Blockade of VIP during neonatal development induces neuronal damage and increases VIP and VIP receptors in brain.
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Aging↗
Biomedical subjects
M Fridkin
Publications and source records attributed to M Fridkin.
Binding of human serum amyloid P component (hSAP) to human neutrophils.
Human serum amyloid P component (hSAP) and human C-reactive protein (hCRP) are normal serum constituents related to the pentraxin family of plasma proteins. hSAP has morphological and immunochemical identity and extensive sequence similarity to the amyloid P (AP) component found in normal tissues and particularly in amyloid deposits. hCRP and its proteolytic products have been previously shown to bind and to interact with various types of human leukocytes. Binding-displacement experiments with 125I-labeled hSAP and hCRP show that both proteins have specific high-affinity binding sites on normal human polymorphonuclear leukocytes (PMN) and each can compete efficiently with the binding of the other. Scatchard analysis of hSAP-displacement curves reveals a heterogeneous population of hSAP-binding sites existing on the PMN cells, among them about 300,000 low-affinity binding sites with Kd < or = 5 x 10(-6) M and about 30,000 high-affinity binding sites with Kd < or = 5 x 10(-8) M. hAP was found to be degraded by enzymes from human neutrophils to yield a mixture of low-molecular-mass peptides, similarly to the case of CRP reported previously. The binding of hSAP can be efficiently inhibited by this peptide mixture. The results suggest that both hCRP and hSAP, together with related peptides, may participate in vivo in an unknown mechanism of regulation of human neutrophils.
Binding, Competitive↗
Inhibition of cell adhesion to glycoproteins of the extracellular matrix by peptides corresponding to serum amyloid A. Toward understanding the physiological role of an enigmatic protein.
Synthetic peptides related to amino acid residues 29-42 of human serum amyloid A (SAA), Tyr-Ile-Gly-Ser-Asp-Lys-Tyr-Phe-His-Ala-Arg-Gly-Asn-Tyr, were found to inhibit the adhesion of human T-lymphocytes and of mouse M4 melanoma cells to surfaces coated with the major cell adhesive glycoproteins of the extracellular matrix, laminin or fibronectin. Correspondingly inhibitory activity was manifested by the entire 14-residue peptide, by its YIGSD laminin-related domain, and by RGN, the fibronectin-related domain. Intact recombinant SAA (rSAA) and its 1-76 fragment, an amyloid A (AA) protein, also inhibited cell adhesion. The peptides did not inhibit collagen and ADP-induced aggregation of human platelets. Proteolysis of SAA by lysosomal enzymes originating from human neutrophils led to generation of specific peptide segments some of which pertain to the 29-42 domain. It is suggested that the acute-phase protein SAA might be involved, either directly or via its peptide fragments, in inhibition of inflammatory reactions or metastatic processes which depend on integrin and possibly other extracellular-matrix-specific receptors mediated specific recognition and interactions with immobilized components of blood-vessel walls.
Amino Acid Sequence↗
Sequential degradation of the neuropeptide gonadotropin-releasing hormone by the 20 S granulosa cell proteasomes.
The decapeptide gonadotropin-releasing hormone (GnRH) is degraded by the 20 S multicatalytic proteinase complex (proteasome EC 3.4.99.46), purified from ovarian granulosa cells, at the Tyr5-Gly6 bond and to a lesser extent at the Gly6-Leu7 bond, when incubated for 2 h at 37 degrees C. Further cleavage, at Trp3-Ser4 and Ser4-Tyr5 bonds of the neurohormone occurs only subsequently to the appearance of the initial N-terminal degradation products, (1-5)GnRH and (1-6)GnRH. Our results suggest that the sequential degradation of GnRH can serve as an important mechanism for the rapid termination of its biological activity in target cells.
Amino Acid Sequence↗
The gene for the neuropeptide gonadotropin-releasing hormone is expressed in the mammary gland of lactating rats.
