Immune privilege of the testis for islet xenotransplantation (rat to mouse).
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Animals↗
Biomedical subjects
R B Harris
Publications and source records attributed to R B Harris.
The effects of duration of diabetes and number of transplanted islets on streptozotocin-diabetic rats.
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Animals↗
Heparin-von Willebrand factor binding as assessed by isothermal titration calorimetry and by affinity fractionation of heparins using synthetic peptides.
The ability of proteins to bind heparin, a heterogeneous sulfated glycosaminoglycan, likely depends on the conformational uniqueness of specific binding domains. Based on the motif of a consensus heparin-binding synthetic peptide, a 23-residue sequence (Y565-A587) of human von Willebrand factor (vWF) was previously identified which binds heparin with affinity comparable to that of the native protein (Sobel, M., Soler, D. F., Kermode, J. C., and Harris, R. B. 1992 J. Biol. Chem. 267, 8857-8862). This peptide undergoes a conformational change upon binding heparin. Isothermal titration calorimetry has now been used to further quantify this binding reaction. In experiments done at 25 degrees C, Y565-A587 bound heparin with about the same affinity (Kd = 9.0 x 10(-7) M) as a "core" sequence peptide encompassing residues K569-I580. Binding between these peptides and heparin is overwhelmingly enthalpically favored and is dependent on the formation of productive electrostatic bonds; hydrophobic interactions do not play a significant role in mediating binding. Furthermore, when immobilized on Sepharose in a manner which does not compromise essential cationic residues, the vWF domain peptides are effective affinity ligands. They bind a species of heparin which possesses significantly enhanced affinity for native vWF. The Kd for binding between the high-affinity heparin and Y565-A587 is about threefold lower than that determined with crude, unfractionated heparin. Thus, the vWF peptides are a useful model for studying the physiological role of heparin binding to the native protein.
Amino Acid Sequence↗
Potential role of two novel elastase-like enzymes in processing pro-transforming growth factor-alpha.
Transforming growth factor-alpha (TGF-alpha) is a mitogenic peptide produced by tumor cells and by virally and chemically transformed cells in culture. TGF-alpha is almost certainly derived from its precursor protein (pro-TGF-alpha) by limited proteolysis, but the physiologically relevant processing enzyme(s) is(are) unknown. We now report that oncogenically transformed rat liver epithelial cells (known to secrete TGF-alpha) and Schwann cells in culture transfected with SV40 T-antigen (which are now reported to express mRNA encoding pro-TGF-alpha) contain membrane associated, neutral pH, serine proteinases which are elastase-like in their substrate specificity, but elastase is not known to be associated with these cell types. In both cell types, the enzyme is associated with a subcellular fraction enriched for microsomes and plasma membranes. Furthermore, the enzyme appears to be specifically induced 4-fold in the transformed epithelial cells as compared with the level of enzyme present in the nontransformed parental cells. The enzymes have been purified approximately 20,000-fold to near homogeneity (50-60 units/mg) and are virtually identical with regard to their molecular weights (38,000) and other physiochemical properties. Results obtained with numerous synthetic peptide substrates show the enzymes prefer nonpolar residues such as Ala and Val in the P1 and P2 positions, but promiscuity of cleavage specificity observed with long-chain peptide substrates is attributed to the absence of structure in these peptides. Thus, although these enzymes may be involved in processing pro-TGF-alpha at the plasma membrane of the cell, it is just as likely that these enzymes play other physiological roles in the parental and/or transformed cells and that there is no specific endoproteolytic processing enzyme of pro-TGF-alpha.
Amino Acid Sequence↗
Conformational analysis and proteolytic processing of synthetic pre-pro-GnRH/GAP protein.
Homogeneous pre-pro-GnRH/GAP protein was recently synthesized in 100 mg quantities by solid-phase methods and surprisingly, the synthetic pre-pro-protein, which normally does not escape the endoplasmic reticulum, was found to inhibit the release of prolactin from cultured pituitary cells. This is the first demonstration of significant biological activity associated with a precursor protein and provides the rationale for its further study. We now report the results of our initial examination of the conformational properties of pre-pro-GnRH/GAP protein as a prelude to solving its solution phase conformation by homonuclear 1H-NMR protocols. Thermal and pH titration fluorescence and circular dichroism spectroscopies reveal that the protein is resistant to thermal-induced conformational changes but is particularly sensitive to pH-induced conformational changes; while Asp/Glu and Arg residues may contribute to structural stability, His and Lys residues predominate. Pre-pro-GnRH/GAP is about 30% helix in the range of 2-40 degrees C; however, even at 90 degrees C, the peptide retains nearly 50% of its helix character. There is no evidence for a cooperative transition; for this reason, differential scanning calorimetry failed to yield a defined transition thermogram. Pre-pro-GnRH/GAP apparently does not pass through a transition state as a function of temperature but appears to flex and retain a high percentage of helix structure, resulting in subtle changes in secondary structure. There is no discernible isodichroic point. On either side of the neutral pH range, however, there are dramatic changes in structure that result in nonreversible denaturation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
GAP-releasing enzyme is a member of the pro-hormone convertase family of precursor protein processing enzymes.
