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Biomedical subjects

M Fridkin

Publications and source records attributed to M Fridkin.

At least 127 records · Page 7Linked to original sources
PubMed1992

Augmentation of the antibacterial activity of magainin by positive-charge chain extension.

Novel analogs of the broad-spectrum antimicrobial peptide magainin-2 were obtained by extension of its chain through addition of segments of positively charged amino acids to either its N or its C terminus and by increasing its helicity. The activity of magainin-2 toward American Type Culture Collection strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus was most considerably enhanced by these modifications, whereas, in general, its low hemolytic capacity was not or was only slightly affected. The antibacterial potencies of magainin-2 and its derivatives were more evident following decreases of pH from 7.2 to 6 and 5.

Amino Acid Sequence↗
PubMed1992

A fatty neuropeptide. Potential drug for noninvasive impotence treatment in a rat model.

Vasoactive intestinal peptide (VIP), a key penile neurotransmitter, induces erection after local injection in man. To augment the therapeutic potential of VIP for impotence treatment and circumvent difficulties of direct penile injections, a strategy was designed to increase peptide hydrophobicity. This was accomplished by the synthesis of a conjugate of VIP and stearic acid (stearyl-VIP). Upon penile topical application, stearyl-VIP, in contrast to native VIP, significantly increased sexual function as measured by copulatory activity and penile reflexes (erections) in testosterone-treated, castrated rats. In addition, stearyl-VIP penetrated the body in amounts severalfold greater than VIP. Pharmacokinetic studies demonstrated 10-fold higher penile concentrations of stearyl-VIP, as compared with that measured in the blood 15 min after application, with a gradual decrease thereafter. The peak of incorporation into peripheral tissues that was observed 30 min after administration was 1,000-fold less than that found in the penile tissue. Tissue extraction and chromatographic analysis revealed that stearyl-VIP remained essentially intact for greater than or equal to 15 min and was cleared after 1 h. Thus, topically administered stearyl-VIP had increased bioavailability in comparison with VIP without apparent toxicity, suggesting significant therapeutic potential.

Animals↗
PubMed1992

C-reactive protein (CRP) peptides inactivate enolase in human neutrophils leading to depletion of intracellular ATP and inhibition of superoxide generation.

The nature and the biochemical mechanism of inhibition of neutrophil membrane-associated oxidative metabolism by two synthetic peptides p77-82 and p201-206 (amino acid sequences Val-Gly-Gly-Ser-Glu-Ile and Lys-Pro-Gln-Leu-Trp-Pro respectively, from the primary amino acid sequence of C-reactive protein) have been ascertained. Preincubating neutrophils for 15 min with 50 microM of p77-82 or p201-206 resulted in superoxide generation by opsonized zymosan stimulated neutrophils being inhibited by 34 +/- 2% (P less than 0.005) and 29 +/- 2% (P less than 0.005) respectively. With a 60-min preincubation period 6.25 microM of p77-82 or p201-206 was effective in inhibiting this superoxide generation by 12 +/- 2% (P less than 0.01) and 10 +/- 1% (P less than 0.01) respectively. Neither peptide inhibited neutrophil arachidonic acid release, transmembrane potential or transductional events preceding superoxide generation. Inhibition of neutrophil functions was found to be due to the ability of each peptide (50 microM) following a 15-min preincubation period to inhibit both neutrophil glycolysis and ATP generation by approximately 30%. The inhibition of ATP generation and glycolysis in neutrophils is attributable to the ability of these peptides to inhibit uncompetitively the glycolytic enzyme enolase. Using purified enolase the relative Ki values for p77-82 and p201-206 were 27 and 19 microM respectively. Inhibition of neutrophil function by the peptides is concluded to be due to effective interference of neutrophil energy metabolism.

Adenosine Triphosphate↗
PubMed1991

A VIP antagonist distinguishes VIP receptors on spinal cord cells and lymphocytes.

