Biomedical subjects

A M Felix

Publications and source records attributed to A M Felix.

At least 37 records · Page 2Linked to original sources

PubMed1992

Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor.

Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15-200 microM) have been isolated and fully characterized. The cleavage product, GRF(4-44)-NH2, and the isomerization product, [beta-Asp3]GRF(1-44)-NH2, from the degradation of GRF(1-44)-NH2 in acidic solution and the corresponding products, GRF(4-29)-NH2 and [beta-Asp3]GRF(1-29)-NH2, from the degradation of GRF(1-29)-NH2 have been isolated and characterized. The products, [beta-Asp8]GRF(1-44)-NH2 and [Asp8]GRF(1-44)-NH2, from the deamidation of GRF(1-44)-NH2 at pH 8.0 and the corresponding products, [beta-Asp8]GRF(1-29)-NH2 and [Asp8]GRF(1-29)-NH2, from the deamidation of GRF(1-29)-NH2 have been isolated and characterized. All the degradation products of GRF(1-44)-NH2 and GRF(1-29)-NH2 were evaluated for biological activity and found to have much lower in vitro potencies than the parent peptides. Degradation occurs at Asp3 and Asn8 and the kinetics of these various transformations versus pH and temperature have been studied. GRF is most stable at pH 4-5. At pH below the pKa of the Asp3 side-chain (pH less than 4), cleavage at Asp3-Ala4 is the major route of degradation. For pH greater than 4, isomerization of Asp3 to beta-Asp3 (iso-Asp3) predominates. The rates of cleavage and isomerization are simple first order and vary with pH, independent of buffer concentration, such that the protonated (COOH) form of Asp3 undergoes cleavage while the ionized (COO-) form isomerizes. The more rapid deamidation of Asn8 to generate beta-Asp8 and Asp8 in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH greater than or equal to 5 and is general base-catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn8 in GRF(1-29)-NH2 at pH less than or equal to 2 to give exclusively [Asp8]GRF(1-29)-NH2. The deamidation of Asn8 in GRF(1-29)-NH2 at pH 8.0, 22-55 degrees C, is relatively insensitive to temperature for T less than 37 degrees C, possibly due to conformational constraints. Asp25 and Asn35 are sterically, conformationally, or otherwise hindered with respect to these changes as no degradation at these sites was observed under the conditions employed.

Amino Acid Sequence↗

PubMed1991

In vitro stability of growth hormone releasing factor (GRF) analogs in porcine plasma.

The stability of growth-hormone releasing factor (growth regulating factor; GRF) analogs in porcine plasma was examined. GRF analogs were incubated in porcine plasma at 37 degrees C, extracted and subsequently analyzed using high performance liquid chromatography (HPLC). GRF(1-29)-NH2 was rapidly broken down in the plasma with a degradation rate of t1/2 = 13 min. The primary degradation product was identified as GRF(3-29)-NH2. Substitution of Gly15 by Ala15 slightly prolonged the plasma half-life (t1/2 = 17 min) and the major degradative fragment was found to be [Ala15]GRF(3-29)-NH2. The cleavage between the 2 and 3 position of the peptide was not inhibited by trasylol at a concentration of 1,000 KIU/ml but was dramatically reduced by the combined use of diprotin A and trasylol. Absence of the free amino group at the N-terminus and/or substitution of a D-amino acid residue at the penultimate position completely prevented cleavage between the 2 and 3 position in the structural linear GRF analogs. Side-chain to side-chain cyclization between Asp8 and Lys12 amino acid residues significantly improved the stability of GRF in plasma with t1/2 greater than 2 hr. An additional stability was provided by substitution of D-Ala2 for Ala2 in the structural cyclic analog. Cyclization between Lys21 and Asp25 also improved the stability of the GRF peptide in the plasma. Stability was further enhanced by the presence of D-Ala2 or by forming a dicyclic analog through an additional linkage between Asp8 and Lys12.

