Mutation of the α2 domain disulfide bridge of the class I molecule HLA-A0201 Effect on maturation and peptide presentation

A combination of saturation and site-directed mutagenesis was utilized to disrupt the α₂ domain disulfide bridge of HLA-A^*0201. Mutation of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine (C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58–66) were recognized poorly by HLA-A^*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A^*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476–484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A^*0201 using a minigene coding for either the influenza A matrix peptide 58–66, or HIV-1 pol peptide 476–484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface.     

Functional role of Alix in HIV-1 replication

Retroviral Gag proteins encode small peptide motifs known as late domains that promote the release of virions from infected cells by interacting directly with host cell factors. Three types of retroviral late domains, with core sequences P(T/S)AP, YPX_nL, and PPPY, have been identified. HIV-1 encodes a primary P(T/S)AP-type late domain and an apparently secondary late domain sequence of the YPX_nL type. The P(T/S)AP and YPX_nL motifs interact with the endosomal sorting factors Tsg101 and Alix, respectively. Although biochemical and structural studies support a direct binding between HIV-1 p6 and Alix, the physiological role of Alix in HIV-1 biology remains undefined. To elucidate the function of the p6–Alix interaction in HIV-1 replication, we introduced a series of mutations in the p6 Alix binding site and evaluated the effects on virus particle production and virus replication in a range of cell types, including physiologically relevant primary T cells and macrophages. We also examined the effects of the Alix binding site mutations on virion morphogenesis and single-cycle virus infectivity. We determined that the p6–Alix interaction plays an important role in HIV-1 replication and observed a particularly severe impact of Alix binding site mutations when they were combined with mutational inactivation of the Tsg101 binding site.     

Induction of V3-Specific Cytotoxic T Lymphocyte Responses by HIVgagParticles Carrying Multiple Immunodominant V3 Epitopes of gp120

Efforts to develop a vaccine to prevent infection of human immunodeficiency virus (HIV) have focused on the induction of neutralizing antibodies. In our previous study, we reported that chimericgag–envvirus-like particles (VLPs) induce neutralizing antibodies which block HIV infection. In addition to the neutralizing antibodies, the cytotoxic T-lymphocyte (CTL) response is considered to be another major immune defense mechanism required for recovery from many different viral infections. In the present study, we have constructed chimeric fusion proteins using HIV-2gagprecursor protein with (1) four neutralizing epitopes from HIV-1 gp160; (2) three tandem copies of consensus V3 domain, which have been derived from 245 different isolates of HIV-1 and carries both the principal neutralizing determinant (PND) and CTL epitopes; and (3) V3 domains from HIV-1_IIIB, HIV-1_MN, HIV-1_RF, and HIV-1_SF2. These chimeric fusion proteins were expressed in a large quantity within insect cells, and released as VLPs into the cell culture medium. The purifiedgag–envVLPs from all three constructs appear to be spherical particles similar to immature HIV but slightly larger than thegagVLPs. Immunoprecipitation analysis showed that the chimeric proteins were recognized not only by HIV-1 positive patient sera, but also by monoclonal and polyclonal antisera raised against V3 peptides of HIV-1_IIIB, HIV-1_MN, HIV-1_RF, and the gp120 antiserum against HIV-1_SF2. Balb/C mice immunized with these chimeric VLPs successfully induced CTL activity against V3 peptide-stimulated target cells. In addition, a high degree of cross-reactivity was observed among the four different strains of HIV-1 V3 domain, indicating that the tandem multiple consensus V3 peptide sequence carried by HIV-2gagcan be used as a potential HIV vaccine against various HIVs.     