Vasoactive intestinal peptide (VIP) is a neuropeptide which also interacts with cells of the immune system. The paucity of specific VIP receptor antagonists has hampered studies of possible receptor heterogeneity and of VIP function. To aid in achieving these goals, a new VIP antagonist, a hybrid between neurotensin and VIP, has been synthesized. This peptide interacted with VIP receptors on spinal cord cells with an affinity 10-fold greater than VIP itself. In contrast, 1000-fold higher concentrations of the antagonist were required to displace labeled VIP from its receptor on lymphoid cells as compared to VIP itself, suggesting VIP receptor heterogeneity between immune and spinal cord cells.

Amino Acid Sequence↗
PubMed1991

Glyco-tuftsin derivatives modulate interleukin-1 and tumor necrosis factor production.

Six Thr1 (O-glyco)-derivatives of the "phagocytosis stimulating peptide" tuftsin, H-Thr-Lys-Pro-Arg-OH and the N-glycosylated undecapeptide H-Thr-Lys-Pro-Arg-Glu-Gln-Gln-Tyr-Asn(beta-D-GlcNAc)-Ser-Thr-OH, which correspond to the "tuftsin-region" at the Fc-domain of immunoglobulin G (amino acid residues 289-299), were evaluated in comparison with tuftsin and rigin, H-Gly-Gln-Pro-Arg-OH, for their capacity to evoke the release of interleukin-1 and tumor necrosis factor from mouse peritoneal macrophages and from human monocytes. Several glycosylated tuftsin derivatives were found to modulate, in a rather dose-dependent manner, the release of the two cytokines from both cell types.

Amino Acid Sequence↗
PubMed1991

An antagonist to vasoactive intestinal peptide affects cellular functions in the central nervous system.

A vasoactive intestinal peptide (VIP) antagonist was synthesized and used to investigate the interactions of VIP with its receptors present in the central nervous system (CNS). The VIP antagonist is a hybrid peptide consisting of a portion of VIP and a portion of neurotensin, designed to change the membrane permeability of the VIP portion. The hybrid antagonist displaced 80 to 90% of [125I]VIP binding to cell cultures from cerebral cortex, hippocampus or spinal cord. The displacement curve was biphasic, suggesting two binding sites. In the case of cortical astrocytes, the antagonist had a Ki of 45 pM at one site and a Ki of 74 nM at the other. At the lower affinity binding site, the antagonist was about 10-fold more potent than VIP in displacing radiolabeled VIP. The accumulation of cyclic AMP (cAMP) in VIP-stimulated cortical glia cultures was decreased by the new antagonist (EC50, 59 nM). This decrease in cAMP was greater than that achieved in the presence of other putative VIP antagonists. Finally, the addition of 1 nM hybrid antagonist to dissociated spinal cord cultures resulted in a 42% reduction in neuronal cell counts as compared with controls, and the EC50 of this effect was about 30 pM, which corresponded closely to the Ki of antagonist displacement of [125I]VIP binding at the high-affinity site. The antagonist appears to be a competitive blocker for both VIP-mediated increases in cAMP formation or VIP-associated maintenance of neuronal survival in spinal cord cultures. Thus, we describe a potent VIP antagonist which interacts with two functionally distinct VIP receptors in the CNS.

Amino Acid Sequence↗
PubMed1991

Hypothalamic hormones in milk.

Following a multistep extraction and fractionation of sheep and human milk, immunoreactive and biologically active somatostatin (SS) was demonstrated as a constituent of milk. Milk, in contrast to serum, contains only SS-14-like material and not SS-28. HPLC-purified sheep milk somatostatin was found to inhibit both basal and prostaglandin-induced release of growth hormone from cultured rat pituitary cells. The effect was dose-dependent and similar to that of synthetic SS. In order to determine whether SS or vasoactive intestinal peptide (VIP) can be synthesized by the mammary gland we have looked for SS and VIP messenger RNA in rat glands extracted on different days of lactation. No messages for SS or VIP were detected in the mammary gland, whereas, as expected, hybridization signals for alpha-casein were evident. These results suggest that somatostatin and VIP are not synthesized by the mammary gland and are probably concentrated from blood. In other experiments we have demonstrated that one hour after oral administration of labelled somatostatin to rat pups, about 95% of the neuropeptide, extracted from the stomach, is eluted exactly at the same position as the marker peptide. These results suggest that, in contrast to the adult, somatostatin remains intact in the stomach of the neonate and therefore may be absorbed in a biologically active form.

