Process optimization for the production of sugar for the bioethanol industry from Tapioca,a non-conventional source of starch

A commercial preparation of -amylase, Biotempase, obtained from Biocon India Pvt. Ltd., and crude glucoamylase produced from Aspergillus sp. NA21 were used to hydrolyse tapioca powder, a non-conventional starchy substrate. Among various concentrations of starch (15–35%, dry weight/volume) tried for maximum liquefaction; slurry made with 25% substrate concentration proved optimal. An economical process of liquefaction was carried out using steam under pressure (0.2–0.3 bar, 104–105 °C) to liquefy a 25% slurry in just 45 min, contrary to a slower process carried out at 95 °C in a water bath. For liquefaction of starch a pH of 5.0 proved to be optimum. The dose of Biotempase as prescribed by the supplier could be reduced by 33% achieving the same degree of liquefaction, by addition of CaCl₂ to the starch slurry at the concentration of 120 mg/l. The conditions for the saccharification of liquefied starch were optimized to be 60 °C and pH 5.0, producing 90% saccharification in 24 h. Supplementation of divalent ions Ca²⁺, Mg²⁺ and Zn²⁺ in the process of saccharification showed no effect. Finally glucose was found to be the main hydrolysis product in the saccharification of tapioca starch.     

Cloning and sequencing of complete tau-crystallin cDNA from embryonic lens of Crocodylus palustris

τ-Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete τ-crystallin cDNA from the embryonic lens ofCrocodylus palustris and establish it to be identical to the α-enolase gene from non-lenticular tissues. Quantitatively, the τ-crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile τ-crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5′- and 3′-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete τ-crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5′- and 3′-ends respectively. Further, it was found to be identical to its putative counterpart enzyme α-enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of τ-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.     

Thalidomide suppresses NF-kappa B activation induced by TNF and H2O2, but not that activated by ceramide, lipopolysaccharides, or phorbol ester

Thalidomide ([+]-alpha-phthalimidoglutarimide), a psychoactive drug that readily crosses the blood-brain barrier, has been shown to exhibit anti-inflammatory, antiangiogenic, and immunosuppressive properties through a mechanism that is not fully established. Due to the central role of NF-kappaB in these responses, we postulated that thalidomide mediates its effects through suppression of NF-kappaB activation. We investigated the effects of thalidomide on NF-kappaB activation induced by various inflammatory agents in Jurkat cells. The treatment of these cells with thalidomide suppressed TNF-induced NF-kappaB activation, with optimum effect occurring at 50 microg/ml thalidomide. These effects were not restricted to T cells, as other hematopoietic and epithelial cell types were also inhibited. Thalidomide suppressed H(2)O(2)-induced NF-kappaB activation but had no effect on NF-kappaB activation induced by PMA, LPS, okadaic acid, or ceramide, suggesting selectivity in suppression of NF-kappaB. The suppression of TNF-induced NF-kappaB activation by thalidomide correlated with partial inhibition of TNF-induced degradation of an inhibitory subunit of NF-kappaB (IkappaBalpha), abrogation of IkappaBalpha kinase activation, and inhibition of NF-kappaB-dependent reporter gene expression. Thalidomide abolished the NF-kappaB-dependent reporter gene expression activated by overexpression of TNFR1, TNFR-associated factor-2, and NF-kappaB-inducing kinase, but not that activated by the p65 subunit of NF-kappaB. Overall, our results clearly demonstrate that thalidomide suppresses NF-kappaB activation specifically induced by TNF and H(2)O(2) and that this may contribute to its role in suppression of proliferation, inflammation, angiogenesis, and the immune system.     

Transcription corepressor CtBP is an NAD(+)-regulated dehydrogenase

The Lcd1p/Mec1p complex is crucial for normal S phase progression and for signaling DNA damage. We show that Lcd1p/Ddc2p and Mec1p in cell extracts bind to DNA ends. Although Lcd1p binds DNA independently of Mec1p, recruitment of Mec1p to DNA requires Lcd1p. DNA binding by Lcd1p is also independent of Rad9p, Rad17p, and Rad24p. Recombinant Lcd1p binds DNA, and this is impaired by Lcd1p mutations that abrogate its in vivo functions. Furthermore, Mec1p is recruited to cdc13-induced DNA damage and HO endonuclease-induced double-strand breaks in vivo. This requires Lcd1p, and recruitment of Lcd1p/Mec1p to cdc13-induced damage is abolished by Lcd1p mutations that abrogate its in vivo functions. Recruitment of Lcd1p to these lesions is independent of Mec1p and Rad9p/Rad24p. Thus, recruitment of Mec1p to DNA lesions by Lcd1p is crucial for the DNA damage response.     

