Interaction of aldehydes with collagen: effect on thermal, enzymatic and conformational stability

Stabilization of type I rat tail tendon (RTT) collagen by various aldehydes, viz. formaldehyde, gluteraldehyde, glyoxal and crotanaldehyde was studied to understand the effect of each on the thermal, enzymatic and conformational stability of collagen. The aldehydes have been found to increase the heat stability of rat tail tendon collagen fibres from 62 to 77-86 degrees C. The increase in thermal stability was found to be in a species dependent manner. The variation in the thermal stability of collagen brought about by aldehydes was in the order of formaldehyde > gluteraldehyde > glyoxal > crotanaldehdye. The aldehydes also impart a high degree of stability to collagen against the activity of the degrading enzyme, collagenase. The order of enzymatic stability brought about by aldehydes follows the same trend as the thermal stability brought about by them. This shows that the number of cross-links formed influence both the thermal and enzymatic stability in the similar manner. The effect of various aldehydes on the secondary structure of collagen was studied using circular dichroism and it was found that the aldehydes lead to changes in the amplitude of the circular dichroic (CD) spectrum but did not alter the triple helical conformation of collagen. The secondary structure of collagen is not significantly altered on interaction with different aldehydes.     

Progress and recent trends in biotechnological methods for leather processing

Global environmental regulations are changing the leather-processing industry. Pre-tanning and tanning processes contribute 80-90% of the total pollution in the industry and generate noxious gases, such as hydrogen sulfide, as well as solid wastes, such as lime and chrome sludge. The use of enzyme-based products is currently being explored for many areas of leather making. Furthermore, enzymes are gaining increasing importance in the de-hairing process, eliminating the need for sodium sulfide. This review discusses emerging novel biotechnological methods used in leather processing. One significant achievement is the development of a bioprocess-based de-hairing and fiber-opening methodology to reduce toxic waste.     

Apoptosis of lymphocytes induced by chromium(VI/V) is through ROS-mediated activation of Src-family kinases and caspase-3

Mechanistic insights into Cr(VI)-induced carcinogenicity and possible implication of Cr(V) species formed by the redox reactions of chromium-bearing species have attracted interest. We have previously demonstrated that when human peripheral blood lymphocytes are exposed to the Cr(V) complexes, viz., sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[Cr(V)O(ehba)(2)] and sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[Cr(V)O(hmba)(2)], apoptosis and formation of reactive oxygen species (ROS) are observed. The molecular mechanisms involving cellular signaling pathways leading to apoptosis are addressed in the present study. Treatment of lymphocytes with Na[Cr(V)O(ehba)(2)] and K(2)Cr(2)O(7) leads to the activation of the Src-family protein tyrosine kinases namely, p56(lck), p59(fyn), and p56/53(lyn), which then activates caspase-3, both of which are under the partial influence of ROS. Inhibition of the Src-family tyrosine kinases activity by PP2 and of caspase-3 by Z-DEVD-FMK reverses apoptosis, thereby suggesting their importance. Antioxidants only partially reverse the apoptosis induced by Cr(VI/V), suggesting that pathways other than those induced by ROS cannot be ruled out. Although the complex, Na[Cr(V)O(ehba)(2)] is known to be relatively stable in aqueous solutions, previous studies have shown that the Cr(V) complex, Na[Cr(V)O(ehba)(2)] disproportionates to Cr(VI) and Cr(III) forms at pH 7.4 through complex mechanistic processes. Dynamics studies employing EPR data show that the Cr(V) state in Na[Cr(V)O(ehba)(2)] is relatively more stable in RPMI-1640 medium containing plasma. Formation of ROS during the reaction of redox partners with Na[Cr(V)O(ehba)(2)] is an early event and compares favorably in kinetic terms with the reported rate processes for disproportionation. This investigation presents evidence for the direct implication of Cr(V) in Cr(VI)-induced apoptosis of lymphocytes.     