Animals↗
PubMed1990

All-D-magainin: chirality, antimicrobial activity and proteolytic resistance.

All-D-magainin-2 was synthesized to corroborate experimentally the notion that the biological function of a surface-active peptide stems primarily from its unique amphiphilic alpha-helical structure. Indeed, the peptide exhibited antibacterial potency nearly identical to that of the all-L-enantiomer. Being highly resistant to proteolysis and non-hemolytic all-D-magainin might have considerable therapeutic importance.

Amino Acid Sequence↗
PubMed1990

Peptides generated from C-reactive protein by a neutrophil membrane protease. Amino acid sequence and effects of peptides on neutrophil oxidative metabolism and chemotaxis.

We have recently provided evidence that C-reactive protein (CRP) could act as an up-regulatable substrate for membrane-associated neutrophil serine protease(s). The resultant degradation of CRP yielded small soluble bioactive peptides that inhibit many of the proinflammatory functions of activated neutrophils and could oppose the tissue destructive potential of these cells. We report on the reverse phase HPLC separation of the small TCA-soluble peptides obtained when CRP is degraded with nonstimulated or PMA-stimulated neutrophils and purified neutrophil membranes. The amino acid sequence of seven peptides isolated from the CRP digest has been ascertained and synthetic peptides homologous to these sequences have been synthesized. Three of the synthetic peptides corresponding to residues 201-206 (CRP-III), 83-90 (CRP-IV), and 77-82 (CRP-V) of the intact protein were identified to significantly inhibit superoxide production from activated neutrophils at 50 microM whereas CRP-III and CRP-V in addition inhibited neutrophil chemotaxis at this concentration. These peptides act additively and their action likely involves the signal transduction pathways for neutrophil activation.

Amino Acid Sequence↗
PubMed1990

Tuftsin induces tissue factor-like activity in human mononuclear cells and in monocytic cell lines.

Normal human monocytes and macrophages generate potent procoagulant activity (PCA) resembling tissue factor (TF) in response to various stimuli. In this study we show that tuftsin, a natural stimulator of many functions of monocytes and macrophages, also stimulates a potent PCA in mixed mononuclear cells and monocytes, and a mild PCA in lymphocytes and cell lines of monocytic origin (U937 and THP). No activity was generated by several lymphoid cell lines and HL-60 cells. The PCA resembled TF in that it accelerated clotting through the extrinsic coagulation pathway and was inhibited by concanavalin-A and by monoclonal anti-TF antibodies. The induction of TF-like activity by tuftsin was dose- and time-dependent. It was located in the cell membrane and did not require T cells for expression. Generation of TF-like activity was prevented by actinomycin D, while cytarabine had no effect on this process, suggesting that expression of the activity depends on protein synthesis. Studies with various tuftsin analogs suggest that tuftsin stimulates generation of TF-like activity, as well as other functions of monocytes via the same receptors. The results with the monocytic cell lines show that tuftsin affects mainly mature cells. The induction of TF-like activity in mononuclear cells by tuftsin constitutes an important link between mononuclear cells and the immune and coagulation systems. It may play a major role in the pathogenesis of thromboembolism and fibrin deposition in various inflammatory and immunologic disorders.

Blood Coagulation Factors↗
PubMed1990

Degradation of gonadotropin-releasing hormones in the gilthead seabream, Sparus aurata. I. Cleavage of native salmon GnRH and mammalian LHRH in the pituitary.