Amino Acid Sequence↗

PubMed1991

Effect of human growth hormone-releasing factor and a potent analog on antibody formation in African green monkeys.

African Green monkeys were injected (2 x daily subcutaneously for six months) with human GRF(1-44)-NH2 (10 micrograms/kg BW) or a more potent analog, [desNH2Tyr1,Ala15]-hGRF(1-29)-NH2 (2 micrograms/kg BW) to determine the potential of each peptide to induce antibody formation. Blood samples were taken every two weeks, diluted 1:100 and tested for ability to bind radioiodinated hGRF. One animal in the hGRF(1-44)-NH2 group [N = 6] produced low-titer GRF antibodies by 6 weeks (19% binding) and continued throughout the 24 weeks of treatment (average = 50-60% binding). Similarly, one animal in the hGRF analog group [N = 6] displayed low-titer GRF antibodies by 18 weeks (14% binding), with the highest binding observed at 24 weeks (51% binding). Subsequent dilutions (1:1,000 and 1:3,000) of these bleedings confirmed that higher GRF antibody titers were not masked by antibody excess. Dialyzed sera from these two animals did not affect the abilities of hGRF(1-44)-NH2 or [desNH2Tyr1,Ala15]-hGRF(1-29)-NH2 to stimulate GH secretion by rat pituitary cells in vitro. After 20 weeks of treatment, significant GH responses (increased mean GH area under the curve 2.3-2.5 fold and GH peak 3.5-3.7 fold, that of control) were observed following hGRF or hGRF analog injection. Therefore, the low titer GRF antibodies detected in monkey sera during six months of treatment with hGRF or a potent analog were biologically non-neutralizing.

Animals↗

PubMed1991

Synthesis and biological evaluation of mouse growth hormone-releasing factor.

The recently described mouse growth hormone-releasing factor (mGRF) was synthesized by the solid phase procedure, purified by 2 stages of preparative high performance liquid chromatography and fully characterized. The biologic activity of the 42-amino acid peptide (H-His-Val-Asp-Ala-Ile-Phe- Thr-Thr-Asn-Tyr- Arg-Lys-Leu-Leu-Ser-Gln-Leu-Tyr-Ala-Arg-Lys-Val-Ile-Gln-Asp-Ile-Met-Asn- Lys- Gln-Gly-Glu-Arg-Ile- Gln-Glu-Gln-Arg-Ala-Arg-Leu-Ser-OH) was assessed in primary cultures of both mouse and rat anterior pituitary cells and compared to synthetic rat (rGRF) and human (hGRF) growth hormone-releasing factors. mGRF was equipotent to rGRF in mouse somatotrophs but slightly less potent in rat somatotrophs, while hGRF was 3-5 times less potent in both rodent species.

Amino Acid Sequence↗

PubMed1991

Biosynthesis of human growth hormone-releasing hormone (hGRH) in the pituitary of hGRH transgenic mice.

