Peptide S is a novel potent inhibitor of voluntary and fast-induced food intake in rats

Peptide S (NPS or PEPS) and its cognate receptor have been recently identified both in the central nervous system and in the periphery. NPS/PEPS promotes arousal and has potent anxiolytic-like effects when it is injected centrally in mice. In the present experiment, we tested by different approaches its central effects on feeding behaviour in Long-Evans rats. PEPS at doses of 1 and 10 microg injected in the lateral brain ventricle strongly inhibited by more than 50% chow intake in overnight fasted rats with effects of longer duration with the highest dose (P<0.0001). A similar decrease was observed for the spontaneous intake of a high-energy palatable diet (-48%; P<0.0001). This anorexigenic effect was comparable to that induced by corticotropin-releasing hormone in fasted rats at equimolar doses. However, peptide S did not modify food intake stimulated by neuropeptide Y (NPY) at equimolar doses. It also did not affect the fasting concentrations of important modulators of food intake like leptin, ghrelin, and insulin in circulation. This study therefore showed that peptide S is a new potent anorexigenic agent when centrally injected. Its inhibitory action appears to be independent of the NPY, ghrelin, and leptin pathways. Development of peptide S agonists could constitute a new approach for the treatment of obesity.     

Neurokinin B in a human pheochromocytoma measured with a specific radioimmunoassay

An N-terminally directed antiserum to neurokinin B was raised in rabbits using an immunogen prepared by coupling the free-SH group of neurokinin B extended from its C-terminus by a cysteine residue (NKB-Cys) to an -NH2 group on human serum albumin using a heterobifunctional cross-linking reagent. In radioimmunoassay with 125I-Bolton-Hunter-labelled NKB-Cys as tracer, the antiserum showed no cross-reactivity with other tachykinins. An extract of a human pheochromocytoma, previously shown to contain peptides derived from preprotachykinin A, contained NKB-LI (13 pmol/g wet weight). The retention time of tumor neurokinin on reversed-phase HPLC was the same as that of synthetic neurokinin B. Peptides with the retention times of substance P, neurokinin A, neurokinin A (3-10)-peptide and neuropeptide K were also identified in the tumor extract. NKB-LI was not detected in extracts of a further nine pheochromocytomas or in five carcinoid tumors that expressed the preprotachykinin A gene.     

Succinylated lipid A is a potent and specific inhibitor of endotoxin mitogenicity.

Chemically modified lipopolysaccharides of Salmonella abortus-equi were tested for mitogenicity on mouse spleen cells as well as antagonism of the mitogenicity of intact lipopolysaccharide (LPS). All the lipopolysaccharide preparations deacylated by different alkaline treatments suffered a drastic loss of mitogenicity. The mitogenic activity of lipid A was also lost when succinic residues were introduced on hydroxyl groups. Partially deacylated alkaline-treated preparations (but not completely deacylated preparations) inhibited the activation of splenic B-cells by LPS. They were found to be toxic to spleen cells, however, and to suppress not only the mitogenicity of LPS but that of concanavalin A as well. This inhibitory action was not exhibited when all of the fatty acid was eliminated. Succinylated lipid A, on the other hand, was not toxic to the cells and inhibited the B-cell mitogenicity of lipopolysaccharide (but not the T-cell mitogenicity of concanavalin A). Chemical analysis revealed that about 4.6 mol of succinic acid had been introduced into lipid A by succinylation, and that the fatty acid and phosphate composition was unchanged by this treatment. Macrophages do not seem to participate in this inhibition. Inhibition was observed when succinylated lipid A was added either at the same time or after lipid A mitogen, but optimal inhibition was expressed when it was added to the culture 3 h before LPS. Inhibition was not affected by washing the cells before adding LPS. Inhibition increased as the ratio of suppressor to mitogen increased, suggesting that the succinylated lipid A competes with intact LPS.     

Chelerythrine is a potent and specific inhibitor of protein kinase C

The benzophenanthridine alkaloid chelerythrine is a potent, selective antagonist of the Ca++/phospholopid-dependent protein kinase (Protein kinase C: PKC) from the rat brain. Half-maximal inhibition of the kinase occurs at 0.66 microM. Chelerythrine interacted with the catalytic domain of PKC, was a competitive inhibitor with respect to the phosphate acceptor (histone IIIS) (Ki = 0.7 microM) and a non-competitive inhibitor with respect to ATP. This effect was further evidenced by the fact that chelerythrine inhibited native PKC and its catalytic fragment identically and did not affect [3H]- phorbol 12,13 dibutyrate binding to PKC. Chelerythrine selectively inhibited PKC compared to tyrosine protein kinase, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. The potent antitumoral activity of celerythrine measured in vitro might be due at least in part to inhibition of PKC and thus suggests that PKC may be a model for rational design of antitumor drugs.     