The recent discovery of mammalian endoproteinases which show extensive sequence homology with the yeast Kex 2 gene product (kexin) has lead to the hypothesis that processing enzymes of pro-hormone precursor proteins belong to a family of calcium dependent, subtilisin-like serine proteinases. We previously showed that hypothalamic GAP-releasing enzyme shares these characteristics and possesses the requisite specificity to be considered as a processing enzyme of progonadotropin releasing hormone (pro-GnRH) precursor protein. Thus, GAP-releasing enzyme (and other non-related proteins) were tested for their immunological reactivity with antisera raised against pituitary pro-hormone convertase 1/3 (PC1/3) and insulinoma PC2. On the basis of indirect enzyme-linked immunosorbent (ELISA) and Western blot assays, GAP-releasing enzyme is now shown to be immunologically related to PC1/3. We can conclude that GAP-releasing enzyme is also likely to be a member of the pro-hormone convertase family and should be considered the physiologically relevant processing enzyme of pro-GnRH. It is possible that GAP-releasing enzyme represents bovine hypothalamic PC1/3.
Amino Acid Sequence↗
Factors influencing body weight regulation.
As obesity is a major health problem for both the adult and adolescent populations of the United States there is an increasing need to understand basic mechanisms that control the body weight of an individual. Body fat content in adults is determined by energy balance status which is influenced by both food intake and energy expenditure. Current evidence suggests a dominant role for control of intake although chronic exercise and genetic factors will influence body composition. The hypothalamus appears to be the brain area responsible for determining food selection and intake although it is not yet clear how central mechanisms modulate peripheral feeding and satiety responses.
Animals↗
The impact of high- or low-fat cafeteria foods on nutrient intake and growth of rats consuming a diet containing 30% energy as fat.
The objective of this study was to determine the impact of high-fat and low-fat cafeteria foods on energy intake and body composition of growing rats. Two sets of 30 male Sprague-Dawley rats weighing either 90 g or 280 g, were each divided into two groups of eight and two groups of seven animals, matched for average weight. All animals were offered a semi-purified diet containing 30% kJ fat. After ten days, initial body composition, carcass energy and serum lipids, insulin and glucose were determined in seven rats from each age group. Seven rats in each age group continued to receive the semi-purified diet ad libitum, eight received semi-purified diet plus one high-fat cafeteria food each day and eight received semi-purified diet plus one low-fat cafeteria food each day. After 38 days, body composition, efficiency of energy retention and serum lipids, insulin and glucose were determined. Food and energy intakes were greater for rats offered cafeteria foods compared with controls. Those given low-fat foods ate the greatest quantity of food but those fed high-fat foods had the highest energy intake. High-fat foods increased fat intake to approximately 43% of energy. Low-fat foods decreased fat intake to approximately 24% kJ. There was no effect of treatment on weight gain or lean body mass of either age group. Young rats fed low-fat cafeteria foods had less body fat than their controls. There was no significant increase in body fat content of rats fed high-fat cafeteria foods.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Design and synthesis of a helix heparin-binding peptide.
Elaboration of heparin-protein-binding interactions is necessary to understand how heparin modulates protein function. The heparin-binding domain of some proteins is postulated to be a helix structure which presents a surface of high positive charge density. Thus, a synthetic 19-residue peptide designed to be alpha-helical in character was synthesized, and its interaction with heparin was studied. The peptide was shown to be 75% helix by circular dichroism (CD) spectrometry in neutral pH buffer (at 2 degrees C); helicity increased to nearly 85% under high ionic strength conditions or to nearly 100% in 75% ethanol. Increasing the temperature of the solution caused a change in the spectral envelope consistent with a coil transition of the peptide. The midpoint of the transition (i.e., the temperature at which the helix content was determined to be 50%) was 25 degrees C, and the determined van't Hoff enthalpy change (delta HvH) was 3.2 kcal/mol of peptide. By CD, heparin increases the helix content of the peptide to 100% and increases the apparent thermal stability of the peptide by about 1 kcal/mol. The melting point for the helix/coil transition of the heparin-peptide complex was 50 degrees C. The thermal coefficient of the transition (approximately 300 deg.cm2.dmol-1.degree C-1) was essentially the same for the peptide alone or the peptide-heparin complex. Dissociation of the complex under high ionic strength conditions was also observed in the CD experiment. Biological assays showed less heparin-binding activity than expected (micromolar KD values), but this was attributed to the absence of critical lysyl residues in the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Localization and characterization of a heparin binding domain peptide of human von Willebrand factor.
Human von Willebrand factor, a plasma glycoprotein which plays a critical role in regulating hemostasis, binds heparin, but the physiological importance and mode of this interaction is poorly understood. Using the motif of an amino acid sequence of a consensus heparin binding synthetic peptide, a 23-residue sequence (Tyr565-Ala587) of human von Willebrand factor was identified that retains the consensus motif and binds heparin with affinity comparable with native von Willebrand factor and the consensus peptide. In a fluid phase binding assay, the Tyr565-Ala587 peptide competed effectively with von Willebrand factor for binding heparin. Synthesis and testing of peptides overlapping Tyr565-Ala587, as well as adjacent cationic regions, showed this core sequence to be the optimal linear binding domain. Far ultraviolet circular dichroism spectrometry of the Tyr565-Ala587 peptide suggested that the peptide undergoes conformational change upon binding heparin. The Tyr565-Ala587 peptide thus encompasses part (or all) of a functionally important heparin binding domain of von Willebrand factor. Further study of this and related peptides may be useful for exploring how heparin may influence von Willebrand factor-mediated platelet hemostasis.
Amino Acid Sequence↗
Characterization of homogeneous atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor.
We previously reported the discovery and partial characterization of bovine atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor, which is associated with atrial granule membranes. We now report the physicochemical properties of electrophoretically homogeneous enzyme purified by a series of chromatography steps from a subcellular fraction enriched for atrial granules. The enzyme tends to associate during purification to higher molecular weight species, but SDS-PAGE analysis reveals a single polypeptide chain of molecular weight 70,000. The enzyme is activated 2-3 fold by Ca+2 and 1.5-fold by Mg+2 and is nearly 100% inhibited by Zn+2 or Co+2. Thus, the enzyme can be considered a calcium activated, neutral pH, serine proteinase. Based on the hydrolysis of numerous synthetic peptide substrates, the recognition sequence for the enzyme within the pro-hormone has been mapped to A96PRSLRR102; cleavage occurs at the Arg98-Ser99 bond yielding bioactive atrial natriuretic peptide directly from the pro-hormone. The doublet of basic amino acids is part of the recognition sequence but is not the primary cleavage site. It is our hypothesis that the processing site sequence acts as a recognition element for the endoproteinase and resides at the surface of the pro-hormone and thus contributes to the molecular basis for limited proteolysis.
Amino Acid Sequence↗
Prevalence of self-reported poor sleep in a healthy population aged 50-65.
Many population-based surveys have reported that the prevalence of poor sleep increases with age. Despite the uniformity of findings, it remains unclear to what extent age-related declines in overall physical health are related to those results. One approach to this problem has been to adjust for such confounding variables multivariately. Some prior studies using this approach have not shown the expected age-related increases in the prevalence of poor sleep. Another approach has been the study of sleep in carefully screened, healthy populations. The current study reports the prevalence of disturbed sleep in a population, ages 50-65, carefully screened for physical health as part of an ongoing study of exercise and cardiovascular function. The prevalence of self-reported trouble falling asleep every night or almost every night (1.1% M, 2.6% F), trouble awakening and returning back to sleep (4.4% M, 3.3% F), and use of hypnotic medication at least twice a week (1.6% M, 2.6% F) were consistently lower than in nearly all previous population-based studies of individuals of comparable age. This implies that when overall physical health factors are taken into account a decline in sleep quality is not necessarily an inevitable component of aging per se. As has been shown in other studies, there were small but statistically significant relationships between self-reported depression and poor sleep. Despite the low prevalence of poor sleep, about a third of the population reported feeling not well-rested and/or not getting the sleep they required. The individuals in this study also reported obtaining significantly less sleep relative to normative data from 30 years ago.(ABSTRACT TRUNCATED AT 250 WORDS)
Age Factors↗
Adipocyte insulin responsiveness in female Sprague-Dawley rats fed a low fat diet containing a fat-mimetic carbohydrate.
Two experiments examined the effects of replacing high fat with low fat diets on adipocyte insulin sensitivity and response. Female Sprague-Dawley rats had free access to diets containing 21% (control), 61% (high fat) or 2% (low fat) of energy as fat. In the low fat diet a carbohydrate-based fat-mimetic carbohydrate replaced all but the essential fat present in the high fat diet. Insulin-stimulated glucose utilization by isolated adipocytes was measured after 10, 30 or 50 d. In a second study adipocyte insulin saturation curves were measured after 36 d. Rats fed the high fat diet for 30 d were insulin resistant and adipocyte basal and insulin-stimulated glucose utilization were depressed. The low fat diet initially stimulated glucose utilization of adipocytes but did not change insulin responsiveness. After 50 d there was no difference in glucose utilization between adipocytes from rats fed control and low fat diets. Insulin resistance in rats fed the high fat diet was associated with a nonsignificant reduction in insulin receptor number. These observations do not exclude the possibility of a post-receptor defect in glucose utilization.
Adipose Tissue↗
Insulin insensitivity is rapidly reversed in rats by reducing dietary fat from 40 to 30% of energy.
The objective of this study was to determine the effect of reducing dietary fat content from 40 to 30% of total energy on body composition and insulin sensitivity in rats. Female Sprague-Dawley rats were fed either a control diet (30% energy as fat) or a high fat diet (40% energy as fat) for 10 wk. In vivo glucose tolerance tests indicated that both basal and glucose-stimulated insulin concentrations were elevated in rats fed the high fat diet. Two groups of rats offered the high fat diet were then fed the control diet (HF,C) and two that had received the high fat diet were then fed a diet (30% energy as fat) containing a fat-mimetic carbohydrate (HF,M). Two groups continued to receive the high fat diet and two groups continued to receive the control diet. Rats were killed 1 and 2 wk after the diet switch. In vivo glucose tolerance tests indicated that insulin sensitivity was corrected in HF,C and HF,M rats after 3 d. Body fat content was greater in rats fed the high fat diet and remained high after 7 d of consuming either diet with 30% of energy from fat. There was no effect of diet on liver fatty acid utilization or on diaphragm glucose oxidation. Adipocytes from all treatment groups were insulin resistant, possibly due to feeding status or age of the rats. The results of this study suggest that a moderate reduction in fat intake, from 40 to 30% of energy, can produce a rapid improvement of insulin sensitivity in insulin-insensitive rats, independent of changes in body fat content and irrespective of the means used to reduce dietary fat content.
Adipose Tissue↗
Processing enzyme specificity is a consequence of pro-hormone precursor protein conformation.
Peptide-hormones are synthesized as higher molecular weight, precursor proteins which must initially undergo limited endoproteolysis to yield the bioactive peptide(s). The ability of two different endoproteinases, gonadotropin-associated peptide (GAP)-releasing enzyme and atrial granule serine proteinase (which are likely to be the physiologically relevant processing enzymes of bovine hypothalamic pro-gonadotropin-releasing hormone/gonadotropin-associated peptide and bovine pro-atrial natriuretic factor precursor proteins, respectively), to act at their own recognition sequences within their relevant pro-hormone proteins has now been contrasted with their ability to act at the recognition sequence for the alternate enzyme or to act at their own recognition sequence when it is placed within the protein framework of the alternate precursor protein. The results show that each enzyme acts with specificity at its own recognition sequence even when it is placed within the framework of the alternate pro-hormone. However, the enzymes fail to act (or act in a non-specific manner) at the alternate recognition sequence even if it is placed within the peptide framework of its own pro-hormone protein. Thus, despite the fact that both recognition sequences are similar in sequence and residue composition and that both contain a doublet of basic amino acids, it appears that sequence and the local conformation assumed by the processing site within the pro-hormone protein are essential for each endoproteinase to act with fidelity. As part of our continuing work, we now also report several newly determined physicochemical properties of hypothalamic GAP-releasing enzyme, the processing enzyme of pro-gonadotropin-releasing hormone/GAP protein.
Amino Acid Sequence↗
Mapping the active site of meprin-A with peptide substrates and inhibitors.
The extended substrate-binding site of meprin-A, a tetrameric metalloendopeptidase from brush border membranes of mouse kidney proximal tubules, was mapped with a series of peptide substrates. Previous studies led to the development of the chromogenic substrate Phe5(4-nitro)bradykinin for meprin-A. With this substrate, several biologically active peptides were screened as alternate substrate inhibitors, and, of these, bradykinin (RPPGFSPFR) was found to be the best substrate with a single cleavage site (Phe5-Ser6). Three types of bradykinin analogues were used for a systematic investigation of substrate specificity: (1) nonchromogenic bradykinin analogues with substitutions in the P3 to P3' subsites were used as alternative substrate inhibitors of nitrobradykinin hydrolysis, (2) analogues of nitrobradykinin with variations in the P1' position were tested as substrates, and (3) intramolecularly quenched fluorogenic bradykinin analogues with substitutions in the P1 to P3 sites were tested as substrates. A wide variety of substitutions in P1' had little effect on KM (174-339 microM) but markedly affected kcat (51.5 s-1 = A greater than S greater than R greater than F greater than K greater than T greater than E = 0). Substitutions in P1 had a greater effect on KM (366 microM-2.46 mM) and also strongly affected kcat (98.5 s-1 = A greater than F much greater than L greater than E greater than K = 2.4 s-1). The variety of allowed cleavages indicates that meprin-A does not have strict requirements for residues adjacent to the cleavage site. Substitutions farther from the scissle bond also affected binding and hydrolysis, demonstrating that multiple subsite interactions are involved in meprin-A action.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Calorimetric and spectroscopic examination of the solution phase structures of prekallikrein binding domain peptides of high molecular weight kininogen.
Unique sequence-binding sites are exposed on the surface of high molecular weight kininogen which complex prekallikrein or factor XI with high affinity and specificity. A sequence comprising 31 residues of the mature kininogen molecule (Asp565-Lys595) retains full binding activity for prekallikrein (KD = 20 nM) and assumes a complex folded structure in solution which is stabilized by long-range interactions between N- and C-terminal residues. The sequence Trp569-Lys595 (27 residues) shows only 28% of this binding affinity and lacks the key structural features required for protein recognition (Scarsdale, J. N., and Harris, R. B., J. Prot. Chem. 9, 647-659, 1990). We were thus able to predict that N- or C-terminal truncations of the binding-site sequence would disrupt the conformational integrity required for binding. Two new peptides of 20- and 22- residues have now been synthesized and their solution phase structures examined. These peptides are N- and C-terminal truncations, respectively, of the 27-residue sequence and correspond to the sequences Asp576-Lys595 and Trp569-Asp590 of high molecular weight kininogen. The results of fluorescence emission and circular dichroism (CD) spectroscopies in the range 25-90 degrees C and from differential scanning calorimetry (DSC) all substantiate the idea that the C-terminal truncation peptide binds prekallikrein 35-fold poorer than the 31-residue peptide because it is relatively unordered and possesses a less stable structure. Surprisingly, the N-terminal truncation peptide (20-mer) shows structural stability even at elevated temperatures and, like the 31-residue peptide, undergoes cold-induced denaturation observable in the DSC. 2D-NMR analysis of the 20-residue peptide revealed two distinct structures; one conformer possesses a more compact, folded structure than the other. However, the predicted structures assumed by either conformer are very different from those of either the 31- or 27-residue peptides. Hence, the binding affinity of the 20-residue peptide is 60-fold poorer than that for the 31-residue peptide because it assumes a nonproductive binding conformation(s).
Amino Acid Sequence↗
Characterization of liver epithelial cells transfected with myc and/or ras oncogenes.
While many liver tumors contain activated myc and ras oncogenes, the mechanisms by which these genes contribute to cellular transformation is poorly understood. Activated versions of the cellular oncogenes, c-myc and/or c-H-ras were transfected into normal rat liver epithelial cells to identify cellular pathways that are altered in the cells containing the oncogenes. The results of these and other investigations indicate that the biological properties associated with the transfection of c-myc include immortalization, reduced contact inhibition of growth, activation of phospholipase A2-mediated pathways, increased sensitivity to transformation with a ras gene, and greatly increased sensitivity to growth factors. The biological properties associated with the transfection of the ras gene include morphological transformation, anchorage-independent growth, tumorigenicity, increased phosphatidylinositol metabolism, the induction of growth-factor processing and secretion, which leads to (exogenous) growth factor-independent tumor growth, and a marked resistance to normal inhibitors of growth such as TGF-beta. It is proposed that the complementary actions of the myc and ras genes in cellular transformation may be related to the ras-induced secretion of autocrine growth factors by cells sensitized to their effects by the myc gene. The increased stimulus for growth coupled to a ras-induced insensitivity to growth inhibitors may lead to clonal expansion of these cells and tumor development.
Animals↗