Potent antiobesity effect of a short-length peptide YY-analogue continuously administered in mice

The gastrointestinal peptide, peptide YY_3–36 (PYY_3–36) and its shorter peptide analogues have been reported to reduce appetite by activating the neuropeptide Y2 receptor (Y2R), which is associated with obesity and other metabolic diseases. A 14-amino acid PYY analogue, Ac-[d-Pro²⁴,Cha^27,28,36,Aib³¹]PYY(23–36) (3), showed high binding affinity and agonist activity for the Y2R, similar to that of PYY_3–36, but had weak anorectic activity upon continuous administration in lean mice. Three amino acid substitutions [Pya(4)²⁶, Aib²⁸, Lys³⁰], which contributed to the decreased hydrophobicity of 3, efficiently increased its anorectic activity. The compound containing these three amino acids, Ac-[d-Pro²⁴,Pya(4)²⁶,Cha^27,36,Aib^28,31,Lys³⁰]PYY(23–36) (22), exerted more potent and durable food intake suppression than that by PYY_3–36 in lean mice, as well as excellent Y2R agonist activity (EC₅₀: 0.20 nM) and good subcutaneous bioavailability (66.6%). The 11-day continuous administration of 22 at 1 mg/kg/day successfully produced antiobese and antidiabetic effects, with more than 20% body weight loss in obese and Type 2 diabetes ob/ob model mice.     

A potent neuromedin U receptor 2-selective alkylated peptide

Neuromedin U (NMU) mediates various physiological functions via NMUR1 and NMUR2 receptors. NMUR2 has been considered a promising treatment option for diabetes and obesity. Although NMU-8, a shorter peptide, has potent agonist activity for both receptors, it is metabolically unstable. Therefore, NMU-8 analogs modified with long-chain alkyl moieties via a linker were synthesized. An octadecanoyl analog (17) with amino acid substitutions [αMePhe¹⁹, Nle²¹, and Arg(Me)²⁴] and a linker [Tra-γGlu-PEG(2)] dramatically increased NMUR2 selectivity, with retention of high agonist activity. Subcutaneous administration of 17 induced anorectic activity in C57BL/6J mice. Owing to its high metabolic stability, 17 would be useful in clarifying the physiological role and therapeutic application of NMU.     

Production and Characterization of a Pressure-induced Gel from Freeze-concentrated Milk

A process was developed to produce a characteristic milk gel. Raw and market milk samples were freeze-concentrated using bacterial ice nuclei. The concentrates were kept at 5°C and compressed at 300–600 MPa for 5 min. The combination of the freeze concentration and the pressurization gave a milk gel without adding any gelling agents. The addition of sugar at 10% to the concentrated milk improved its gel strength and viscoelasticity. The gel was characterized by a phase transition at about 62–75°C.     

Participation of peroxisomal beta-oxidation system in the chain-shortening of a xenobiotic acyl compound

A drug, (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid, was metabolized to 4-(1-imidazolylmethyl)benzoic acid in isolated hepatocytes of rats, which was enhanced markedly by the pretreatment of rats with clofibrate. With liver homogenates, the formation of the CoA-ester of this drug and its subsequent chain-shortening were demonstrated. In the series of these reactions, acyl-CoA synthetase, CoA, ATP and NAD were required, whereas cyanide did not inhibit the reaction. These results indicate that peroxisomes are capable of shortening the acyl side-chains of drugs by the beta-oxidation, giving an additional suggestion on the functions of peroxisomes.     

Isolation of lipid glucuronic acid and N-acetylglucosamine derivatives from a rat fibrosarcoma

Incubation of labeled UDP-GlcUA and UDP-GlcNAc with microsomes of a fibrosarcoma yielded labeled glycolipids resistant to hydrolysis with dilute alkali. These compounds have been tentatively identified as lipid-GlcNAc, lipid-GlcNAc-GlcUA and lipid-tetra and hexasaccharides containing both GlcUA and GlcNAc.     

Reversible PEGylation of peptide YY3-36 prolongs its inhibition of food intake in mice

Administration of peptide YY(3-36) (PYY(3-36)) to fasting humans or mice shortly before re-feeding effectively reduced their food intake, but PYY(3-36) exhibited a functional half-life of only approximately 3 h. Attachment of poly(ethylene glycol) to proteins and peptides (PEGylation) prolongs their half-life in vivo, but completely inactivated PYY(3-36). We developed a reversibly PEGylated PYY(3-36) derivative by coupling it to a 40 kDa PEG through a spontaneously cleavable linker. The resulting conjugate (PEG(40)-FMS-PYY(3-36)) gradually released unmodified PYY(3-36) in vivo, exhibiting an eightfold increase in its functional half-life, to approximately 24h. This long-acting PYY(3-36) pro-drug may serve as an effective means for controlling food intake in humans.     

