxin reductase (TrR) from Escherichia coli. AhpF contains an additional 200-residue N-terminal domain possessing a second redox-active disulfide center also required for AhpC reduction. Our studies indicate that this N-terminus contains a tandem repeat of two thioredoxin (Tr)-like folds, the second of which contains the disulfide redox center. Structural and catalytic properties of independently expressed fragments of AhpF corresponding to the TrR-like C-terminus (F[208-521]) and the 2Tr-like N-terminal domain (F[1-202]) have been addressed. Enzymatic assays, reductive titrations, and circular dichroism studies of the fragments indicate that each folds properly and retains many functional properties. Electron transfer between F[208-521] and F[1-202] is, however, relatively slow (4 x 10(4) M(-)(1) s(-)(1) at 25 degrees C) and nonsaturable up to 100 microM F[1-202]. TrR is nearly as efficient at F[1-202] reduction as is F[208-521], although neither the latter fragment, nor intact AhpF, can reduce Tr. An engineered mutant AhpC substrate with a fluorophore attached via a disulfide bond has been used to demonstrate that only F[1-202], and not F[208-521], is capable of electron transfer to AhpC, thereby establishing the direct role this N-terminal domain plays in mediating electron transfer between the TrR-like part of AhpF and AhpC.     

Rationalization of poor solubility of TGF-β3 using MD simulation

Despite sequence and structural similarity, TGF-β3 has low solubility among other isoforms of TGF-β. We used nanosecond of molecular dynamic simulations (MD) with explicit solvent, alone and in presence of urea, to investigate the intermediates resulting from the unfolding process of TGF-β3 and TGF-β1. MD simulations of the full-length proteins show a very early loss of α-helix in TGF-β3 compared to the one in the TGF-β1. MD simulation of a small fragment consisting of H3 α-helix of TGF-β3 shows conversion of this segment to β-sheet. Relative instability of H3 α-helix in TGF-β3 and its propensity to form β-sheet may explain the poor solubility of TGF-β3 compared to TGF-β1. The other reasons for poor solubility of TGF-β3 may be the hydrophobic patches on its surface and low charge over the entire range of pH.     

Discovery of 3-hydroxy-4-cyano-isoquinolines as novel, potent, and selective inhibitors of human 11β-hydroxydehydrogenase 1 (11β-HSD1)

Derived from the HTS hit 1, a series of hydroxyisoquinolines was discovered as potent and selective 11β-HSD1 inhibitors with good cross species activity. Optimization of substituents at the 1 and 4 positions of the isoquinoline group in addition to the core modifications, with a special focus on enhancing metabolic stability and aqueous solubility, resulted in the identification of several compounds as potent advanced leads.     

Mechanisms for variability in a member of the scavenger-receptor cysteine-rich superfamily

 This study reports a molecular analysis of pig WC1, a new member of the scavenger-receptor cysteine-rich (SRCR) superfamily. The pig WC1 contains up to six extra-cellular SRCR domains, highly homologous to other members of the family. However, the striking feature of the WC1 gene, as for its cattle and sheep homologues, is that it is present as a multigene family showing extensive sequence diversity, for both DNA and predicted protein sequence. The basis of this diversity was examined and was shown to be attributable to several different causes. These included single base-pair changes within SRCR domains, the optional usage of whole domains or exons, including a SRCR domain and the proximal “hinge” region, and alternative isoforms of the putative cytoplasmic tail. These results suggest that WC1 may code for a new, though more primitive type of antigen recognition structure specific for γ/δ T cells. Received: 12 November 1996 / Received: 10 March 1997