The high concentration of gonadotropin-releasing hormone (GnRH) in milk of several species implies that the mammary gland is either a site of synthesis for this neuropeptide or that it is efficiently concentrated from plasma by this organ. By PCR amplification of mammary gland cDNA, we have demonstrated expression of the mRNA for GnRH. The GnRH mRNA was present in the mammary gland of pregnant and lactating rats but not of virgin rats, implying that expression of the GnRH gene is activated during pregnancy, probably by prolactin. In contrast, actin mRNA was evident in all the preparations of mammary glands. Since GnRH is also known to be synthesized by the placenta, it is likely that the placenta and the mammary gland are complementary units by which the mother exercises control over the development and the metabolism of the infant during pregnancy as well as after parturition. In addition, GnRH synthesized by the mammary gland may also affect the mother by a paracrine and/or an endocrine mechanism.
Animals↗
CTL induction by a tumour-associated antigen octapeptide derived from a murine lung carcinoma.
Many mouse and human tumours express major histocompatibility complex (MHC) class I-associated antigens that constitute targets for syngeneic cytotoxic T lymphocytes (CTL). Genes encoding such antigens were isolated from a mouse mastocytoma and from human melanomas by genetic methods. Isolation and characterization of MHC class I-associated peptides has enabled specific anchor residues to be identified that are typical of peptides that bind to distinct class I molecules. Moreover, CTL specific to particular MHC-peptide combinations have been used to identify naturally occurring antigenic peptides in cell extracts and enabled them to be sequenced directly. Most known MHC ligands are of viral origin or are self peptides derived from normal proteins. Here we use total acid extraction and repeated fractionation to isolate and sequence Lewis lung carcinoma (3LL)-specific peptide(s), which shows sequence homology to the connexin 37 protein. Synthetic octamers based on these sequences bind to 'empty' H-2Kb molecules on RMA-S cells, sensitize RMA-S cells to lysis by specific anti-3LL CTL, and induce anti-tumour CTL. The tumour-associated peptide originates from mutated connexin 37 expressed in 3LL.
Amino Acid Sequence↗
Activity of two synthetic amphiphilic peptides and magainin-2 against herpes simplex virus types 1 and 2.
The in vitro antiviral activity of two amphiphilic synthetic peptides, modelin-1 (mod-1) and modelin-5 (mod-5), and of the natural antibacterial peptide magainin-2 (mag-2) against herpes simplex viruses type 1 (HSV-1) and 2 (HSV-2) were evaluated. The peptides were incubated with the virus, i.e. direct inactivation, and their effects examined by means of plaque reduction assay and/or reduction in virus yield. Only mod-1 displayed a strong antiviral effect against HSV-1 and HSV-2, with 50% effective dose (ED50) values of 4.6 and 4.1 micrograms/mL, respectively. Mag-2, mod-5 and a mixture of both had no significant inhibitory effect. Addition of mod-1 up to a concentration of 100 micrograms/mL to the culture medium had no significant cytotoxic effect on host vero cells, as measured by the trypan blue-exclusion method. It showed, however, considerable hemolytic activity against human red blood cells. Experiments including acyclovir (ACV) as a reference viral inhibitor indicated that the mode of action of mod-1 is different from that of ACV. In contrast to ACV, the peptide inactivates the virus following a very short incubation before vero cell infection, suggesting some kind of direct interaction of the peptide with the viral envelope, rather than inhibition of viral DNA replication or gene expression. Our results suggest that mod-1 may be an effective topical antiviral agent against herpes viruses.
Amino Acid Sequence↗
Severe microcephaly induced by blockade of vasoactive intestinal peptide function in the primitive neuroepithelium of the mouse.
Vasoactive intestinal peptide (VIP) has potent growth-related actions that influence cell mitosis, neuronal survival, and neurodifferentiation in cell culture. VIP can also produce dramatic growth in postimplantation mouse embryos in vitro, characterized by large increases in cell number. The goal of the present study was to assess the role of VIP on early nervous system development in vivo. Pregnant mice were treated with a specific antagonist to VIP. Prenatal administration of the antagonist early in development (E9-E11) produced severe microcephaly characterized by decreased embryonic brain weight with reduced DNA and protein content. The retardation of growth was disproportionally manifested in the brain compared with the body and was prevented by co-treatment with VIP. Identical treatment with the antagonist later in gestation had no detectable effect on embryonic growth. VIP receptors, which were restricted to the central nervous system during this stage of embryonic development, were increased in the neuroepithelium of antagonist-treated embryos while the number of cells in S-phase was significantly decreased. Thus, VIP regulates brain growth in vivo and inhibition of its action provides new insight into a molecular mechanism for microcephaly.
Animals↗
Stearyl-norleucine-vasoactive intestinal peptide (VIP): a novel VIP analog for noninvasive impotence treatment.
The present report relates to pharmaceutical composition for the treatment of male impotence. The transdermal application of a potent derivative of vasoactive intestinal peptide (VIP) coupled to a suitable hydrophobic moiety (e.g. stearyl-VIP) in a suitable ointment composition (e.g. Sefsol) enhances sexual activity and erection formation in a variety of impotence models in rats (sterile rats, diabetic rats, and animals with high blood pressure). Furthermore, exchange of the methionine in position 17 with norleucine enhances biological activity. Thus, stearyl-Nle17-VIP may be considered useful for the treatment of impotence.
Administration, Cutaneous↗
Vasoactive intestinal peptide: a growth promoter in neuroblastoma cells.
Vasoactive intestinal peptide (VIP) is a neuromodulator, growth regulator and secretagogue for neuronal survival factors. Moreover, VIP has been suggested to be a mitogenic factor for embryonic neurons in the sympathetic nervous system. We now show that VIP had mitogenic activity in a human neuroblastoma cell line (NMB), as measured by cell number and thymidine incorporation. This mitogenic activity was dose dependent and was decreased with culture maturation. Northern blot analysis revealed VIP mRNA transcripts in this cell line suggesting an autocrine role for VIP in neurogenesis.
Blotting, Northern↗
A vasoactive intestinal peptide antagonist inhibits non-small cell lung cancer growth.
The most prevalent lung cancer, non-small cell lung cancer (NSCLC) has receptors for vasoactive intestinal peptide (VIP). Here the effects of a VIP antagonist (VIP-hyb) on NSCLC growth were investigated. In vivo, when VIPhyb (10 micrograms, s.c.) was daily injected into nude mice, xenograft formation was significantly inhibited by approximately 80%. In vitro, VIP (100 nM) stimulated colony formation approximately 2-fold, whereas 1 microM VIPhyb inhibited colony formation by approximately 50% when adenocarcinoma cell line NCI-H838 was used. The attenuation of tumor proliferation is receptor mediated, as VIPhyb inhibited specific 125I-labeled VIP binding to cell lines NCI-H157 and NCI-H838 with an IC50 of 0.7 microM. VIP (10 nM) increased the cAMP levels 5-fold when cell line NCI-H838 was used, and 10 microM VIPhyb inhibited the increase in cAMP caused by VIP. Northern blot analysis and radioimmunoassays have shown VIP mRNA and VIP-like immunoreactivity in NSCLC cells. These data suggest that VIP may be a regulatory peptide in NSCLC and that VIPhyb is a VIP receptor antagonist that inhibits proliferation.
Amino Acid Sequence↗
Structure-function studies of amphiphilic antibacterial peptides.
The synthesis of 11 peptides, ranging in composition from 9 to 17 amino acid residues, by solid-phase methodology was accomplished with the purpose of studying how the amphiphilic and hydrophobic character, the size of the molecule, and the charge distribution modulate the antibacterial activity. It was found that peptides composed of 16 and 17 amino acid residues, with high hydrophobic (mainly due to Trp or Phe) and hydrophilic (due to Lys) character distributed along opposite amphiphilic faces, showed considerable antibacterial activity against clinically isolated bacteria together with Gram positive and Gram negative ATCC bacterial strains. However, the hemolytic capacity of the peptides was also significant. Decreasing the hydrophobic character of the molecule by replacing Trp or Phe with Leu residues while maintaining the basic contribution of Lys drastically reduced the hemolytic activity and only slightly decreased the bioactivity. Peptides composed of 9-10 amino acid residues with high hydrophobic and basic nature possess antibacterial activity but, in general, are less active than the larger counterpart peptides. By replacing all Trp residues of a short peptide by Leu residues, the activity was considerably reduced. Circular dichroism studies and antibacterial assays showed that shorter peptides with very low helical content, and thus deprived of amphiphilic character, still have appreciable bioactivity. This observation, coupled with the fact that due to their small size they cannot span the bacterial outer lipid bilayer, may suggest different mechanisms of action for long-chain vis-a-vis short-chain peptides.