Cycloheximide is not a specific inhibitor of protein synthesis in vivo

Cycloheximide is frequently presumed to inhibit specifically the cytoplasmic protein synthesis of eukaryotes. Although previous investigators have shown that it had other effects on the cells of a variety of organisms, these results were frequently presumed to be secondary effects of the inhibition of protein synthesis. This paper shows that a wide range of deleterious effects are produced by cycloheximide on a single organism, Chlamydomonas reinhardi Dangeard. If, protein synthesis is inhibited by nonpermissive conditions in temperature-sensitive mutants or with other treatments these “secondary” effects are not produced. Instead, cycloheximide appears to have two or three independent inhibitory effects on the cell. Moreover, in contrast to a number of previous investigations, these results show that protein synthesis is not required for RNA synthesis. Instead the rate of RNA synthesis is actually increased by interference with protein synthesis.     

Fluoride is a strong and specific inhibitor of (asymmetrical) Ap4A hydrolases

Fluoride acts as a noncompetitive, strong inhibitor of (asymmetrical) Ap4A hydrolases (EC 3.6.1.17). The Ki values estimated for the enzymes isolated from seeds of some higher plants (yellow lupin, sunflower and marrow) are in the range of 2-3 microM and I50 for the hydrolase from a mammalian tissue (beef liver) is 20 microM. The anion, up to 25 mM, does not affect the following other enzymes which are able to degrade the bis(5'-nucleosidyl)-oligophosphates: Escherichia coli (symmetrical) Ap4A hydrolase (EC 3.6.1.41), yeast Ap4A phosphorylase (EC 2.7.7.53), yellow lupin Ap3A hydrolase (EC 3.6.1.29) and phosphodiesterase (EC 3.1.4.1). None of halogenic anions but fluoride affects the activity of (asymmetrical) Ap4A hydrolases. Usefulness of the fluoride effect for the in vivo studies on the Ap4A metabolism is shortly discussed.     

NADPH is a specific inhibitor of protein import into glyoxysomes

We have studied the import of proteins into glyoxysomes in vitro and show that this process is specifically inhibited by NADPH. NADPH affects both binding and translocation of proteins into glyoxysomes, and inhibition is determined by the ratio of NADP⁺ to NADPH. The site of action of NADPH is most likely within the glyoxysome because (1) pretreatment of glyoxysomes with NADPH, followed by re-isolation of the organelles prior to the import assay, resulted in inhibition of import that could be restored by the addition of NADP⁺; (2) low concentrations of NADPH inhibited binding of proteins to broken glyoxysome membranes. The sensitivity of protein import to inhibition by NADPH declines as glyoxysomes are converted to leaf-type peroxisomes. A model is proposed that speculates on a possible role for NADPH in regulating protein import into plant peroxisomes.     

Ontogeny of hypertonic preabsorptive inhibitory control of intake in neonatal rats

The ontogenetic development of postingestive inhibitory control of ingestion by the osmotic load of a preload was examined in rats. On postnatal days 6 (P6) and 12 (P12), pups were deprived for either 6 or 24 h. Gastric preloads (5% body wt) of water, mannitol (a sugar alcohol that is not absorbed) in six concentrations [from 0.125 M (hypotonic) to 1.0 M (hypertonic)], or sham preloads were administered 5 min before a 30-min intake test. Compared with sham treatment, isotonic mannitol (0.25 M), a probe of volumetric control, significantly reduced intake on P12, but not on P6. Compared with isotonic mannitol, the three highest hypertonic concentrations (0.5, 0.66, and 1.0 M) significantly decreased intake on P12, at both levels of deprivation. On P6, 0.66 and 1.0 M mannitol reduced intake after 24 h, but not after 6 h, of deprivation. Thus, on P6, the hypertonic control was detectable only after prolonged deprivation and the volumetric control was not present. On P12, both controls were observed and the hypertonic control was more potent than on P6.     

Evidence that a collagen-derived nonapeptide is a specific inhibitor of platelet-collagen interaction

The aggregation of platelets by type III collagen is inhibited by the collagen peptide α₁ (III)CB4 and by 3 fragments derived from this peptide. These fragments share a common portion of 9 aminoacids with the sequence [Gly-Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys]. This peptide has been synthetized and its ability to prevent the aggregation of platelets has been studied using various inducers. A marked inhibition of the aggregation by type III collagen was observed, and this response was dose related. The effect was much less pronounced when type I collagen was used. Aggregations by ADP, epinephrin, arachidonic acid, ionophore A 23187 were not altered. The inhibition seems then to be specific for collagen and can be associated to the blocking of platelet membrane sites by this nonapeptide.     

N,N'-dicyclohexylcarbodiimide is a specific, reversible inhibitor of proline-beta-naphthylamidase

N,N'-Dicyclohexylcarbodiimide (DCCD) was found to inhibit the activity of proline-beta-naphthylamidase purified from porcine intestinal mucosa. The inhibition is rapid and reversible, and it is not due to the dissociation of the enzyme subunits. The mode of the inhibition by DCCD is noncompetitive with respect to each of the two substrates tested. Ki values of DCCD for the enzyme were determined to be 1.9 microM with proline-beta-naphthylamide and 12 microM for L-leucine ethyl ester. To our knowledge, this is the first time that DCCD was found to be a potent, reversible inhibitor for an enzyme.     