We have studied the posttranslational processing of prohuman GH-releasing hormone (pro-hGRH) to the mature hormones, hGRH(1-44)-NH2 and hGRH(1-40)-OH, and its carboxyl-terminal peptide (hGCTP) in pituitary cells from transgenic mice bearing a metallothionein-hGRH fusion gene after incubation with [35S]methionine. After separation on HPLC, 35S-labeled and unlabeled hGRH in medium and cell extracts were characterized by RIA and immunoprecipitation with antisera against hGRH and against hGCTP. After a 4-h pulse, unlabeled and [35S]pro-hGRH, hGRH(1-44)-NH2, and hGRH(1-40)-OH were identified in medium and cell extracts by both RIA and immunoprecipitation with anti-hGRH serum. In cell extracts, after a 0.5-h pulse, [35S]pro-hGRH and hGRH(1-44)-NH2 but not [35S]hGRH(1-40)-OH were detectable. After a 0.5-h chase, however, 35S-labeled hGRH(1-40)-OH, pro-hGRH, and [35S]hGRH(1-44)-NH2 were all measurable. After a 4-h chase, comparable levels of [35S]hGRH(1-44)-NH2 and hGRH(1-40)-OH were present, and very little intracellular 35S-pro-hGRH remained. A progressive decrease in the ratio of immunoprecipitable pro-hGRH to mature hGRH peptides and an increase in the ratio of hGRH(1-40)-OH to hGRH(1-44)-NH2 was observed in the two chase periods. In medium, [35S]hGRH(1-44)-NH2 was detectable at all times, whereas only minimal amounts of [35S]hGRH(1-40)-OH were present. Labeled and unlabeled pro-hGRH in cell extracts was also detected with anti-hGCTP serum, and another peak, which coeluted with synthetic hGCTP, was also identified. The low molar ratio of intracellular immunoreactive hGCTP to hGRH (less than 0.02) suggests a more rapid turnover rate of hGCTP than of hGRH. These results demonstrate the processing of hGRH prohormone to both mature forms of hGRH and provide evidence that hGRH(1-40)-OH is derived from hGRH(1-44)-NH2.

Animals↗

PubMed1991

Identification of 11,13-dihydroxy-14-octadecaenoic acid as a circulating Na+/K(+)-ATPase inhibitor.

Two digitalis-like compounds (DLC) were purified to homogeneity from bovine plasma. The purification procedure consisted of organic extractions and batch chromatography followed by three subsequent separations using reverse-phase high-performance liquid chromatography. The presence of a DLC in the different fractions was monitored by their ability to inhibit (a) [3H]ouabain binding to rat brain synaptosomes, and (b) microsomal Na+/K(+)-ATPase activity. Using mass spectrometry and nuclear magnetic resonance spectroscopy the structure of one of the DLCs was identified as 11,13-dihydroxy-14-octadecaenoic acid. It is suggested that this new hydroxy, unsaturated, fatty acid derivative may regulate Na+/K(+)-ATPase activity under some physiological and pathological conditions.

Animals↗

PubMed1991

Comparison of enzymatic semisyntheses of peptide amides: human growth hormone releasing factor and analogs.

Enzymatic semisyntheses of growth hormone releasing factor (GRF), a 44-residue peptide amide hormone, from C-terminal acid precursors, are compared. A recombinant alpha-amidating enzyme was used to convert the glycine-extended precursor, GRF(1-44)-Gly-OH, to GRF(1-44)-NH2 in an essentially quantitative fashion. Trypsin was used to convert the precursors, GRF(1-43)-OH and GRF(1-44)-OH, to GRF(1-44)-NH2 (60 and 15% conversion, respectively) in a 75% v:v N,N'-dimethylacetamide solution containing a large excess of leucine amide. Carboxypeptidase Y catalyzed transpeptidations of the precursors, GRF(1-44)-OH and [Ala44]-GRF(1-44)-OH, to GRF(1-44)-NH2 in aqueous leucine amide solutions were also attempted. The trypsin catalyzed direct amidation of [Ala15]-GRF(1-29)-OH in concentrated ammonium acetate/ammonia buffer (95% 1,4-butanediol cosolvent) to form the superactive analog, [Ala15]-GRF(1-29)-NH2 (ca. 25% conversion at equilibrium), is also described.

Amino Acid Sequence↗

PubMed1990

Prothymosin alpha expression is associated to cell division in rat testis.

Using immunohistochemical methods, we have investigated the cellular distribution of prothymosin alpha (ProT) in adult rat testis. A policlonal antibody raised against thymosin alpha 1 conjugated to keyhole limpet hemocyanin was used. ProT immunoreactivity was observed in the cytoplasm and nucleus of spermatogonia and primary spermatocytes in initial phases of the first meiotic division, preleptotene, leptotene and zygotene. However, in pachytene phase they already showed a weak or negative staining. On the other hand, secondary spermatocytes, spermatids, spermatozoa and Sertoli cells were not stained. Based on this fact we suggest that ProT is present in the proliferative cycle in the final steps of G1 phase, throughout the S and G2 phases and in initial steps of the prophase.