The pattern and kinetics of degradation of native salmon gonadotropin-releasing hormone (sGnRH) and mammalian leuteinizing hormone-releasing hormone (LHRH) by pituitary bound enzymes were studied in the gilthead seabream, Sparus aurata. sGnRH and LHRH were incubated for different periods of time with membrane or cytosolic fractions of pituitary homogenates. At the end of the incubation, the degradation mixture was fractionated on reverse-phase high-pressure liquid chromatography. The degradation products were identified by comparing their retention times to those of synthesized GnRH fragments and by analyzing their amino acid composition. The main GnRH degradative activity resides in the cytosolic fraction of the pituitary homogenate. Both sGnRH and LHRH are rapidly degraded by pituitary cytosol, with 78.3 and 87.7% of the peptides, respectively, cleaved after 3 hr of incubation. Maximal degradation of sGnRH occurred at a pH range of 7 to 8. The main initial products of degradation of sGnRH and LHRH are the 1-5, 6-10, and 1-9 fragments. This suggests the involvement of two site-specific peptidases, a Tyr5-Gly6 endopeptidase and a Pro9-Gly10NH2 peptidase or postproline cleaving enzyme. While the 1-6 and 1-9 fragments undergo rapid secondary degradation, the 1-5 is relatively stable. Competition experiments suggest that the endopeptidase cleaving the sGnRH at the Tyr5-Gly6 bond is not specific to the neuropeptide and is probably a general proteolitic enzyme. However, the cleavage at the 9-10 bond has a high degree of specificity to the Pro9-Gly10NH2 sequence found in sGnRH. The two proposed pituitary peptidases of S. aurata have some characteristics similar to those of rat hypophyseal and hypothalamic GnRH cleaving enzymes. No differences are found in hypophyseal GnRH degradative activity between females with occytes undergoing previtellogenesis or advanced stages of vitellogenesis.

Amino Acids↗
PubMed1990

Degradation of gonadotropin-releasing hormones in the gilthead seabream, Sparus aurata. II. Cleavage of native salmon GnRH, mammalian LHRH, and their analogs in the pituitary, kidney, and liver.

The pattern and kinetics of degradation of native salmon gonadotropin-releasing hormone (sGnRH), mammalian luteinizing hormone-releasing hormone (LHRH), and some of their analogs by cytosolic enzymes of pituitary, kidney, and liver were studied in the gilthead seabream, Sparus aurata. The native peptides sGnRH and LHRH are rapidly degraded by all three tissues, LHRH being degraded faster than sGnRH. The kinetics of production of the peptide fragments suggest that initial cleavage of sGnRH and LHRH in the three studied tissues occurs at the 5-6 and 9-10 bonds. This indicates the initial activity of a Tyr5-Gly6 endopeptidase and a Pro9-Gly10NH2 peptidase or postproline cleaving enzyme. Secondary degradation of the main initial fragments (1-5, 6-10, and 1-9) is more intensive in the kidney than in the pituitary or liver. Substitution of the position 6 amino acid glycine by a dextrorotatory (D) amino acid such as in the D-Trp6-LHRH renders the 5-6 bond resistant to cleavage. However, whereas [D-Trp6]-LHRH is intensively cleaved at the Pro9-Gly10NH2 bond by the pituitary, its cleavage at this site by the kidney and liver is slow. This suggests a low activity of the Pro9-Gly10NH2 peptidase in the kidney and liver as compared to the pituitary. When, in addition to the position 6 substitution, the carboxy terminus Pro9-Gly10NH2 is modified to Pro9NET, such as in the [D-Ala6-Pro9NET]-LHRH and the [D-Arg6-Pro9NET]-sGnRH, the 9-10 cleavage site is also blocked, resulting in GnRH analogs highly resistant to degradation. The relationships between susceptibility of the different forms of GnRH to enzymatic degradation by the pituitary, kidney, and liver and their relative biological activities in S. aurata are discussed. We conclude that increased resistance of GnRH analogs to enzymatic degradation contributes to their superactivity.

Animals↗
PubMed1990

Polytuftsin: a potential precursor for slow release of the phagocytosis stimulating peptide tuftsin.

1. Polytuftsin (Thr-Lys-Pro-Arg)n, was synthesized through polycondensation of an amino-free and carboxyl-activated derivative of tuftsin, H2N-Thr-Lys(Z)-Pro-Arg(Tos)-OSu, following suitable deprotection and fractionation steps. 2. Digestion of polytuftsin by trypsin, as well as by normal human serum, at 37 degrees C, yielded free tuftsin. 3. Polytuftsin affected the decreased formation of lung-metastasis, in B16 melanoma treated mice and prolonged the survival of animals more efficiently than tuftsin. 4. Tuftsin was found to be totally degraded by serum enzymes within approximately 60 min at 37 degrees C.

Amino Acid Sequence↗
PubMed1990

Thiolysis of the 3-nitro-2-pyridinesulfenyl (Npys) protecting group. An approach towards a general deprotection scheme in peptide synthesis.

