Ram Sharma

A human gene encoding morphine modulating peptides related to NPFF and FMRFamide

FMRFamide‐related peptides have been isolated from both invertebrates and vertebrates and exhibit a wide range of biological effects in rats. We show here that in humans 2 FMRFamide‐related peptides are encoded by a single gene expressed as a spliced mRNA. The larger predicted peptide (AGEGLNSQFWSLAAPQRFamide) differs from the peptide isolated from bovines (AGEGLSSPFWSLAAPQRFamide) by the substitutions of 2 amino acids. The shorter predicted peptide (NPSF, SQAFLFQPQRFamide) is 3 amino acids longer than the bovine 8 amino‐acid NPFF (FLFQPQRFamide) or the human NPFF peptide isolated from serum [5] , suggesting that the encoded protein is subject to cleavage by a tripeptidyl peptidase or by a novel processing mechanism. On rat spinal cord, the larger peptide is indistinguishable in activity from the equivalent bovine peptide whereas the smaller extended peptide is inactive.

Hydrophobic mismatch and the incorporation of peptides into lipid bilayers: a possible mechanism for retention in the Golgi.

Preferential interaction of trans-membrane alpha-helices whose hydrophobic length matches the hydrophobic thickness of the lipid bilayer could be a mechanism of retention in the Golgi apparatus. We have used fluorescence methods to study the interaction of peptides Ac-K2-G-Lm-W-Ln-K2-A-amide (Pm+n) with bilayers of phosphatidylcholines with chain lengths between C14 and C24. The peptide P22 (m = 10, n = 12) incorporates into all bilayers, but P16 (m = 7, n = 9) does not incorporate into bilayers when the fatty acyl chain length is C24 and only partly incorporates into bilayers where the chain length is C22. The strongest binding is seen when the hydrophobic length of the peptide matches the calculated hydrophobic thickness of the bilayer. It is suggested that a too-thin bilayer can match to a too-long peptide both by stretching of the lipid and by tilting of the peptide. However, a too-thick bilayer can only match a too-thin peptide by compression of the lipid, which becomes energetically unfavorable when the difference between the bilayer thickness and the peptide length exceeds about 10 A. The presence of cholesterol in the bilayer leads to a marked reduction in the incorporation of P16 into bilayers where the chain length is C18. Hydrophobic mismatch could explain retention of proteins with short trans-membrane alpha-helical domains in the Golgi, the effect following largely from the low concentration of cholesterol in the Golgi membrane compared to that in the plasma membrane.

Mechanistic studies on the phosphorylation of photoexcited rhodopsin

The mechanism of the photophosphorylation of rhodopsin was studied using several synthetic peptides corresponding to the sequence of the phosphorylation domain. It was found that the decapeptide (residues 339–348) was effectively phosphorylated by rhodopsin kinase only when incubation was performed in the presence of both rhodopsin and light. These results are interpreted to suggest that in the dark‐adapted state rhodopsin kinase exists in an inactive conformation and that this is converted into a catalytically competent form only after interaction with metarhodopsin II (Rho*)

Morphometric analysis of intestinal mucins under different dietary conditions and gut flora in rats.

Elucidation of the mechanisms that alter the biosynthesis, turnover, and degradation of intestinal mucins is relevant to the understanding of both the normal gut ecosystem and various intestinal diseases. In this study image analysis was used to quantify the effects of diet and microbial flora on the mucin composition of goblet and deep crypt cells, the number and volume density of mucin-containing cells, and the staining density of their stored mucins in the small and large intestine of germ-free and conventionally maintained rats fed two different diets. One was a coarsely ground commercial rodent diet containing crude fiber of cereal origin and the other a purified diet composed of finely powdered ingredients, including cellulose as a source of fiber. The changes in mucin production were also analyzed in germ-free rats colonized with a human flora. Feeding a commercial diet reduced the volume density of cells containing neutral and sulfomucins in the jejunum of conventional rats and the staining density of neutral and acidic mucins in the germ-free rats. Both rat and human floras reduced the number of cells containing acidic and sulfomucins and the staining density of neutral mucins in the small intestine of animals fed on a purified diet. However, inoculation of human flora increased the staining density of stored neutral and sulfated mucins in the cells of the large intestine. The results demonstrate that the dietary changes are influential in modifying the amount and proportion of mucins in the small intestine and the microbial flora in the large intestine.Elucidation of the mechanisms that alter the biosynthesis, turnover, and degradation of intestinal mucins is relevant to the understanding of both the normal gut ecosystem and various intestinal diseases. In this study image analysis was used to quantify the effects of diet and microbial flora on the mucin composition of goblet and deep crypt cells, the number and volume density of mucin-containing cells, and the staining density of their stored mucins in the small and large intestine of germ-free and conventionally maintained rats fed two different diets. One was a coarsely ground commercial rodent diet containing crude fiber of cereal origin and the other a purified diet composed of finely powdered ingredients, including cellulose as a source of fiber. The changes in mucin production were also analyzed in germ-free rats colonized with a human flora. Feeding a commercial diet reduced the volume density of cells containing neutral and sulfomucins in the jejunum of conventional rats and the staining density of neutral and acidic mucins in the germ-free rats. Both rat and human floras reduced the number of cells containing acidic and sulfomucins and the staining density of neutral mucins in the small intestine of animals fed on a purified diet. However, inoculation of human flora increased the staining density of stored neutral and sulfated mucins in the cells of the large intestine. The results demonstrate that the dietary changes are influential in modifying the amount and proportion of mucins in the small intestine and the microbial flora in the large intestine.

Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer's patches.

The nature of cell-associated carbohydrates in the human intestine that may mediate transepithelial transport of bacterial and dietary lectins and their processing by the lymphoid cells of Peyers patches is not known. Because the cell surface carbohydrate receptors for lectins may vary in different species, the glycoconjugates of human and mouse follicle-associated epithelium and gut-associated lymphoid tissue were compared. A panel of 27, mainly recently isolated, lectins were used to identify glycoconjugate expression in M-cells, enterocytes, goblet cells, lymphocytes and macrophages in mouse and human intestine. Mouse M-cells were exclusively labelled by fucose-specific lectins but in human follicle-associated epithelium no distinct M-cell staining pattern was observed. In the human Peyers patches,Bryonia dioica lectin bound selectively to paracortical T-lymphocytes andChelidonium majus lectin to germinal centre B-cells. Certain mannose-specific lectins (Galanthus nivalis, Hippeastrum hybrid) stained the tingible body macrophages in the germinal centre of human Peyers patches but labelled the macrophages in the paracortical T-cell region of the mouse. The results indicate distinct differences in glycosylation between mouse and human Peyers patches and their associated lymphoid cells. When considering cell surface glycoconjugates as target molecules for the gut immune system, care has to be taken to choose the appropriate lectin for each species.

Topoisomerase enzymes as therapeutic targets for cancer chemotherapy.

The topoisomerase enzymes are essential for DNA metabolism, where they act to adjust the number of supercoils in DNA, a key requirement in the cellular processes of transcription and replication. Their enzymatic mechanism creates transient nicks (type I) or breaks (type II) in the double stranded DNA polymer, allowing DNA to be converted between topological isomers. Humans possess both types of topoisomerase enzymes, however the two types utilize very different enzymatic mechanisms. Both type I and type II topoisomerases have been identified as clinically important targets for cancer chemotherapy and their inhibitors are central components in many therapeutic regimes. Over the course of the last 30 years inhibitors with extensive structural diversity have been developed through a combination of drug screening and rational design programs. Simultaneously much emphasis has been placed upon establishing the mechanisms of action of both classes of topoisomerase enzyme. Crucial structural insights have come from the crystal structure of topoisomerase I, while modelling comparisons are beginning to map out a possible framework for topoisomerase II action. This review discusses these recent advances in the fields of enzyme mechanism and inhibitor design. We also address the development of drug resistance and dose-limiting side effects as well as cover alternative methods in drug delivery.

Modulation of the receptor binding affinity of amphiregulin by modification of its carboxyl terminal tail

Amphiregulin (AR), a heparin-binding, epidermal growth factor (EGF) receptor ligand has homology with EGF but exhibits a lower affinity for the EGF receptor than EGF. As the mature form of AR is truncated at the C terminus and lacks a conserved leucine residue known to be essential for high affinity binding of EGF to the EGF receptor, wild-type AR (AR1-84), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR1-90) and a similarly extended construct with a Met86 to Leu substitution (AR1-90(leu86)) were expressed as recombinant proteins in yeast, purified by heparin affinity and C18 reverse phase chromatography and their relative biological activities determined. The growth factors were tested in mitogenesis and EGF receptor autophosphorylation assays and their relative order of potencies was found to be leu86 > met86 > wt. The AR1-90(leu86) construct was found to be 50- to 100-fold more active than wild type AR1-84 consistent with previously reported studies of the role of the equivalent C-terminal leucine in EGF or TGF alpha. Significantly, the C-terminally extended form of AR, AR1-90, which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR1-84. This difference in activity of the C-terminally extended form of AR may be of biological significance since differential proteolytic processing of the AR precursor in vivo could result in production of multiple forms of the growth factor with differing affinities for the EGF receptor and hence differing biological potencies.

Evidence for the cytoplasmic location of the N- and C-terminal segments of sarcoplasmic reticulum (Ca2+-Mg2+)-ATPase.

Antibodies were produced against 5 peptides corresponding to segments of the (Ca2+-Mg2+)-ATPase of fast-twitch rabbit skeletal muscle sarcoplasmic reticulum (SR) including the N- and C-terminal regions. With the exception of antibodies directed against the peptide corresponding to residues 567-582 all antibodies bound strongly to the ATPase in intact SR vesicles, indicating that the epitopes were located on the cytoplasmic face of the SR. When the vesicles were disrupted, by solubilisation in SDS, binding of these antibodies was unchanged, further supporting the idea that these epitopes were located on the cytoplasmic face of SR. This is the first demonstration of the location of the N- and C-terminal regions of SR (Ca2+-Mg2+)-ATPase. These observations are discussed in the light of current structural models of the ATPase.

Lectin Histochemistry Reveals the Appearance of M-cells in Peyer's Patches of scid Mice After Syngeneic Normal Bone Marrow Transplantation

Peyers patches in the intestinal mucosa are characterized by the presence of several lymphatic follicles and interfollicular T-cell regions. Luminal antigens are transported across the intestinal epithelium to stimulate the Peyers patch pre-B-cells in the follicles that proliferate and migrate to distant sites. Evidence suggests that antigen priming of B-lymphocytes is responsible for the number and location of Peyers patches during postnatal life, but little is known about the histogenesis of Peyers patches and their overlying membranous (M) cells. To examine whether T- and B-lymphocytes in Peyers patches have an influence on M-cell generation, we studied the development of Peyers patches and M-cells in severe combined immunodeficient (scid) mice reconstituted with bone marrow cells from normal syngeneic mice. Our experiments demonstrate that the donor bone marrow cells in the host scid mice repopulate to form single (primary) follicles and aggregates of lymphoid nodules, the Peyers patches. Use of the lectins Anguilla anguilla (AAA) and Ulex europaeus I (UEA-I) as positive markers of mouse Peyers patch M-cells revealed that M-cells develop in the dome epithelium overlying the single primary follicles and Peyers patches of reconstituted scid mice. This experimental system therefore allows the study of the histogenesis of Peyers patches and M-cells.

Carbohydrate expression in the intestinal mucosa

UNLABELLED The understanding of mechanisms responsible for alterations in mucin synthesis and secretions is essential in elucidating the aetiology of intestinal disorders. The existence of distinct mucins and M-cell transport mechanisms, and their beneficial effects, have long been recognised. Since nutritional and bacterial factors alter the mucin characteristics and are relevant to planning preventative strategies for intestinal diseases, the studies described in this monograph were designed to test the hypothesis that mucin composition and endocrine status of the intestinal tract are altered by dietary constituents and microbial flora. A study of gut-associated lymphoid tissue (GALT) was undertaken to test the hypothesis that lymphocyte-epithelial interactions influence the glycosylation of cells overlying Peyers patches. The effects of diet and microbial flora were analysed by comparing the data from male Wistar germ-free rats, with conventional or human flora. Such rats were fed either a commercial diet, containing crude fibre, or a purified diet. Bone marrow transfers from syngeneic Balb/c mice to severe combined immunodeficient (SCID) mice were made to induce the formation of GALT. A comparison was made of the glycoconjugate profile of Peyers patches in the guts of mice and humans. The results of the study showed that feeding a fibre-rich diet to germ-free rats resulted in increased villus-crypt lengths, decreased carboxylated mucin content of goblet cells, increased N-acetylglucosamine and sialic acid residues in the surface goblet cells, and a reduction in the number of endocrine cells in the small intestine. The lectin markers revealed M-cell development in the dome epithelium of the reconstituted SCID mice. Mouse M cells were labelled by fucose-specific lectins but in human Peyers patches no distinct M-cell-staining pattern was observed. CONCLUSIONS (1) the interactions between diet and flora alter the mucosal architecture and the activity of endocrine cells; (2) the dietary changes are influential in modifying the epithelial mucin predominantly in the small intestine while the microbial flora influences the mucosal architecture predominantly in the large intestine; (3) the use of bone marrow transplantation from syngeneic mice into SCID mice along with lectin markers for M cells can be used to study the histogenesis of Peyers patches; (4) the distinct differences between mouse and human Peyers patches suggest that when considering cell surface glycoconjugates as target molecules appropriate lectin should be used for each species. A future challenge in intestinal epithelial cell biology is identifying the nature and distribution of cell surface receptors for specific dietary components and bacteria.