Animals↗

PubMed1990

Defining minimal requirements for antibody production to peptide antigens.

The role that individual determinants play in modulating the immune response of an organism to a pathogen is often obscured because of the complexity of the pathogen. In order to gain a better appreciation of the role of individual determinants in the immune response, a pathogen may be dissociated into smaller components, for example peptides representing specific epitopes. These isolated components are often poorly immunogenic and historically have required the use of adjuvants to stimulate antibody production. This report defines the minimal essential requirements for antibody production to a peptide in this system. These are the ability to stimulate both B- and T-helper lymphocytes, anchorage in a phospholipid complex and multivalency within the complex. When these conditions are met, no additional adjuvants are necessary. This procedure has allowed us to identify three distinct T-helper cell epitopes from HIV gp160. In addition, this information has been used to produce a simple, totally synthetic and highly immunogenic preparation for the production of antibodies to peptides.

Amino Acid Sequence↗

PubMed1990

A novel Fmoc-based anchorage for the synthesis of protected peptides on solid phase.

A novel bifunctional compound, 9-(hydroxymethyl)-2-fluoreneacetic acid, was synthesized, coupled to benzhydrylamine-resin, and evaluated for its application to the solid phase synthesis of protected peptide fragments. Anchor-bond cleavage was achieved with 15% piperidine/DMF. A protected heptapeptide, Boc-Val-Val-Ser(Bzl)-His(Tos)-Phe-Asn-Lys-(Z)-OH, corresponding to the sequence (1-7) of rat-transforming growth factor-alpha, was synthesized using this new support with an overall yield of 46%.

Amino Acids↗

PubMed1990

Cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen.

By means of immunohistochemical methods, we have investigated the cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen, using specific antibodies raised against thymosin alpha-1 and against parathymosin. We observed prothymosin alpha immunoreactivity in lymphoid cells both in thymus and spleen. In the thymus, prothymosin alpha staining was more marked in cortex than in medulla. In the spleen, prothymosin alpha was found in lymphocytes of the periarteriolar lymphatic sheaths and was especially prominent in the germinal centers. Parathymosin immunoreactivity in the thymus was mainly localized in the medulla; positive cells were reticuloepithelial cells from the thymic reticulum and the blood barrier. Thymocytes were negative. In spleen, parathymosin was found in reticular cells arranged in a ring between the periarteriolar lymphatic sheath and the marginal zone. Our results do not support an exclusive role for these peptides as immune system hormones or cytokines.

Animals↗

PubMed1990

Localization and growth hormone (GH)-releasing activity of rat testicular GH-releasing hormone-like peptide.

The testis contains many peptides originally described as originating in the central nervous system. The physiological function of these factors in the testis is generally unknown. We previously reported that the rat testis contains both a peptide with GH-releasing hormone-like immunoactivity (tGHRH-LI) and a mRNA species that cross-hybridizes with a hypothalamic cDNA for rat GHRH (rGHRH). The current study was designed to further characterize tGHRH-LI by determining its location within rat testis, and to evaluate whether tGHRH-LI and hypothalamic GHRH share similar biological and electrophoretic properties. Partially purified tGHRH is capable of stimulating GH secretion from cultured anterior pituitary cells in a dose-dependent manner. Testicular GHRH and rGHRH have different HPLC retention times and significantly different electrophoretic properties by Western gel analysis. The estimated size of tGHRH-LI is approximately 3.7 times that of synthetic rGHRH. Using immunohistochemistry, tGHRH-LI is localized to mature sperm forms in rat testis. We conclude that rat tGHRH-LI and rGHRH share some structural and functional properties and are probably related peptides. However, the difference in electrophoretic mobility and HPLC retention time indicates that they are not identical. The presence of tGHRH-LI in rat sperm, within the confines of the blood-testis barrier, which is generally impermeable to peptides, leads us to speculate that tGHRH serves a paracrine or autocrine role in testicular physiology.

Animals↗

PubMed1990

The antagonism of glucocorticoid inhibition of wound healing in rats by growth hormone-releasing factor.

Daily therapeutic injections of cortisone to rats will cause weight loss and impaired wound healing. Weight loss is attributed to the catabolic effect of steroid, whereas impaired healing is associated with reductions in fibroplasia and connective tissue deposition. As the major structural protein component of connective tissue is collagen, its absence is responsible for the retarded gain in wound breaking strength. Cortisone also blocks wound closure by inhibiting wound contraction. An anabolic agent such as growth hormone may antagonize the effect of cortisone on the wound healing process. Endogenous GH can be released from the pituitary by exogenous injections of growth hormone-releasing factor (GRF). Two synthetic GRF peptides, a natural 44-amino acid peptide of the human GRF sequence, GRF-44, and an N-terminally substituted analog 29 residues, GRF-29A, were studied. Each was given twice daily with a single daily injection of cortisone for a 7-day period. Concurrent administration of GRF-44 or GRF-29A and cortisone to rats had no effect on restored body weight loss or inhibited wound contraction. While GRF-44 restored collagen deposition and caused restored wound breaking strength, GRF-29A was ineffective in restoring either. GRF-44, a synthetic peptide that stimulates pituitary release of growth hormone, antagonized some of the inhibiting effect of steroid on wound repair by promoting fibroplasia and collagen deposition.

Animals↗

PubMed1989

Evidence for the monomeric nature of thymosins.

According to gel-filtration experiments, alpha- and beta-thymosins appear to form oligomers, which are 4-5-fold larger than the corresponding polypeptides. However, on analysis by sedimentation equilibrium ultracentrifugation, prothymosin alpha and thymosin beta 4 showed relative molecular masses of 12,800 and 4600, which are close to the values calculated from their amino acid sequences, confirming their existence in solution as discrete monomeric entities.

Animals↗

PubMed1989

Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma.

The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRH[GRH(1-44)-NH2 and GRH(1-40)-OH], and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH2] were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(3-44)-NH2 was unaffected by an aminopeptidase inhibitor, indicating that this metabolite was generated from a single step cleavage by a dipeptidylpeptidase (DPP) rather than sequential aminopeptidase cleavages. Conversion to GRH(3-44)-NH2 was blocked by diprotin A, a DPP type IV (DPP IV) competitive inhibitor. D-Amino acid substitution at either position 1 or 2 also prevented hydrolysis, characteristic of DPP IV. Analysis of endogenous plasma GRH immunoreactivity from a human GRH transgenic pig revealed that the major peak coeluted with GRH(3-44)-NH2. Native GRH exhibited trypsin-like degradation at the 11-12 position but cleavage at the 12-13 site occurred only with GRH(1-32)-NH2 and GRH(1-29)-NH2. Formation of these metabolites was independent of prior DPP IV hydrolysis but was greatly reduced by trypsin inhibitors. Evaluation of plasma stability of potential GRH super analogues, designed to resist degradation by these enzymes, confirmed that GRH degradation in plasma occurs primarily by DPP IV, and to a lesser extent by trypsin-like enzyme(s).

Amino Acid Sequence↗

PubMed1988

Expression of an enzymatically active Yb3 glutathione S-transferase in Escherichia coli and identification of its natural form in rat brain.