Engineering the binding properties of the T cell receptor:peptide:MHC ternary complex that governs T cell activity

The potency of a T cell is determined in large part by two interactions, binding of a cognate peptide to the MHC, and binding of the T cell receptor (TCR) to this pepMHC. Various studies have attempted to assess the relative importance of these interactions, and to correlate the corresponding binding parameters with the level of T cell activity mediated by the peptide. To further examine the properties that govern optimal T cell activity, here we engineered both the peptide:MHC interaction and the TCR:pepMHC interaction to generate improved T cell activity. Using a system involving the 2C TCR and its allogeneic pepMHC ligand, QL9–L^d, we show that a peptide substitution of QL9 (F5R), increased the affinity and stability of the pep–L^d complex (e.g. cell surface t_1/2-values of 13 min for QL9–L^d versus 87 min for F5R–L^d). However, activity of peptide F5R for 2C T cells was not enhanced because the 2C TCR bound with very low affinity to F5R–L^d compared to QL9–L^d (K_D = 300 μM and K_D = 1.6 μM, respectively). To improve the affinity, yeast display of the 2C TCR was used to engineer two mutant TCRs that exhibited higher affinity for F5R–L^d (K_D = 1.2 and 6.3 μM). T cells that expressed these higher affinity TCRs were stimulated by F5R–L^d in the absence of CD8, and the highest affinity TCR exhibited enhanced activity for F5R compared to QL9. The results provide a guide to designing the explicit binding parameters that govern optimal T cell activities.     

Inhibition of M-tropic HIV-1 infection by the fd phage-gene 3 protein with MIP-1α-binding activity

CCR5 is a chemokine receptor with seven transmembrane-domains. It is expressed on T cells and macrophages and functions as the principal co-receptor for macrophage (M)-tropic strains of HIV-1. The anti-CCR5 monoclonal antibody (mAb) 2D7 inhibits the binding and chemotaxis of the three natural β-chemokine ligands of CCR5, macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES, to CCR5⁺ cells. The mAb also efficiently blocks the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro.In this study, we attempted to determine the peptide motif recognized with the 2D7 mAb. We isolated phage clones by panning a phage display library using 2D7 and identified three peptide motifs. One of these phage clones (M23) showed a marked inhibitory activity on HIV-1 infection. The unique sequence of 15 amino acids with an internal disulfide bond was inserted in the g3p of the M23 phage clone (M23-g3p). The M23-g3p was purified by fast-performance liquid chromatography (FPLC). We show here that (1) M23-g3p was specifically recognized with anti-CCR5 mAb; (2) M23-g3p showed inhibitory activity on the infectivity of M-tropic but not T-tropic HIV-1 strains; (3) M23-g3p bound to MIP-1α, MIP-1β, and RANTES but not MCP-1. These results suggested that the M23-g3p might mimic the CCR5-binding domain shared by β-chemokines, MIP-1α, MIP-1β, and RANTES as well as the HIV-1 infection.     

Synthesis, characterization, and preliminary assessment of anti-Flt1 peptide–hyaluronate conjugate for the treatment of corneal neovascularization

Anti-Flt1 peptide of GNQWFI has been reported to inhibit vascular endothelial growth factor receptor 1 (VEGFR1) – mediated endothelial cell migration and tube formation. In this work, a protocol to synthesize anti-Flt1 peptide–hyaluronate (HA) conjugate was successfully developed for the treatment of corneal neovascularization. Using tetrabutyl ammonium salt of HA (HA-TBA), water-insoluble anti-Flt1 peptide could be conjugated with HA in dimethyl sulfoxide (DMSO) by the amide bond formation between carboxyl groups of HA and N-terminal amine groups of GGNQWFI. The formation of anti-Flt1 peptide–HA conjugate was confirmed by ¹H NMR and fluorometric analyses. The average number of grafted peptide molecules in anti-Flt1 peptide–HA conjugates could be controlled from 3 to 30 per single HA chain by changing the feeding amount of peptide for the conjugation reaction. According to in vitro biological activity tests, anti-Flt1 peptide–HA conjugate exhibited a significant inhibition effect on the binding of Flt1-Fc to VEGF₁₆₅ coated on the well. Furthermore, in vivo biological activity of anti-Flt1 peptide–HA conjugate was confirmed from the inhibitory effect on corneal neovascularization in silver nitrate cauterized corneas of SD rats. The VEGF receptor 2 expression was also reduced after treatment with anti-Flt1 peptide–HA conjugate. The water-soluble anti-Flt1 peptide–HA conjugate was thought to have a potential to be developed as anti-angiogenic therapeutics for the treatment of corneal neovascularization.     