The hydroxylic side-chain functional groups of serine, threonine, hydroxproline and tyrosine, the alpha and epsilon-amino moieties of lysine and the thiol group of cysteine were masked by the 3-nitro-2-pyridinesulfenyl (Npys) protecting group. Deprotection was mildly affected by thiolysis with either 2-mercaptopyridine and 2-mercaptomethyl imidazole (O- and N-Npys) or with 3-mercaptoacetic acid and 2-mercaptoethanol (S-Npys). Thiolysis was monitored spectrophotometrically and was completed in a rather short time. Incorporation of the Npys group into a whole and single thiolyzable deprotection scheme is suggested.

Chemical Phenomena↗
PubMed1989

Antibodies directed against phosphothreonine residues as potent tools for studying protein phosphorylation.

Here we report the development of novel antibodies which specifically react with phosphothreonine residues [anti-(P-Thr)antibodies]. The specificity of the antibodies was assessed in radioimmunoassays where we could demonstrate that half-maximal and maximal binding of the antibodies to plates coated with BSA - P-Thr occurred at serum dilutions of 1:4000 and 1:1000, respectively. P-Thr inhibited antibody binding with a half-maximal effect at 40 microM. P-Ser was 200-fold less potent while P-Tyr was essentially ineffective. Anti-(P-Thr) antibodies could specifically bind to phosphothreonine-containing proteins on Western blots. Using such a procedure we could demonstrate enhanced threonine phosphorylation of the EGF receptor upon treatment of intact unlabeled A431 cells with EGF. We could further demonstrate antibodies binding to proteins present in extracts of rat hepatoma cells (Fao). P-Thr at 10 microM completely inhibited antibody binding while P-Ser, P-Tyr, Thr or Ser, each present at tenfold higher concentrations, had no such inhibitory effect. Anti-(P-Thr) antibodies were also capable of specifically immunoprecipitating 32P-labeled phosphoproteins present in Triton extracts of Fao cells. Immunoprecipitation of proteins of 38 kDa, 55 kDa, 85 kDa, 100 kDa and 155 kDa was inhibited by 1 mM P-Thr but not by P-Tyr. These findings suggest that anti-(P-Thr) antibodies could be powerful tools in studies aimed at monitoring alterations in threonine phosphorylation of specific proteins as they occur under physiological conditions in response to various extracellular stimuli. Identification of such proteins can be conveniently monitored by immunoblotting.

Animals↗
PubMed1989

Human peritoneal macrophage activity is increased by tuftsin.

Peritonitis caused by Candida albicans is a major complication of continuous ambulatory peritoneal dialysis (CAPD). Increasing the activity of the peritoneal macrophages--the predominant cell type found in the peritoneal cavity--may be of great importance in the prevention and therapy of peritonitis. Therefore, the activating effect of tuftsin was studied on human peritoneal macrophages from CAPD patients. Tuftsin induced a biphasic effect on macrophage activity within a range of 2 X 10(-9)-2 X 10(-6) M, with a maximal activity of 2 X 10(-7) M. At this concentration, tuftsin enhanced by twofold cell association with radiolabelled candida (from 2 +/- 0.2 to 4 +/- 0.2 candida per macrophage) and superoxide anion production in response to exposure to candida (from 150 +/- 20 to 300 +/- 20 nmoles/mg). These results suggest the potential use of tuftsin as a therapeutic drug.

Adult↗
PubMed1989

Estrogen regulation of vasoactive intestinal peptide mRNA in rat hypothalamus.

The participation of gonadal steroid hormones in regulation of the vasoactive intestinal peptide (VIP) gene expression in the hypothalamus was studied using a quantitative densitometric hybridization assay. In the female rat the levels of VIP mRNA were found to be significantly decreased following ovariectomy (4.41 +/- 0.7 arbitrary units of absorbance vs. 8.52 +/- 0.18). This decrease was largely reversed after three days of treatment with estradiol dibenzoate. In contrast to the female rats, no significant change in VIP mRNA levels was observed in the male rats, following orchidectomy. These results suggest a sexual dimorphism with regard to the steroid regulation of hypothalamic VIP gene expression in the rat.

Animals↗