RNA recognition via the SAM domain of Smaug

The Nanos protein gradient in Drosophila, required for proper abdominal segmentation, is generated in part via translational repression of its mRNA by Smaug. We report here the crystal structure of the Smaug RNA binding domain, which shows no sequence homology to any previously characterized RNA binding motif. The structure reveals an unusual makeup in which a SAM domain, a common protein-protein interaction module, is affixed to a pseudo-HEAT repeat analogous topology (PHAT) domain. Unexpectedly, we find through a combination of structural and genetic analysis that it is primarily the SAM domain that interacts specifically with the appropriate nanos mRNA regulatory sequence. Therefore, in addition to their previously characterized roles in protein-protein interactions, some SAM domains play crucial roles in RNA binding.     

Monomeric midkine induces tumor cell proliferation in the absence of cell-surface proteoglycan binding

Midkine (MK), a retinoic acid-inducible heparin-binding protein, is a mitogen which initiates a cascade of intracellular protein tyrosine phosphorylation mediated by the JAK/STAT pathway after binding to its high affinity p200(+)/MKR cell surface receptor in the G401 cell line [Ratovitski, E. A. (1998) J. Biol. Chem. 273, 3654-3660]. In this study, we determined the biophysical characteristics of purified recombinant murine MK and analyzed the requirements for ligand multimerization and cell surface proteoglycan binding for the G401 cell mitogenic activity of MK. Our studies indicate that the secreted form of MK (M = 13 kDa) exists in solution as an asymmetric monomer with a frictional coefficient of 1. 48 and a Stokes radius of 23.7 A. By constructing bead models of MK using the program AtoB and the program HYDRO to predict the hydrodynamic properties of each model, our data suggest that MK has a dumb-bell shape in solution composed of independent N- and C-terminal domains separated by an extended linker. This asymmetric MK monomer is a biologically active ligand with mitogenic activity on G401 cells in vitro. Neither heparin-induced formation of noncovalent MK multimers nor tissue transglutaminase II covalent multimerization of MK enhanced MK mitogenic activity in this system. Since neither heparin competition nor cell treatment with chondroitinase ABC or heparinase III abolished the mitogenic effects of MK on G401 cells, cell-surface proteoglycan binding by MK does not appear to be a requirement for its observed mitogenic effects. These results provide strong evidence that the MK-specific p200(+)/MKR has distinctive biochemical properties which distinguish it from the receptor tyrosine phosphatase cell-surface proteoglycan PTPzeta/RPTPbeta and support the hypothesis that the diverse biological effects of MK are mediated by multiple cell-specific signal transduction receptors.     

Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1. 5 A resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar alpha/beta core, but in BglII, the core is augmented by a beta-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein-DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.     

Immunocytochemical demonstration of vasopressin binding in rat kidney

We investigated the immunoperoxidase demonstration of vasopressin (VSP) bound to paraffin-embedded sections of rat kidney and the effects of various fixatives. Slices of rat kidney from normal and 4-day water-deprived rats were incubated with 10(-7) M VSP, fixed, and embedded in paraffin. Hydrated sections of these tissues were again incubated with 10(-7) M VSP or 10(-7) M VSP and 10(-5) M oxytocin (OXY). VSP bound to the sections was demonstrated using rabbit anti-Arg8 VSP antiserum and peroxidase-labeled second antibody. In sections of kidney from both normal and water-deprived rats, immunoperoxidase labeling was most intense in the renal papilla and was restricted to the cells of the ducts of Bellini and loops of Henle. In the medulla, the collecting ducts and medullary thick ascending limbs of Henle were moderately stained. In the normal kidney sections there was no staining of the proximal tubules, distal convoluted tubules (DCT), and only slight staining of the cortical collecting ducts (CCD). However, in the water-deprived rats there was a considerable increase in the staining of the DCT and CCD. Simultaneous incubation in OXY and VSP resulted in reduced immunoperoxidase labeling of the tubules. Omission of VSP incubation led to a similar decrease in stain intensity, indicating a specificity for the sites of VSP binding. This technique allows the identification of cells responsible for the binding of VSP in the kidney.     

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.