A synthetic peptide which specifically inhibits heat-treated interleukin-8 binding and chemotaxis for neutrophils

Interleukin-8 (IL-8) is a peptide which is secreted by stimulated human monocytes and which is chemotactic for human neutrophils. We synthesized three overlapping peptides spanning the amino-terminal region of the IL-8 sequence. None of the peptides retained the chemotactic activity of the native molecule. One of the peptides, IL-8(_3–25), inhibited the neutrophil chemotactic activity of recombinant IL-8 (rIL-8) which had been preheated to 40°C but did not reduce neutrophil chemokinesis, or the chemotactic activity of unheated rIL-8, FMLP, C5a or LTB₄. Interleukin-8 exhibited similar binding kinetics and chemotaxis for neutrophils regardless of whether it had been pretreated at 40°C.In addition, IL-8(_3–25) was also able to decrease the binding of prehead IL-8 to neutrophils. IL-8(_3–25), which can self-associate, binds directly to receptors on the neutrophil. The data suggest that heat-treated, but not untreated, IL-8 causes the IL-8(_3–25) multimers to disaggregate, allowing the monomeric peptide to directly bind to the IL-8 receptor and thus inhibiting IL-8/receptor binding.     

The calcitonin gene-related peptide (CGRP) antagonist CGRP8–37 blocks vasodilatation in inflamed rat skin: involvement of adrenomedullin in addition to CGRP

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent microvascular vasodilator in rat skin and effects are antagonised by CGRP_8–37. In this study, CGRP_8–37 significantly (P<0.05) inhibited the time-dependent (3–5 h) increase in skin blood flow measured in the anaesthetised rat, after intradermal administration of the inflammatory cytokine interleukin-1β (3 pmol/site), indicating the involvement of CGRP1 receptors. The CGRP-related peptide adrenomedullin (ADM) is also a potent vasodilator in rat skin, with effects antagonised by CGRP_8–37. We show that ADM mRNA expression is increased in rat skin after treatment with IL-1β and that the IL-1β-induced blood flow is blocked by a selective ADM antibody (P<0.05). Thus ADM is expressed locally in the inflamed cutaneous microvasculature where it can, in addition to, or as an alternative to CGRP, contribute to IL-1β-induced vasoactive effects.     

Neutralizing antibodies as a prognostic indicator in the progression of acquired immune deficiency syndrome (AIDS)-related disorders: A double-blind study

A double-blind longitudinal study for the presence of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies (NAb) in the sera of 36 patients with acquired immune deficiency syndrome (AIDS), 149 prodromal homosexual subjects, and 33 heterosexual subjects has been carried out. All AIDS patients and 68% of prodromal homosexual subjects (101/149) were found to be HIV-1 antibody positive by Western blot assay. All heterosexual subjects were HIV-1 antibody negative. Neutralizing antibody(s) was determined by testing the protective activity of sera against HIV-1 infection of human T-cell line H9. Study subjects were divided into NAb(+) (antibody titer, >1:40) and NAb(–) (antibody titer, <1:40) groups. During the 24-month observation period 2 of 80 (3%) HIV-1(+) NAb(+) individuals progressed to AIDS and died, as compared to 5 of 21 (24%) of HIV-1(+) NAb(–) subjects who progressed to AIDS. Similarly, among the NAb(+) AIDS patients 8 of 23 (35%) died, while 10 of 13 (77%) of the NAb(–) patients died during the course of the study. In addition, the absence or reduction of HIV-1 p17 and p24 antibodies directed against HIV-1 antigens as well as the low titer or absence of NAb appears to be closely related to the clinical progression of the disease. These studies suggest that a decrease in the virus neutralization capacity of the sera and a decrease or complete loss of HIV-1 p17 and p24 antibodies may be useful as prognostic indicators for the progression of disease in HIV-1-seropositive patients.     

Human Immunodeficiency Virus Type 1 Disease Progression, CCR5 Genotype, and Specific Immune Responses

The correlation among the presence of a 32-bp deletion in the CC-chemokine receptor 5 (CCR5) gene, disease progression, and human immunodeficiency virus type 1 (HIV-1)-specific immune responses was analyzed for a cohort of 79 Caucasian HIV-1-infected patients. The CCR5 genotype (CCR5/CCR5 = wild type/wild type or Δ32CCR5/CCR5 = 32-bp deletion/wild type) in peripheral blood mononuclear cells was determined by PCR, followed by sequencing of both wild-type and Δ32CCR5 gene fragments. HIV-1-specific humoral responses to gp41 and V3_MN peptides were determined by enzyme immunoassays. The prevalence of the Δ32CCR5 allele was lower among 37 patients with rapid progression (progression to AIDS or to a CD4 cell count of <200 × 10⁶/liter in less than 9 years; P < 0.01) compared to that for 42 patients with slow progression (no AIDS and CD4 cell count of >200 × 10⁶/liter after at least 9 years from infection) or to that for 25 non-HIV-1-infected Swedish blood donors (P < 0.05). No differences were observed in the wild-type CCR5 sequences between the different groups of patients. For three analyzed patients, the 32-bp Δ32CCR5 gene deletions were identical. The antibody titers against gp41 and a V3_MN peptide in patients with the Δ32CCR5/CCR5 genotype were not significantly different from those in pair-matched CCR5/CCR5 controls. However, in 13 analyzed patients, a stronger serum neutralizing activity was associated with the Δ32CCR5/CCR5 genotype. Thus, a CCR5/CCR5 genotype correlates with a shortened AIDS-free HIV-1 infection period and possibly with a worse neutralizing activity, without an evident influence on the antibody response to two major antigenic regions of HIV-1 envelope.     

Analogues of peptide 9–21 of glycoprotein D of herpes simplex virus and their binding to group VII monoclonal antibodies

Summary Several analogues of the amino acid sequence of peptide 9–21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were synthesized and investigated for reactivity with different group VII monoclonal antibodies, Mabs LP14, ID3, 170, HD4, A16, E_II-24 and E_V-10, in a competition enzyme-linked immunosorbent assay (ELISA). Replacement of Arg at position 16 by His resulted in a loss of binding with the group VII Mabs. Substitution of Pro at residue 14 by Leu gave a reduced binding for a number of Mabs and loss of binding for Mab A16. Substitution of Lys at position 10 by Glu gave reduced binding for three out of the seven Mabs. In addition substitutions of Met at position 11 by norleucine and oxidized Met were studied. The boundaries of the epitope cluster were mapped by studying synthetic variants of peptide 9–21 which were shorter either at the C-terminus or at the N-terminus, or both. Peptide 10–18 and peptide 9–17 were the shortest peptides, which were still reactive with the group VII Mabs. Mab HD4 requires the N-terminus of peptide 9–21 for effective binding, while for binding of other Mabs contribution of the residues in the C-terminal part of this peptide is more important.     

Effect of prolonged physical exercise on the fibrinolytic system

Summary The effect of a test marathon race on plasma fibrinolytic activity (FA) was studied in 16 endurance athletes before, immediately after, 3 h, and 31 h after the run. Tissue plasminogen activator (t-PA) activity increased about 31-fold immediately after the run. Similar increases were found in t-PA antigen concentration. Plasminogen activator inhibitor (PAI) was not detectable immediately after the race and was significantly decreased 3 h (P < 0.05) and 31 h (P < 0.01) later. B_15–42 peptide increased by 0.63 pmol · ml^–1 (P<0.001),d-dimer by 68.3 ng · ml^–1 (P< 0.05). Euglobulin lysis time (ELT) was reduced from 109 to 18 min (P<0.001). The increased t-PA activity and t-PA antigen concentration disappeared in the course of the first 3 h after exertion. ELT also reached its pre-exercise levels at this time. Thirty-one hours after the race ELT and t-PA antigen levels were slightly but significantly reduced (P<0.05), whereas B_15–42 peptide remained increased (P<0.05). t-PA activity was unchanged compared with pre-exercise values. It seems that the exercise-induced FA is mainly caused by the marked increase of t-PA antigen and t-PA activity.     

Angiotensin-(1–7) stimulates water transport in rat inner medullary collecting duct: evidence for involvement of vasopressin V2 receptors

The peptide angiotensin-(1–7) [Ang-(1–7)] is known to enhance water transport in rat inner medullary collecting duct (IMCD). The aim of this study was to determine the mechanism of the Ang-(1–7) effect on osmotic water permeability (P _f). P _f was measured in the normal rat IMCD perfused in vitro in presence of agonists [Ang-(1–7), arginine vasopressin (AVP) and Ang-(3–8)], and antagonists of the angiotensin and the vasopressin cascade. Ang-(1–7), but not Ang-(3–8), increased P _f significantly. The effect of Ang-(1–7) on P _f was abolished by its selective antagonist, A-779, added before or after Ang-(1–7). Prostaglandin E₂ and the protein kinase A inhibitor H8 also blocked the Ang-(1–7) effect. Blockade of vasopressin V₁ receptors by antagonists did not change the Ang-(1–7) effect, but pre-treatment with a V₂ antagonist abolished the effect of Ang-(1–7) on P _f. Similarly, pre-treatment with A-779 inhibited AVPs effect on P _f. Forskolin-stimulated P _f was blocked both by A-779 and by the V₂ antagonist. Finally, Ang-(1–7) increased cAMP levels in fresh IMCD cell suspensions whilst the forskolin-stimulated cAMP synthesis was decreased by A-779 and the V₂ antagonist. These data provide evidence that Ang-(1–7) interacts via its receptor with the AVP V₂ system through a mechanism involving adenylate-cyclase activation.     

Tumoral synthetic parathyroid hormone related peptide inhibits amiloride-sensitive sodium transport in cultured renal epithelia

The amino-terminal fragment of a tumoral parathyroid hormone-related peptide (PTHrP (1–34)) produced by a human squamous cell carcinoma of the lung was recently synthetized. In the present work its effect on the amiloride-sensitive sodium transport, taken as an estimate of the Na⁺/H⁺ exchanger activity of cultured opossum kidney (OK) epithelia was compared to that of synthetic bovine parathyroid hormone (bPTH (1–34)). Both PTHrP (1–34) and bPTH (1–34) inhibited the initial rate of amiloride-sensitive²²Na transport. Half maximal inhibitory activity was obtained at about 10^–11 M for both PTHrP (1–34) and bPTH (1–34). In conclusion, tumoral PTHrP (1–34) appears to be as effective as bPTH (1–34) for inhibiting the amiloride-sensitive Na transport, and presumably for decreasing the activity of the Na⁺/H⁺ exchanger present in the apical membrane of kidney epithelial cells.     

Comparison of NucliSens and Amplicor Monitor Assays for Quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Plasma of Persons with HIV-1 Subtype A Infection in Abidjan, Côte d’Ivoire

We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, Côte d’Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (n = 67 [96%]; mean RNA levels, 5.2 log₁₀ HIV-1 RNA copies/ml) than the NucliSens assay (n = 56 [80%]; 4.3 log₁₀ HIV-1 RNA copies/ml) or the standard HIV Monitor assay (n = 44 [63%]; mean RNA levels, 3.8 log₁₀ HIV-1 RNA copies/ml) (all P values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (r = 0.76; P < 0.001) and the standard HIV Monitor assay (r = 0.57; P < 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (r = 0.60; P < 0.001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (r = −0.47 for the NucliSens assay, −0.45 for the standard HIV Monitor assay, and −0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.     