Glutathione S-transferases containing Yb3 subunits are relatively uncommon forms that are expressed in a tissue-specific manner and have not been identified unequivocally or characterized. A cDNA clone containing the entire coding sequence of Yb3 glutathione S-transferase mRNA was incorporated into a pIN-III expression vector used to transform Escherichia coli. A fusion Yb3-protein containing 14 additional amino acid residues at its N terminus was purified to homogeneity. Recombinant Yb3 was enzymatically active with both 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates but lacked glutathione peroxidase activity. Substrate specificity patterns of recombinant Yb3 were more limited than those of glutathione S-transferase isoenzymes containing Yb1- or Yb2-type subunits. Peptides corresponding to unique amino acid sequences of Yb3 as well as a peptide from a region of homology with Yb1 and Yb2 subunits were synthesized. These synthetic peptides were used to raise antibodies specific to Yb3 and others that cross-reacted with all Yb forms. Immunoblotting was utilized to identify the natural counterpart of recombinant Yb3 among rat glutathione transferases. Brain and testis glutathione S-transferases were rich in Yb3 subunits, but very little was found in liver or kidney. Physical properties, substrate specificities, and binding patterns of the recombinant protein paralleled properties of the natural isoenzyme isolated from brain.

Amino Acid Sequence↗

PubMed1988

A radioimmunoassay for parathymosin alpha using antibodies to synthetic N-terminal peptide 1-30.

Antibodies against the N-terminus of rat parathymosin alpha have been raised in rabbits by conjugating parathymosin alpha (1-30) to hemocyanin. A radioimmunoassay for parathymosin alpha was established by utilizing antibodies against the above polypeptide and parathymosin alpha(1-12)[Tyr] as tracer. The useful range was 5-450 pmol for parathymosin alpha. An epitope was located in the amino acid sequence 1-12. The antiserum failed to crossreact with the same molar concentrations of the partly homologous thymosin alpha 1 or prothymosin alpha. With this radioimmunoassay, parathymosin alpha was isolated from calf thymus after separation from prothymosin alpha by reversed phase HPLC. Endogenous proteases did not appear to generate N-terminal fragments of parathymosin alpha in rat liver extracts in a similar fashion to that observed for prothymosin alpha. Parathymosin alpha has a ubiquitous distribution in the human tissues examined, with levels ranging from 93 (brain) to 1043 (liver) ng of parathymosin alpha(1-30) equivalents/g (wet weight).

Adult↗

PubMed1988

The identification of prothymosin alpha-like material in vertebrate lymphoid organs by a radioimmunoassay for the N-terminal decapeptide.

A radioimmunoassay (RIA) is described for the detection and quantitation of prothymosin alpha (ProT alpha), and its N-terminal fragments containing as a minimum the first ten amino acid residues. This range of peptides includes thymosins alpha 1 (T alpha 1) and alpha 11 (T alpha 11). Antibodies against T alpha 1 and the tracer T alpha 1(1-10)Tyr11(125I), an analogue of the major epitope, were utilized in this RIA. 50% displacement of the ligand was observed with 1.3 pmol of T alpha 1 and 6.4 pmol of ProT alpha. The partially homologous parathymosin alpha (ParaT alpha) showed less than 2% crossreactivity with ProT A. Sephacryl S-200 gel filtration separation of the peptides of calf thymus, chicken spleen and trout spleen extracts prepared by a method eliminating proteolysis, combined with the above RIA, showed the presence of a major immunoreactive peak. Its elution volume corresponded to that of rat ProT alpha (apparent mol. weight 36,000) for both calf (37,000) and chicken (35,000) tissues. In trout it corresponded to a significantly higher molecular weight (62,000). No detectable levels of shorter fragments, including T alpha 1, were observed in any of the above species. The levels of ProT alpha-like peptides in calf thymus, chicken spleen and trout spleen were found to be 246, 8.6 and 7.7 micrograms respectively, of rat ProT alpha equivalents per gram of fresh tissue. The significance of the presence of ProT alpha-like polypeptides in vertebrate species as distant as fish and mammals, the absence of short T alpha 1-like fragments, and the relative conservation of the N-terminus as suggested by the RIA is discussed.

Animals↗