Caspase-3: Its potential involvement in Cr(III)-induced apoptosis of lymphocytes

In this study, we have examined the role of caspase-3 in apoptosis of lymphocytes induced by the chromium(III) complexes viz. tris-(1,10-phenanthroline)chromium(III) chloride (Cr(III)-phen) and trans-diaqua[1,3-bis(salicylideneamino)propanechromium(III)] perchlorate (Cr(III)-salprn). Evidence for caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in lymphocytes exposed to Cr(III) complexes is revealed through Western blotting analysis. Blocking the activity of caspase-3 with z-DEVD-fmk, prevents apoptosis as evidenced through [3H]-thymidine incorporation, DNA fragmentation assay and measurement of sub-G1 cells by flow cytometry. Pretreatment of lymphocytes with free radical scavengers completely attenuates the activity of caspase-3 suggesting that reactive oxygen species (ROS) are upstream activators of caspase-3. Preincubation of lymphocytes with PP2, a selective Src-family tyrosine kinase inhibitor, abolishes the activation of caspase-3 indicating that Src-family tyrosine kinases viz. p56lck, p59fyn and p53/56lyn are mediators of caspase-3 activation during Cr(III) exposure. Collectively, our findings support a plausible mechanism in which Cr(III) mediates ROS generation that precedes the up-regulation of p56lck, p59fyn and p53/56lyn which eventually activates caspase-3 to promote apoptotic cell death of lymphocytes. To our knowledge, this is the first report suggesting the importance of Src-family tyrosine kinases for the activation of caspase-3 in metal-induced apoptotic cell death.     

Basic and applied research in India: Present and future

Models for supporting organized applied research programs Position of India in the global scenario of basic research New policy initiatives and programs Conclusion     

Chromium(III)-induced apoptosis of lymphocytes: death decision by ROS and Src-family tyrosine kinases

Apoptosis is an active process induced by a variety of physiological and external stimuli, in which elimination of damaged cells are effected through a genetically controlled process. In this study, we have examined the mechanism of chromium(III) [Cr(III)]-induced cytotoxicity with respect to its relationship to oxidative stress. Morphology, flow cytometry, and DNA fragmentation studies show that tris-(1,10-phenanthroline)chromium(III) [Cr(III)-phen], tris-(2,2′-bipyridyl)chromium(III) [Cr(III)-bpy], trans-diaqua[1,2-bis(salicylideneamino)ethanechromium(III)] [Cr(III)-salen], and trans-diaqua[1,3-bis(salicylideneamino)propanechromium(III)] [Cr(III)-salprn] induced apoptosis of lymphocytes. Pentaammineaquachromium(III) [Cr(III)-hpa] does not induce apoptosis. Apoptosis induced by these complexes involves the generation of reactive oxygen species (ROS) as seen by increased fluorescence of dichloroflourescein (DCF) observed through flow cytometry. Pretreatment of lymphocytes with antioxidants completely abrogate apoptosis. Cr(III) treatment also increased the expression and activation of Src-family tyrosine kinases viz. p56lck, p59fyn, and p53/56lyn, as seen by immunoblotting and immune complex kinase assay. PP2, a selective Src-family tyrosine kinase inhibitor, abolishes apoptosis, indicating that Src-family tyrosine kinases are directly involved in eliciting apoptosis. Interestingly, a one-to-one correlation between the expression of Src-family tyrosine kinases and ROS is observed, since antioxidants pretreatment inhibits the expression and the activation of these kinases. These results further indicate that Cr(III)-induced apoptosis is mediated through production of ROS, which in turn activates the Src-family tyrosine kinases. The increased activation of Src-family tyrosine kinases may be a mechanism involved in apoptosis of lymphocytes elicited by various other physiological stimuli that exploit ROS as a second messenger.     

Molecular dynamic simulation studies on the effect of one residue chain staggering on the structure and stability of heterotrimeric collagen-like peptides with interruption

A systematic molecular dynamics (MD) simulation has been carried out on collagen-like peptides with different combinations of interruptions in the Gly-X(AA) -Y(AA) repeats. Although experimental studies have been carried out to elucidate the structural consequences of homotrimeric collagen-like peptides, this is the first report on the structural effect on the heterotrimeric models with G4G and G1G breaks present simultaneously in the constituent chains with difference in one residue chain staggering. The results reveal that the axial registry of the interrupted region changes significantly from that of conventional triple helical peptide without interruption. Further, results from MD simulations show the formation of a kink in the interrupted region of the triple-helical peptides. The conformational analysis reveals that the interruption in the Gly-X(AA) -Y(AA) pattern in these peptides induces β-strand conformation in triple helical peptides. The conventional hydrogen bonds in the interrupted triad are affected and new nonconventional H-bonds are formed in the triple helical structure, and as a result interrupted region becomes locally fragile. MM-PBSA calculations on the different systems clearly suggest that the binding affinity varies marginally due to one residue staggering. However, it is found from the structural parameters that hydrogen-bonding pattern differs significantly due to the difference in the staggering of chains.