Amino Acid Sequence↗
Growth factor function of vasoactive intestinal peptide in whole cultured mouse embryos.
Factors controlling central nervous system (CNS) growth immediately after neurulation are mostly unknown. Vasoactive intestinal peptide (VIP) receptors are widely distributed in the embryonic nervous system, and VIP has trophic and mitogenic properties on embryonic neural tissues but inhibits growth and mitosis in certain tumours. To address the potential effects of VIP on embryonic growth, we used whole postimplantation embryo cultures. After a 4-h incubation, VIP stimulated growth, increasing somite number, embryonic volume, DNA and protein content, and number of cells in S-phase. A VIP antagonist substantially inhibited these VIP-mediated increments in growth. The VIP antagonist completely suppressed VIP-stimulated mitosis in the CNS while decreasing the same in non-neuronal tissues by 38%. In vitro autoradiography revealed GTP-sensitive and GTP-insensitive VIP receptors which were differentially regulated in VIP antagonist-treated embryos. The present study suggests that VIP acts as a growth factor on early postimplantation embryos through multiple VIP receptors that exhibit tissue-specific responses.
Analysis of Variance↗
Synthetic peptides derived from the sequence around the plasmin cleavage site in vitronectin. Use in mapping the PAI-1 binding site.
A series of 8 peptides derived from the amino acid sequence accommodating the plasmin cleavage site in vitronectin were synthesized and used to map its binding site for the type I plasminogen activator inhibitor (PAI-1). This mapping assigned the inhibitor binding site to the K348-R370 region with high affinity recognition elements within the K348-R357 sequence. These results account for our previous finding that cleavage of the R361-S362 bond by plasmin significantly reduces the affinity between PAI-1 and vitronectin, since it splits the PAI-1 binding site in two. Furthermore, in the case of the two-chain form of vitronectin, this cleavage detaches the S362-R379 peptide which provides some of the affinity elements for the binding of PAI-1.
Amino Acid Sequence↗
Synthesis and biological activity of [L-hydroxyproline]3-tuftsin analogue and its alpha- or beta-O-D-glucosylated derivatives.
Syntheses are described of the Hyp3-tuftsin analogue and of its derivatives alpha- or beta-O-glycosylated at the side chain function of the hydroxyproline residue. The carbohydrate-free tetrapeptide was prepared by reacting Z-Thr-Lys(Z)-OH with H-Hyp-Arg(NO2)-OBzl by the mixed anhydride procedure. In the synthesis of the alpha-glycosylated analogue the O-glycosyl amino acid was incorporated by reacting Boc-(Glc alpha+beta)Hyp-OH with H-Arg(NO2)-OBzl through the same procedure. The alpha-glucosylated dipeptide was isolated from the diastereomeric mixture, selectively deblocked, and acylated with Z-Thr-Lys(Z)-OH by the mixed anhydride procedure. In the preparation of the beta-glucosylated analogue the BOP procedure was used for reacting Boc-[Glc(Ac)4 beta]Hyp-OH with H-Arg(NO)2-OBzl was well as for the final coupling to tetrapeptide. Removal of protecting groups from crude tetrapeptides was achieved by catalytic hydrogenation. Deacetylation of the sugar moiety of the beta-glucosylated tetrapeptide was achieved by treatment with sodium methoxide in methanol. The synthetic compounds were isolated by ion exchange chromatography, and characterized by elemental analysis, amino acid analysis, optical rotation and proton NMR. Their capacity to evoke the release of interleukin 1 from mouse peritoneal macrophages and to modulate immunogenic activity of antigen-fed cells was evaluated, in comparison with tuftsin and rigin. All of the analogues were found to possess tuftsin-like activity.
Amino Acid Sequence↗
Learning impairment following intracerebral administration of the HIV envelope protein gp120 or a VIP antagonist.
The external envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV) has been shown to be toxic to neurons in culture. To further investigate the neurological effects of gp120, the involvement of this protein with the acquisition of spatial discrimination was assessed. Both native and recombinant gp120 were administered into the cerebral ventricles of adult rats and performance was evaluated in the Morris swim maze. Gp120 treatment retarded acquisition after daily administration of 12 ng. The specificity of this impairment was demonstrated in that the performance of animals given the same amount of gp160 from recombinant baculovirus was not different from animals given saline. Vasoactive intestinal peptide (VIP) has been shown to block gp120-induced neurotoxicity in culture and a VIP receptor antagonist has displayed toxic properties to neurons in culture. We show here that this antagonist, which competitively inhibits VIP binding and blocks VIP-mediated functions in cell cultures from the CNS, also produced an impairment of performance. This retardation was attenuated by cotreatment with VIP, supporting the specificity of the observed impairment. Thus, gp120 and the VIP antagonist produced similar retardation of spatial discrimination, suggesting that both may impair memory for spatially related stimulus control.
Animals↗
Localization of sequence-determined neoepitopes and neutrophil digestion fragments of C-reactive protein utilizing monoclonal antibodies and synthetic peptides.
We recently described 17 anti-CRP mAb, seven to native- (or conformational) and 10 to neo- (or sequence-determined) epitopes, including several anti-neo-CRP mAb specific for CRP peptide 199-206. In the present study, four new anti-native- and four new anti-neo-CRP mAb were generated and characterized by ELISA reactivity with native and modified human and rabbit CRP, as well as binding to pronase fragments of human CRP in Western blots. Assays with 17 synthetic CRP peptides identified anti-neo-CRP mAb specific for peptides 1-16, 14-24 and 137-152, respectively. The anti-neo-CRP mAb were reacted with fragments obtained by digesting CRP with multiple additional enzymes, including Staphylococcal V8 protease, trypsin, elastase, plasmin, thrombin and alpha-chymotrypsin. Native CRP was remarkably resistant to enzymic digestion, particularly in the presence of calcium, but was readily cleavable upon denaturation. Twenty-three informative fragments served to further distinguish mAb reactivity with at least four additional neo-CRP epitopes, which presumptively included residues in the regions of amino acids 22-45, 41-61, 114-121 and 130-138, respectively. The eight epitopes identified corresponded well with predicted regions of CRP antigenicity. In addition, at least six distinct native or conformation-determined epitopes were delineated. Reactivity of the anti-neo-CRP mAb with fragments of CRP generated by PMN enzymes indicated that regions sensitive to cleavage by neutrophil enzymes are located at approximately 3, 10 and 16 kD from the amino terminus of the CRP subunit. We expect that the anti-CRP mAb described and mapped herein will be useful tools for the elucidation of CRP structure and function.
Antibodies, Monoclonal↗
Characterization of neutrophil-mediated degradation of human C-reactive protein and identification of the protease.
Human C-reactive protein (CRP) is shown to mediate a release of enzymatic activity from neutrophils which promotes its own degradation in the extracellular medium. This egress of proteolytic activity, which was upregulated by phorbol 12-myristate 13-acetate (PMA), was found to occur from both the cytoskeleton and membrane fractions of neutrophils and was dependent on the time of incubation of CRP with the cells and the concentration of CRP. Neutrophil kinases activated by PMA are found to be involved in upregulating the activity of the CRP-degrading protease. The apparent molecular weight of the CRP-degrading protease associated with the conditioned medium from PMA-stimulated neutrophils and neutrophil membrane and cytoskeleton preparations, was found by size exclusion chromatography to be 600 kD and migrated on 3-13% SDS-PAGE as four discrete bands to positions corresponding to apparent molecular weights of 209 kD, 316 kD, 398 kD and 501 kD.
C-Reactive Protein↗