Vitamin C status of vitamin A-deficient rats

IT was initially suggested that vitamin A-deficiency leads to an interference in the biosynthesis of ascorbic acid, because depletion of vitamin A was found to cause a fall in the tissue-levels of ascorbate and diminished urinary ascorbic acid excretion in animals1-3. Mapson4, however, concluded that lowered ascorbic acid-levels in vitamin A-deficient rats is due to inanition only, because he was able to show that following chloretone treatment vitamin A-deficient and pair-fed vitamin A normal rats excrete comparable amounts of ascorbic acid in their urine and that restriction of food intake reduces the urinary ascorbate even in the chloretone-treated normal rats. Results of our preliminary experiments reported here clearly indicate that the synthesis of ascorbic acid in rats is markedly reduced during vitamin A-deficiency.

Potential functions for hydrogen bond interactions - III. Empirical Potential Function for the Peptide N-H...O=C Hydrogen Bond

A careful comparison of the distribution in the (R, θ)-plane of all NH ... O hydrogen bonds with that for bonds between neutral NH and neutral C=O groups indicated that the latter has a larger mean R and a wider range of θ and that the distribution was also broader than for the average case. Therefore, the potential function developed earlier for an average NH ... O hydrogen bond was modified to suit the peptide case. A three-parameter expression of the form {Mathematical expression}, with △ = R - Rmin, was found to be satisfactory. By comparing the theoretically expected distribution in R and θ with observed data (although limited), the best values were found to be p1 = 25, p3 = - 2 and q1 = 1 × 10-3, with Rmin = 2·95 Å and Vmin = - 4·5 kcal/mole. The procedure for obtaining a smooth transition from Vhb to the non-bonded potential Vnb for large R and θ is described, along with a flow chart useful for programming the formulae. Calculated values of ΔH, the enthalpy of formation of the hydrogen bond, using this function are in reasonable agreement with observation. When the atoms involved in the hydrogen bond occur in a five-membered ring as in the sequence[Figure not available: see fulltext.] a different formula for the potential function is needed, which is of the form Vhb = Vmin +p1△2 +q1x2 where x = θ - 50° for θ ≥ 50°, with p1 = 15, q1 = 0·002, Rmin = 2· Å and Vmin = - 2·5 kcal/mole. © 1971 Indian Academy of Sciences.

An optimum criterion for eccentrically loaded R. C. columns

An analysis of eccentrically loaded short reinforced concrete columns using a variable failure strain criterion is presented. The method dispenses with the usual procedure of assuming a fixed value for the ultimate strain in concrete. The analysis is based on the use of a simple, single equation for the complete stress-strain curve of concrete and the adoption of a process of maximisation of moment with respect to extreme fibre concrete compressive strain. Columns of rectangular section and loaded eccentrically along one axis only are considered in this paper. A good agreement is observed between the theoretical and experimental values of some test results.

Confined concrete—Its application in R.C. beams and frames

Stress-strain characteristics of concrete confined in steel binders have been determined. A new factor “confinement index” has been introduced for a quantitative measure of the confinement and using these results a “stress-block” has been developed. Tests have been made on simply supported reinforced concrete beams with spiral binder confinement and analysed on the basis of the proposed stress-block. Tests have also been made oon reinforced concrete portal frames and continuous beams with spiral binder confinement at sections of possible plastic hinge formation. An analysis of these tests indicates that a full redistribution of moments has taken place at ultimate.

Limit analysis of rectangular R.C. slabs on ground

A method is presented for obtaining lower bound on the carrying capacity of reinforced concrete foundation slab-structures subject to non-uniform contact pressure distributions. Functional approach suggested by Vallance for simply supported square slabs subject to uniform pressure distribution has been extended to simply supported rectangular slabs subject to symmetrical non-uniform pressure distributions. Radial solutions, ideally suited for rotationally symmetric problems, are shown to be adoptable for regular polygonal slabs subject to contact pressure paraboloids with constant edge pressures. The functional approach has been shown to be well suited even when the pressure is varying along the edges.

Heterocyclic Steroids .7. Rearrangement Of 1-Diethylamino-5-(Met-Methoxyphenoxy)-Pent-2-Yne

Thermal rearrangement of diethylamino5-(m methoxyphenoxy)-pent-2-yne (3) gives 1-(m-methexyphenoxy)-pent-3,4-diene (14) in about 8% yield. Hydration of the latter yields 1-(m-methoxyphenoxy)-pentan-4-one (6), which has been synthesised by an unambiguous route. A mechanism of formation of the allene (14) from the amine (3) has been suggested.

The Critical Role of N- and C-Terminal Contact in Protein Stability and Folding of a Family 10 Xylanase under Extreme Conditions

Background: Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive. Methodology: In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature,alkali pH, and protease and SDS treatment. Based on crystal structure,an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the Nand C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stabilityunder poly-extreme conditions. Conclusion: A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly,substitution of Phe4 with Trp increased stability in SDS treatment.Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as DF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N-and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.

Selective inhibition of cyclooxygenase-2 by C-phycocyanin, a biliprotein from Spirulina platensis

We report data from two related assay systems (isolated enzyme assays and whole blood assays) that C-phycocyanin a biliprotein from Spirulina platensis is a selective inhibitor of cyclooxygenase-a (COX-2) with a very low IC50 COX-2/IC50 COX-1 ratio (0.04). The extent of inhibition depends on the period of preincubation of phycocyanin with COX-2, but without any effect on the period of preincubation with COX-1. The IC50 value obtained for the inhibition of COX-2 by phycocyanin is much lower (180 nM) as compared to those of celecoxib (255 nM) and rofecoxib (401 nM), the well-known selective COX-2 inhibitors. In the human whole blood assay, phycocyanin very efficiently inhibited COX-2 with an IC50 value of 80 nM. Reduced phycocyanin and phycocyanobilin, the chromophore of phycocyanin are poor inhibitors of COX-2 without COX-2 selectivity. This suggests that apoprotein in phycocyanin plays a key role in the selective inhibition of COX-2. The present study points out that the hepatoprotective, anti-inflammatory, and anti-arthritic properties of phycocyanin reported in the literature may be due, in part, to its selective COX-2 inhibitory property, although its ability to efficiently scavenge free radicals and effectively inhibit lipid peroxidation may also be involved. (C) 2000 Academic Press.

Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (N alpha-Fmoc-Ser-[Ac-4,-beta-D-Gal-(1,3)-Ac(2)alpha-D-GalN(3)]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D H-1 NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, C alpha H chemical shift perturbations, (3)J(NH:C alpha H) couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.

Probing Fluorine Interactions in a Polyhydroxylated Environment: Conservation of a C-F center dot center dot center dot H-C Recognition Motif in Presence of O-H center dot center dot center dot O Hydrogen Bonds

Three conformationally locked fluorinated polycyclitols have been specially crafted on a rigid trans-decalin backbone, employing a surprisingly facile pyridine-poly(hydrogen fluoride)-mediated stereospecific epoxide ring opening as the key reaction. Molecula design of the three fluorinated probes under study focused on providing an efficient platform for (a) evaluating the ability of covalently bonded fluorine, vis-a-vis the isosteric hydroxy group, to act as a H-bond acceptor and (b) examining the possibility for an organic fluorine moiety, placed suitably in a spatially invariant position, to engage an 1,3-diaxial OH functionality in a purported intramolecular O-H center dot center dot center dot F hydrogen bond. The present endeavour reveals that C(sp(3))-F center dot center dot center dot H-C(sp(3)) hydrogen bonds, though weak and lesser investigated, can indeed be observed and supramolecular recognition motifs, involving such interactions, can be conserved even in crystal structures laden with stronger O-H center dot center dot center dot O hydrogen bonds.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn2+, at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent crosslinking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine

Members of the receptor-guanylate cyclase (rGC) family possess an intracellular catalytic domain that is regulated by an extracellular receptor domain. GC-C, an intestinally expressed rGC, was initially cloned by homology as an orphan receptor. The search for its Ligands has yielded three candidates: STa (a bacterial toxin that causes traveler's diarrhea) and the endogenous peptides uroguanylin and guanylin. Here, by performing Northern and Western blots, and by measuring [I-125]STa binding and STa-dependent elevation of cGMP levels, we investigate whether the distribution of GC-C matches that of its endogenous ligands in the rat intestine. We establish that 1) uroguanylin is essentially restricted to small bowel; 2) guanylin is very low in proximal small bowel, increasing to prominent levels in distal small bowel and throughout colon; 3) GC-C messenger RNA and STa-binding sites are uniformly expressed throughout the intestine; and 4) GC-C-mediated cGMP synthesis peaks at the proximal and distal extremes of the intestine (duodenum and colon), but is nearly absent in the middle (ileum). These observations suggest that GC-C's activity may be posttranslationally regulated, demonstrate that the distribution of GC-C is appropriate to mediate the actions of both uroguanylin and guanylin, and help to refine current hypotheses about the physiological role(s) of these peptides.