The effect of acetylcholine on chloride transport across the mouse lacrimal gland acinar cell membranes

The mechanisms of Cl^– transport and the effects of acetylcholine (ACh) and electrochemical Cl^– potential changes across the basolateral plasma membrane on intracellular Cl^– activity in the acinar cells of isolated mouse lacrimal glands were studied using double-barreled Cl^–-selective microelectrodes. In the resting state, the basolateral membrane potential (V _m) was about –40 mV and intracellular Cl^– activity was about 35 mmol/l. Addition of ACh (10^–910^–6 mol/l) hyperpolarizedV _m and decreased the Cl^– activity in a dose-dependent manner. ACh (10^–6 mol/l) hyperpolarizedV _m by 20 mV and decreased the cytosolic Cl^– activity with an initial rate of 16.0 mmol/l · min. Reduction of the perfusate Cl^– concentration to 1/9 control depolarizedV _m and decreased cytosolic Cl^– activity at a rate of 1.9 mmol/l · min. AV _m hyperpolarization of 20 mV produced by DC injection to the adjacent cell decreased Cl^– activity at a rate of 4.6 mmol/l · min. DIDS (1 mmol/l) hyperpolarizedV _m by 8 mV with little change in Cl^– activity and increased the input resistance of the cells by 25%. DIDS decreased the rate of change in Cl^– activity induced by low-Cl^– Ringer to 35% of control, but had no effect on the ACh-evoked decrease in the Cl^– activity. Furosemide (1 mmol/l) slightly hyperpolarizedV _m and decreased Cl^– activity at a slow rate but affected Cl^– movements induced by ACh or low-Cl^– Ringer only slightly. Cl^– uptake into the cells was inhibited partially by furosemide. The present results showed that ACh induces an increase in the Cl^– permeability across the luminal plasma membrane and that the basolateral membrane possesses a DIDS-sensitive Cl^– conductance pathway and a furosemide-sensitive Cl^– uptake mechanism.     

Relative reactivity of the V3 loop PND of HIV-1 subtypes A,B, C,D, and F with sera from selected Ugandan localities

Summary Synthetic peptides comprising the predicted principal neutralizing determinant (PND) in new African and North American HIV-1 clones were tested in ELISA for reactivity with ninety six serum samples from asymptomatic donors in six selected localities in Uganda. Irrespective of the geographical origin of the samples, the majority of the test sera cross-reacted at high intensities with the peptides derived from the novel North American clone, BRT3.6 (Group B), the Ugandan clone, CUG045, (Group C), and the Romanian clone, FRMA (Group F). The frequency of reactivity of the peptides from BRT3.6, CUG045, and FRMA were within the ranges of 57–100%, 50–100%, and 57–100%, respectively, for the sera collected from these disparate localities. In contrast to these findings, the V3 peptides derived from the other Ugandan isolates showed a more restricted pattern of reactivity with the same serum samples: AUG06c (1–63%), DUG23c (2%), and DUG044 (38–87%). The results from ELISA inhibition assay indicated that the V3 peptide from BRT 3.6, CUG045, and FRMA express closely related antigenic specificities quite distinct from those in AUG06c and DUG044. The residues comprising the PND in BRT 3.6, CUG045, and FRMA appear to be well conserved in the HIV-1 subtypes prevalent in the selected Ugandan locales.     

Vasoactive intestinal peptide stimulation of renal adenylate cyclase and antagonism by (4Cl-d-Phe6Leu17)VIP

The effect of vasoactive intestinal peptide (VIP) and related peptides [glucagon, secretin, PHI 1-27 (peptide with N-terminal histidine and C-terminal isoleucine)] on renal adenylate cyclase (AC) has been determined in several species. The largest stimulation (4.1±0.5-fold basal) of AC by 1 mol · l^–1 VIP was observed in feline cortical plasma membranes. In rabbit and guinea-pig, VIP increased AC activity 1.5±0.3- and 1.8±0.3-fold respectively but glucagon had no such action. Conversely in the rat glucagon stimulated AC some 3-fold over basal activity whereas VIP had little effect. In dog, cat and mouse both peptides were effective in increasing AC activity. For cat, half-maximal stimulation of cortical plasma membrane AC by VIP was seen at 27.0±9.0 nmol · l^–1 (SEN=9 animals). VIP also increased AC activity in both outer (red) and inner (white) medulla. In feline cortical membranes VIP and PTH (parathyroid hormone) when added in combination were fully additive. However for VIP and glucagon in combination there was no cumulative increase in AC activity, indeed the resultant activity was less than that attained by VIP alone. The VIP analogue (4Cl-d-Phe⁶Leu¹⁷)VIP at 10 mol · l^–1 produced a right shift in the VIP-dose response curve and increased the EC₅₀ from 17.2±5.8 nmol · l^–1 to 132.0±22.2 nmol · l^–1 VIP (SEN=4). There was no reduction in the maximum response elicited by VIP consistent with a competitive type of antagonism by this analogue. PHI-stimulated AC was also reduced by (4Cl-d-Phe⁶-Leu¹⁷)VIP resulting in a similar right shift in the dose response curve. However, this analogue of VIP had no effect on glucagon- or secretin-stimulated AC, indicated by no change in EC₅₀ values.     

Ontogeny of cholecystokinin-8 and glutamic acid decarboxylase in cerebral neocortex of macaque monkey

Summary Concentration of cholecystokinin-8 and the activity of glutamic acid decarboxylase were determined in the various cerebral cortical subdivisions of Japanese monkey (Macaca fuscata fuscata) at three different ages (embryonic 4 months, full-term and adult). The CCK-8 immunoreactive material extracted with 90% methanol from the cerebral cortex of the adult and foetal monkey were shown to be identical with synthetic cholecystokinin-8 by the criterion of co-elution on gel filtration chromatography (Sephadex G-50). The peptide concentration increased dramatically by about 30–80 fold (in terms of protein) and 17–28 fold (in terms of wet weight) between embryonic 4-month-old and full-term monkeys, while the level decreased 1/6–1/16 (protein) and 1/4–1/10 (wet weight) between full-term and adult monkeys. In adults, the highest levels of the peptide was observed in the association cortex, orbital prefrontal cortex and posterior parietal cortex. Glutamic acid decarboxylase activity, on the other hand, gradually increased about 4–10 fold (protein) between embryonic 4-month-old and adult animals and there was little variation in the increase rate among the cerebral subdivisions. In contrast to cholecystokinin-8, no reduction in the enzyme activity occurred between full-term and adult animals. The high level of cholecystokinin-8 in the embryonic period suggests that the peptide may participate in the regulation of the development of primate cerebral cortex.     

Action of the SP2–11 and SP3–11 fragments of substance P on rat peritoneal mast cells

The ability of the SP fragments SP_2–11 and SP_3–11 to release histamine from rat peritoneal mast cells has been compared with that of the whole peptide. SP_1–11 was found to be about 3.4 times more active than SP_2–11 and about 10.4 times more active than SP_3–11. The substance P antagonist [D-Pro⁴, D-Trp^7,9,10] SP_4–11 was equally effective at antagonizing the histamine releasing action of SP_1–11, SP_2–11 and SP_3–11. Benzalkonium chloride was found to be a competitive antagonist of SP and SP_3–11: the dissociation constants for the benzalkonium chloride-receptor interaction being about the same when either SP_1–11 or SP_3–11 was used as the agonist.