Enhanced food and water intake in renin transgenic rats.

In short term experiments angiotensin II (Ang II) is a potent stimulant of thirst, however it is not known whether prolonged activation of the renin-angiotensin system is associated with chronic alteration of water or food intake. Renin transgenic rats TGRmRen(2)27 (TGR) exhibit significant elevation of AngII in the brain regions involved in regulation of body fluid balance. The purpose of the present study was to find out whether TGR rats manifest also different water (WI) and food (FI) intake and renal excretory functions in comparison to their parent Sprague Dawley (SD) strain. To this end 24 h WI and FI as well as urine excretion (Vu) and urinary outputs of solutes (Cosm), sodium (UNaV) and potassium (UKV) were compared under baseline conditions in 16 TGR and 15 SD rats having free access to water and food. In 15 TGR and 17 SD rats effect of 24 h dehydration on water intake was investigated. Under baseline conditions TGR rats consumed significantly greater amount of food and water than SD rats. Vu, UNaV and UKV were not significantly different in both strains. Cumulative water intakes in SD and TGR rats subjected to 24 h dehydration did not differ. The results reveal that under baseline conditions TGR rats manifest greater food and water intakes than SD rats whereas stimulation of thirst by water deprivation is similar in both strains. The results suggest that the ingestive behavior may be chronically altered by upregulation of the renin-angiotensin system.     

Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.     

Anthraflavic acid is a potent and specific inhibitor of cytochrome P-448 activity

Consideration of the computer-optimised dimensions of anthraflavic acid indicates that it is essentially a planar molecule with a large area/depth ratio, that would preferentially interact with the polycyclic aromatic hydrocarbon-induced family of cytochrome P-450 proteins (cytochromes P-448). Anthraflavic acid was a potent inhibitor of the O-deethylations of ethoxycoumarin and ethoxyresorufin, both catalysed primarily by cytochromes P-448, in Arochlor-1254-induced hepatic microsomes. Similarly anthraflavic acid markedly inhibited the mutagenicity of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-I) in the Ames test. In contrast, it has no effect on the dealkylation of pentoxyresorufin, a reaction catalysed primarily by the phenobarbital-induced cytochromes P-450, and NADPH-dependent reduction of cytochrome c. It is concluded that anthraflavic acid is a potent and specific inhibitor of cytochrome P-448 activity.     

Azidocytidine is a specific inhibitor of deoxyribonucleotide synthesis in 3T6 cells

2'-Azidocytidine is a specific inhibitor of DNA synthesis in cultured 3T6 mouse fibroblasts. Earlier work (Akerblom, L., Pontis, E., and Reichard, P. (1982) 257, 6776-6782) indicated that the nucleoside, after phosphorylation, acted by inhibiting both ribonucleotide reduction and DNA strand elongation. We now demonstrate that the effect on strand elongation was due to a contamination of azidocytidine with less than 0.3% of arabinosyl cytosine. Pure azidocytidine inhibits specifically ribonucleotide reductase and its effects on DNA synthesis are secondary to this inhibition. The results with azidocytidine concerning the size and turnover of deoxyribonucleoside triphosphate pools parallel those of hydroxyurea and are distinct from those of arabinosyl cytosine. Together with hydroxyurea, azidocytidine is a useful compound in studies aiming at a specific block of the production of deoxynucleoside triphosphates in intact cells. Comparisons of the effects of azidocytidine and arabinosyl cytosine complement earlier studies with hydroxyurea and aphidicolin concerning inter-relations between dNTP synthesis and DNA replication.     

Trypsin inhibitor and maternal reunion increase plasma cholecystokinin in neonatal rats.

Intragastric soybean trypsin inhibitor increased plasma CCK bioactivity by 87% in nondeprived, 9-12-day-old rat pups. Reunion with the dam for 1 h after overnight maternal deprivation also increased plasma CCK significantly. These results demonstrate that CCK can be released from the small intestine of rats as early as postnatal day 9.     

YacG from Escherichia coli is a specific endogenous inhibitor of DNA gyrase

We assign a function for a small protein, YacG encoded by Escherichia coli genome. The NMR structure of YacG shows the presence of an unusual zinc-finger motif. YacG was predicted to be a part of DNA gyrase interactome based on protein-protein interaction network. We demonstrate that YacG inhibits all the catalytic activities of DNA gyrase by preventing its DNA binding. Topoisomerase I and IV activities remain unaltered in the presence of YacG and its action appears to be restricted only to DNA gyrase. The inhibition of the enzyme activity is due to the binding of YacG to carboxyl terminal domain of GyrB. Overexpression of YacG results in growth inhibition and alteration in DNA topology due to uncontrolled inhibition of gyrase.     

A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase

The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme. I33 inhibited both in vitro IN activities, i.e. 3' end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33. Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an alpha-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN.