ATP-dependent peptide release from mitochondria of reticulocytes

ATP-dependent release of TCA-precipitable peptides from mitochondria-containing stroma (MCS) is described. The process is independent of ubiquitin, but is sensitive to hemin and to heat treatment. Neither chloramphenicol nor EGTA inhibit. 50% of the activity is dependent on charged tRNA. The peptides released from MCS possess a molecular mass of about 1–5 kDa and are degraded to TCA-soluble compounds by a cytosolic protease system (fraction II) without ubiquitin.     

Role of calcium ions in the formation and release of low-molecular-weight substances from optic nerve terminals

Retinal proteins were labeled by intraocular injections of radioactive amino acids. Tissue slices of the superior colliculus (SC) were prepared 18–20 hr later, i.e., when the rapid phases of the axonal transport had reached the SC terminals. The effect of depolarizing pulses of high K and of Ca withdrawal on the secretion of radioactivity was studied in a perfusion system. The effluents were separated into a trichloroacetic acid (TCA) precipitable fraction and a TCA-soluble fraction. High K evoked a release of TCA-soluble radioactivity when [³H]glycine, [³H]leucine, or [³H]proline were used as protein precursors. Small changes occurred for TCA-precipitable fractions. The evoked release of radioactivity was Ca dependent and particularly prominent after labeling with [³H]glycine. Ca withdrawal increased the efflux of exogenous GABA, primary amines, and TCA-precipitable radioactivity but not of TCA-soluble radioactivity when normal media were used. The formation of TCA-soluble radioactivity was measured by incubating combined homogenates of SC and the lateral geniculate body (LGB), containing labeled proteins transported by the slow or rapid phase. The proteolytic activity was highly Ca dependent, for the rapidly transported proteins the half maximum was at 0.1 mM Ca. The formation of TCA-soluble radioactivity was inhibited byp-chloromercuriphenylsulfonic acid (PCMS). Other divalent cations could not substitute for Ca. The rate of formation of TCA-soluble radioactivity and the influence of Ca ions was smaller when proteins of the slow phase were used as substrate.     

Backbone cyclic pheromone biosynthesis activating neuropeptide (PBAN) antagonists: inhibition of melanization in the moth Spodoptera littoralis (Insecta, Lepidoptera)

Antagonistic and agonistic activities of backbone cyclic (BBC) pheromone biosynthesis activating neuropeptide (PBAN) analogues were evaluated in an attempt to identify potent melanotropic antagonists, to gain an insight into their structure-activity relationship (SAR), and to discover molecules with selective and non-selective melanotropic and pheromonotropic properties. Eight potent melanotropic BBC antagonists and seven agonists were disclosed. SAR studies revealed that the structural requirements of the melanotropic and pheromonotropic agonists and antagonists are different. The cyclic structure of the BBC peptides was unimportant for antagonistic activity, and linearization retained their melanotropic and pheromonotropic antagonistic properties. Comparison of the antagonistic activities of the BBC and precyclic peptides with respect to both functions revealed eight selective antagonists (six that were selective melanotropic antagonists and two selective pheromonotropic antagonists) and four non-selective (melanotropic and pheromonotropic) antagonists. The selective melanotropic antagonists exhibited both, pure or mixed agonistic/antagonistic activities. The selective pheromonotropic compounds were pure antagonists. All non-selective compounds were pure antagonists. Comparison of the agonistic activities of the BBC peptides with respect to both functions revealed six selective melanotropic agonists and one non-selective agonistic compound. All compounds (whether selective or non-selective) exhibited pure agonistic activity. Discovery of the selective compounds hints at the possibility that the receptors that mediate the respective activities may have different properties.     

αMSH-like peptides in rat brain: Identification and changes in level during development

MSH-like peptides were extracted from rat brains and separated by high pressure liquid chromatography. Using C and N terminally directed antibodies, and a bioassay for melanotropic activity, two major melanotropic peptides were detected. One peptide was identified as αMSH and the other as des-acetyl αMSH, a form which has not been previously reported in brain. Analysis of the level of αMSH-like peptides in brain and pituitary gland during development, showed steady increases of pituitary αMSH from day 18 fetuses to adults, whereas in brain, significant increases were not observed until one day post partum. This difference in the time of onset of αMSH production in the two tissues suggests the presence of biosynthetically independant pools of αMSH-like peptides in pituitary and brain.     

In vitro effect of short-term exposure to two synthetic peptides,alone or in combination with clarithromycin or rifabutin,on Cryptosporidium parvum infectivity

The viability of Cryptosporidium parvum after exposure to peptide antibiotics was studied by two different methods, a cell culture system and a double fluorogenic staining. The peptides KFFKFFKFF and IKFLKFLKFL exerted high cytotoxic effects on sporozoites, as demonstrated by cell cultures (complete inhibition after 60 min at 100 microg/ml) and flow cytometry (30% after 20 min at 100 microg/ml), but did not affect consistently the oocysts. Clarithromycin and rifabutin demonstrated less activity against sporozoites but higher activity against oocysts (30% after 180 min at 10 microg/ml). The combination between peptides and azithromycin or rifabutin exerted the highest activities.     

Study of frog (Rana esculenta) proopiomelanocortin processing in the intermediate pituitary. Identification of alpha-melanotropin, beta-melanotropin, Lys-gamma-melanotropin, and corticotropin-like intermediate lobe peptide.

The proteolytic processing of frog (Rana esculenta) proopiomelanocortin in melanotropic cells of the intermediate pituitary gland has been examined through purification of the mature fragments by reverse-phase high-pressure liquid chromatography and microsequencing of isolated peptides. alpha-Melanotropin, beta-melanotropin, Lys-gamma-melanotropin, corticotropin-like intermediate lobe peptide, and hinge peptide have been isolated and chemically characterized. The results show a high preservation in the processing sites of frog proopiomelanotropin when compared to bovine counterparts. They reveal also a great conservation of the processing enzyme equipment of melanotropic cells in tetrapods species. Identification of Lys-gamma-melanotropin suggests the occurrence of an endopeptidase able to cleave between two basic residues. On the other hand alpha-melanotropin does not appear to be N-acetylated, as previously found in the clawed-toad Xenopus laevis, and this feature might distinguish amphibian from mammalian proopiomelanocortin processing.     

Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

Isolation,Purification and Characterization of Antimicrobial Peptides Produced from <Emphasis Type="Italic">Saccharomyces boulardii</Emphasis>

Saccharomyces boulardii was used for antimicrobial peptides production. Separation process of produced antimicrobial peptides was conducted using ultrafiltration technique through dialysis membranes with porous 10 (MWCO) kDa. The inhibition activity was determined against four bacterial isolates. As a result, higher inhibition zone against Bacillus cereus were 26, 29 and 33 mm after adding 50, 75 and 100 µL of concentrated peptide, respectively. After that, peptide passed through the Sephadex G-50 column to achieve purified peptide using gel filtration. The high activity of purified peptide was confirmed based on the second peak reaching to 37 mm of bacterial inhibition zone while other peaks did not show any inhibition against tested bacteria. Some of the important characteristics of purified bioactive peptide were applied. Antimicrobial peptides stability was studied and found to be stable at pH range from 5 to 7 values studied in addition to its inhibition activity reached to 100%. Regarding thermal stability, it was observed that the peptide was fully activity at a both 60–80 °C for 30 min. Moreover, molecular weight of a peptide was identified using electrophoresis technique with SDS measured at 5792 Dalton.     

Absence of coupling between release and biosynthesis of peptide hormones in insect neuroendocrine cells

Adipokinetic hormone (AKH)-producing cells in the corpus cardiacum of the insect Locusta migratoria represent a neuroendocrine system containing large quantities of stored secretory peptides. In the present study we address the question whether the release of AKHs from these cells induces a concomitant enhancement of their biosynthesis. The effects of hormone release in vivo (by flight activity) and in vitro (using crustacean cardioactive peptide, locustamyoinhibiting peptide, and activation of protein kinase A and C) on the biosynthetic activity for AKHs were measured. The intracellular levels of prepro-AKH mRNAs, the intracellular levels of pro-AKHs, and the rate of synthesis of (pro-)AKHs were used as parameters for biosynthetic activity. The effectiveness of in vitro treatment was assessed from the amounts of AKHs released. Neither flight activity as the natural stimulus for AKH release, nor in vitro treatment with the regulatory peptides or signal transduction activators appeared to affect the biosynthetic activity for AKHs. This points to an absence of coupling between release and biosynthesis of AKHs. The strategy of the AKH-producing cells to cope with variations in secretory stimulation seems to rely on a pool of secretory material that is readily releasable and continuously replenished by a process of steady biosynthesis.     

Peptide boronic acids, substrate analogs, inhibit chymase, and histamine release from rat mast cells

Peptide boronic acids, such as methoxysuccinyl-Ala-Ala-Pro-(L)boro-Phe-OH, its pinacol ester, and t-butyloxycarbonyl-Phe-Pro-(L)boro-Phe-pinacol, inhibited the activity of chymase from connective tissue mast cells approximately 40- to 80-fold more than atypical chymase from mucosal mast cells, and did not inhibit trypsin. Only peptide boronic acids containing "L" forms of boronic acids were inhibitory. The Ki values of these peptide boronic acids for chymase were in the 60-170 nM concentration range, like those of the natural inhibitors tested, but all the natural inhibitors tested except Eglin C and chymostatin inhibited both chymase and trypsin. Thus these peptide boronic acids should be useful for selective inhibition of chymase with less inhibitory activity for atypical chymase and without inhibition of trypsin. These peptide boronic acids markedly inhibited histamine release induced by anti-rat immunoglobulin E, suggesting that chymase in connective tissue mast cells plays some role in the process of histamine release. These peptides are assumed to be therapeutically useful for treatment of allergic inflammations catalyzed by chymase.     

The Melanotropic Peptide, [Nle4, d-Phe7]α-MSH,Stimulates Human Melanoma Tyrosinase Activity and Inhibits Cell Proliferation

Seventeen human melanoma cell (HMC) lines, both melanotic and amelanotic, were incubated in the continuous presence of a potent melanotropic peptide hormone analog, [Nle⁴,d -Phe⁷]α-MSH, for 72 hr with daily changes of medium. Only one cell line (HD, melanotic) consistently responded to the hormone analog by increased tyrosinase activity. Three (one melanotic, two amelanotic) of the HMC lines also failed to respond to the peptide by either increased or decreased enzyme activity when incubated continuously in the presence of the peptide for longer periods of time (6,15,27,43 days). The HD cell line, however, again responded with increasingly enhanced basal enzyme activity the longer the cells were incubated in the presence of the melanotropin. One amelanotic cell line (C8161) responded with enhanced enzyme activity when grown to confluency in the continuous presence of the peptide. Basal tyrosinase activity of the C8161 cell line may have increased as cell density in the flasks increased. These results suggest that under conditions of increased cell number, phenotypic expression of tyrosinase activity in so called “amelanotic” (tyrosinase-negative) cells is increased and can be enhanced further by stimulation with a melanotropic peptide. Under conditions of increased cell number, the presence of [Nle⁴,d -Phe⁷]α-MSH caused morphological differentiation (shape change); the cells became enlarged and very dendritic. The number of cells in monolayer (surface of the flask) and in the medium were drastically reduced in both melanotic and “amelanotic” cell lines incubated with [Nle⁴,d -Phe⁷]α-MSH. The data support other published reports that melanotropic peptides inhibit human melanoma cell growth (proliferation) in vitro, most likely through a cytostatic mechanism. [Nle⁴,d -Phe⁷]α-MSH also exhibited a prolonged (residual) inhibitory action on HD cell proliferation. In other words, inhibition of cell growth (proliferation) of the HMCs was evident even several days after removal of the melanotropic peptide from the incubation medium.     

PBAN selective antagonists: inhibition of PBAN induced cuticular melanization and sex pheromone biosynthesis in moths

A D-Phe scan (sequential D-Phe replacement) library of linear peptides, synthesized on the basis of a slightly modified active sequence of PBAN (YFSPRL-amide) was employed to detect potential inhibitors of cuticular melanization in Spodoptera littoralis larvae and to compare their stimulatory and inhibitory melanization activity with their pheromonotropic agonistic and antagonistic activities. A quantitative melanotropic assay was used to monitor the extent of cuticular melanization elicited by Hez-PBAN1-33NH2 in S. littoralis larvae in the presence and absence of the D-Phe peptides. The data revealed the presence of two partial melanotropic antagonists, and disclosed the presence of selective pure melanotropic agonists and pure pheromonotropic antagonists indicating differences in the inhibitory and stimulatory patterns of the library with respect to both activities. The differences between the pheromonotropic and melanotropic inhibitory patterns of the peptides hints at the possibility that sex pheromone biosynthesis in the pheromone gland of Heliothis peltigera females and induction of cuticular melanization in S. littoralis may be mediated by different receptors (that may result either from presence of different receptor sub-types or may reflect species differences in receptor structure and/or properties) despite the fact that they are induced by the same peptide (PBAN1-33NH2).     

Differential accumulation of catecholamines, proenkephalin- and chromogranin A-derived peptides in the medium after chronic nicotine stimulation of cultured bovine adrenal chromaffin cells

We have used an antiserum to a synthetic peptide fragment of bovine chromogranin A (ChrgA)[Tyr0] bovine ChrgA (306-313): YLSKEWEDA, together with antibodies to proenkephalin-derived peptides, to measure the release of immunoreactive peptides from nicotine-stimulated cultured bovine adrenal chromaffin cells. Over a period of 6 hr the accumulation of YLSKEWEDA immunoreactivity and Met-enkephalin Arg6Gly7Leu8 (MERGL) immunoreactivity in the medium of 10 microM nicotine-stimulated cells was shown to be biphasic; the initial phase occurred in the first 15-30 min and the second phase reached a peak after 4 hr. In contrast, catecholamine release occurred monophasically over the initial 15-30 min. Investigation of the second phase of peptide accumulation revealed that it was due in part to nicotine-evoked exocytosis and in part to extracellular processing of high molecular weight precursor proteins.     

Valproate and copper accelerate TRH-like peptide synthesis in male rat pancreas and reproductive tissues

Treatment with valproate (Valp) facilitates the synthesis of TRH-like peptides (pGlu-X-Pro-NH(2)) in rat brain where "X" can be any amino acid residue. Because high levels of TRH-like peptides occur in the pancreas and pGlu-Glu-Pro-NH(2) (Glu-TRH) has been shown to be a fertilization promoting peptide, we hypothesized that these peptides mediate some of the metabolic and reproductive side effects of Valp. Male WKY rats were treated with Valp acutely (AC), chronically (CHR) or chronically followed by a 2 day withdrawal (WD). AC, CHR and WD treatments significantly altered TRH and/or TRH-like peptide levels in pancreas and reproductive tissues. Glu-TRH was the predominant TRH-like peptide in epididymis, consistent with its fertilization promoting activity. Glu-TRH levels in the epididymis increased 3-fold with AC Valp. Phe-TRH, the most abundant TRH-like peptide in the pancreas, increased 4-fold with AC Valp. Phe-TRH inhibits both basal and TRH-stimulated insulin release. Large dense core vesicles (LDCV's) contain a copper-dependent enzyme responsible for the post-translational processing of precursors of TRH and TRH-like peptides. Copper (500 microM) increased the in vitro C-terminal amidation of TRH-like peptides by 8- and 4-fold during 24 degrees C incubation of homogenates of pancreas and testis, respectively. Valp (7 microM) accelerated 3-fold the processing of TRH and TRH-like peptide precursors in pancreatic LDCV's incubated at 24 degrees C. We conclude that copper, an essential cofactor for TRH and TRH-like peptide biosynthesis that is chelated by Valp, mediates some of the metabolic and reproductive effects of Valp treatment via acceleration of intravesicular synthesis and altered release of these peptides.     

Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle,Tenebrio molitor (Coleoptera,Tenebrionidae)

The genome of Tribolium castaneum encodes two allatostatin [AS type B; W(X)₆Wamide and AS type C; PISCF‐OH] and one allatotropin (AT) precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing Consortium, 2008: Nature 452:949–955). Here we studied the activity (in vitro) of peptides derived from these precursors on the synthesis/release of juvenile hormone (JH) III. The corpora cardiaca‐corpora allata (CC‐CA) complexes of adult females of another tenebrionid beetle, the mealworm Tenebrio molitor, were used. Incubating the gland complexes in a medium containing Trica‐AS B3 peptide, we showed that the peptide has allatostatic function in T. molitor. The activity of the type C AS depended on the age of the test animals and their intrinsic rate of JH III biosynthesis. The Trica‐AS C peptide inhibited the JH release from CA of 3‐day‐old females with a high intrinsic rate of JH synthesis, but activated JH release from the CA of 7‐day‐old females with a lower intrinsic rate of JH production. The allatotropin peptide (Trica‐AT) also activated the JH release from the CA of 7‐day‐old females in a dose‐dependent and reversible manner. Unexpectedly, a type A AS derived from the precursor of the American cockroach Periplaneta americana (Peram‐AS A2b) inhibited the JH release from the CA of younger and older females in the concentration range of 10^?8 to 10^?4 M, and the effects were fully reversible in the absence of peptide. These data suggest a complex role of allatoactive neuropeptides in the regulation of JH III biosynthesis in beetles. © 2010 Wiley Periodicals, Inc.     

Cardiac nonmyofibrillar proteins in copper-deficient rats

Cardiac nonmyofibrillar proteins from copper-deficient rats appear to have diminished quantity of selected peptides. Identification of some of these peptides was the objective of the present study. Male weanling Long-Evans rats were fed either copper-adequate (n=6) or copper-deficient (n=6) diets for 5 wk. At the end of 5 wk, the rat hearts were removed, quick frozen in liquid nitrogen, and non-myofibrillar proteins separated using sodium-dodecyl-sulfate poly-acrylamide gel electrophoresis (SDS-PAGE). A peptide in the 16-kDa mol-wt region was diminished in copper-deficient rats. Blotting of gels to an Immobilon-P membrane and subsequent sequencing of the amino acids identified the peptide as the δ subunit of mitochondrial ATP synthase. Blotting of gels to nitrocellulose followed by Western blot assay for cytochrome C oxidase using antibodies against the enzyme complex revealed decreased protein content in the copper-deficient rat for this enzyme, primarily the nuclear encoded subunits.     

Stress, neuropeptides, and feeding behavior: a comparative perspective

Stress inhibits feeding behavior in all vertebrates. Data frommammals suggest an important role for hypothalamic neuropeptides,in particular the melanocortins and corticotropin-releasinghormone (CRH)-like peptides, in mediating stress-induced inhibitionof feeding. The effects of CRH on food intake are evolutionarilyancient, as this peptide inhibits feeding in fishes, birds,and mammals. The effects of melanocortins on food intake havenot been as extensively studied, but available evidence suggeststhat the anorexic role of neuronal melanocortins has been conserved.Although there is evidence that CRH and the melanocortins influencehypothalamic circuitry controlling food intake, these peptidesmay have a more primitive role in modulating visuomotor pathwaysinvolved in the recognition and acquisition of food. Stressrapidly reduces visually guided prey-catching behavior in toads,an effect that can be mimicked by administration of CRH, whilecorticosterone and isoproterenol are without effect. Melanocortinsalso reduce prey-oriented turning movements and, in addition,facilitate the acquisition of habituation to a moving prey item.The effects of these neuropeptides are rapid, occurring within30 min after administration. Thus, changes in neuroendocrinestatus during stress may dramatically influence the efficacywith which visual stimuli release feeding behavior. By modulatingvisuomotor processing these neuropeptides may help animals makeappropriate behavioral decisions during stress.     

An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect

A linear pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) antagonist lead (RYF[dF]PRLa) was structurally modified to impart amphiphilic properties to enhance its ability to transmigrate the hydrophobic cuticle of noctuid moth species and yet retain aqueous solubility in the hemolymph to reach target PK/PBAN receptors within the internal insect environment. The resulting novel PK/PBAN analog, Hex-Suc-A[dF]PRLa (PPK-AA), was synthesized and evaluated as an antagonist in a pheromonotropic assay in Heliothis peltigera against 4 natural PK/PBAN peptide elicitors (PBAN; pheromonotropin, PT; myotropin, MT; leucopyrokinin, LPK) and in a melanotropic assay in Spodoptera littoralis against 3 natural PK/PBAN peptide elicitors (PBAN, PT, LPK). The analog proved to be a potent and efficacious inhibitor of sex pheromone biosynthesis elicited by PBAN (84% at 100 pmol) and PT (54% at 100 pmol), but not by MT and LPK. PPK-AA is a selective pure antagonist (i.e., does not exhibit any agonistic activity) as it failed to inhibit melanization elicited by any of the natural PK/PBAN peptides. The analog was shown to transmigrate isolated cuticle dissected from adult female Heliothis virescens moths to a high extent of 25-30% (130-150 pmol), representing physiologically significant quantities. PPK-AA represents a significant addition to the arsenal of tools available to arthropod endocrinologists studying the endogenous mechanisms of PK/PBAN regulated processes, and a prototype for the development of environmentally friendly pest management agents capable of disrupting the critical process of reproduction.     

Peptide transmitters of primary sensory neurons: similar actions of tachykinins and bombesin-like peptides

Previous studies have demonstrated that two peptides, substance P (SP) and substance K (SK), are contained in a common prohormone--beta-preprotachykinin. Both peptides are cleaved from the prohormone and appear to coexist throughout the brain. This study evaluated the behavioral activity of SK and compared it to the activities of SP, bombesin (BN), and structurally related peptides. After intraspinal injection, all of the peptides induced "bite/scratch" behaviors, which differed in durations of action. The specific rank order of these durations of action were: BN greater than gastrin releasing peptide (GRP) = ranatensin (RT) greater than neuromedin B (NMB) greater than kassinin (KASS) = SK = SP and ranged from dose-dependent maxima of approximately 2 min (SP) to approximately 100 min (BN). To examine the possibility that differences in durations of action are due to differences in rates of proteolytic degradation, each peptide was incubated in spinal cord homogenates at 37 degrees C, and the degradation rates were monitored by radioimmunoassay (RIA) and by bioassay. The lengths of incubation time required to produce approximately 90% degradation of peptide immunoreactivity varied across peptides from less than 5 min (SP) to more than 60 min (BN and RT). Degradation of bioactivity generally paralleled degradation of immunoreactivity. The results of this study suggest that durations of nociceptive effects produced by the peptides tested are due, in part, to their resistance to proteolytic degradation.