Analysis of the local dynamics of human insulin and a rapid-acting insulin analog by hydrogen/deuterium exchange mass spectrometry

Human insulin and insulin lispro (lispro), a rapid-acting insulin analog, have identical primary structures, except for the transposition of a pair of amino acids. This mutation results in alterations in their higher order structures, with lispro dissociating more easily than human insulin. In our previous study performed using hydrogen/deuterium exchange mass spectrometry (HDX/MS), differences were observed in the rates and levels of deuteration among insulin analog products, which were found to be related to their self-association stability. In this study, we carried out peptide mapping of deuterated human insulin and lispro to determine the regions responsible for these deuteration differences and to elucidate the type of structural changes that affect their HDX reactivity. We identified A3–6 and B22–24 as the 2 regions that showed distinct differences in the number of deuterium atoms incorporated between human insulin and lispro. These regions contain residues that are thought to participate in hexamerization and dimerization, respectively. We also determined that over time, the differences in deuteration levels decreased in A3–6, whereas they increased in B22–24, suggesting a difference in the dynamics between these 2 regions. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

The comparative effects of 5′-methylthioadenosine and some of its analogs on cells containing,and deficient in, 5′-methylthioadenosine phosphorylase

The antiproliferative effects of 5′-methylthioadenosine and the 5′-methylthioadenosine analogs, 5′-isobutylthioadenosine, 5′-deoxyadenosine and 5′-methylthiotubercidin were examined using two mouse cell lines, one 5′-methylthioadenosine phosphorylase-deficient the other containing 5′-methylthioadenosine phosphorylase. All of the compounds were found to be growth inhibitory to both cell lines, demonstrating that these compounds need not be degraded to exert their inhibitory effects. A correlation was observed between the potency of the growth inhibitory effect and the ability of the cells to degrade these compounds. 5′-Methylthioadenosine, 5′-deoxyadenosine and 5′-isobutylthioadenosine, all of which are substrates for the 5′-methylthioadenosine phosphorylase in vitro, were more growth inhibitory to the 5′-methylthioadenosine phosphorylase-deficient cells than to the 5′-methylthioadenosine phosphorylase-containing cells, whereas, the 7-deaza analog, 5′-methylthiotubercidin, a nondegradable inhibitor of the 5′-methylthioadenosine phosphorylase, was a more potent inhibitor of the 5′-methylthioadenosine phosphorylase-containing cell line. Due to the inhibition by 5′-methylthiotubercidin on 5′-methylthioadenosine phosphorylase in vitro the disposition of cellularly-synthesized 5′-methylthioadenosine was explored using both cell types. 5′-Methylthiotubercidin inhibited the accumulation of exogenous 5′-methylthioadenosine from 5′-methylthioadenosine phosphorylase-deficient cells with no effect on intracellular 5′-methylthioadenosine. In contrast, 5′-methylthiotubercidin caused a large accumulation of extracellular 5′-methylthioadenosine with a concomitant smaller increase intracellularly in 5′-methylthioadenosine phosphorylase-containing cells. That cellularly-synthesized 5′-methylthioadenosine as well as the cellular excretion of this nucleoside are altered in response to treatment with 5′-methylthiotubercidin suggests two possible sites at which 5′-methylthiotubercidin may exert its effect.     

Action mechanism of PEGylated magainin 2 analogue peptide

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic β-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160-1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the α-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.     

Evidence that Fe2+ complexes of 3-aminopicolinate and 3-mercaptopicolinate activate and inhibit phosphoenolpyruvate carboxykinase

3-Mercaptopicolinic acid is known to be an inhibitor of phosphoenolpyruvate carboxykinase and 3-aminopicolinic acid permits Fe²⁺ to activate the enzyme. The potency of mercaptopicolinate is increased by incubating the enzyme with Fe²⁺ prior to assaying for activity. In the present work, the average combining ratio of either pyridine carboxylate with Fe²⁺ at pH 7.5 was determined to be 2:1 when measured by the method of continuous variation of Job or by elemental analysis of the isolated pyridine carboxylate-Fe²⁺ complexes. The ratio of 3-mercaptopicolinate or 3-aminopicolinate to Fe²⁺ that caused the greatest inhibition or activation of purified phosphoenolpyruvate carboxykinase was 2:1. In the absence of Fe²⁺, neither pyridine carboxylate altered the activity of the enzyme. These results indicate that the two pyridine carboxylates can interact with phosphoenolpyruvate carboxykinase as Fe²⁺ coordination complexes.     

Covalent modification of stathmin by CCNU determined by FTMS analysis of modified proteins and tryptic peptides

Chemical modification of proteins is often carried out to generate protein-small molecule conjugates for various applications. The high resolution and mass accuracy of a Fourier transform mass spectrometer is particularly useful for assessing the extent or sites of covalent modifications. As protein-small molecule reactions often produce products with variable numbers of the compound incorporated at different sites, a direct mass analysis of the reaction products at times yields mass spectra hard to interpret. Chromatographic separation at protein level could reduce the complexity of a sample, thus allowing more accurate mass spectrometric analysis. In this report, we demonstrate the utility of reversed-phase protein chromatography and FT-ICR mass spectrometry in analyzing CCNU (lomustine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitroso-urea, MW: 233.7 Da) modification of stathmin. With this combined approach, we determined the stoichiometry as well as sites of CCNU incorporation into the protein, demonstrating differential reactivity of several lysyl residues to CCNU alkylation.     

Application of hexafluoroacetone as protecting and activating reagent in amino acid and peptide chemistry

Summary Using hexafluoroacetone as protecting and activating reagent, multifunctional amino acids like aspartic acid can be functionalized regioselectively. This strategy offers i.a. a two-step synthesis for aspartame and preparatively simple access to multifunctional natural and unnatural amino acids, like 4-oxo-L-amino acids, 5-diazo-4-oxo-L-amino acids, 4-substituted L-proline derivatives and various heterocyclic L-amino acids. On application of this strategy to amino diacetic acid N-substituted glycines become readily available.     

Synthesis and in vitro evaluation of novel rhodanine derivatives as potential cholinesterase inhibitors

Based on a broad spectrum of biological activities of rhodanines, we synthesized aromatic amides and esters of 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (rhodanine-3-acetic acid) via carbodiimide- or PCl₃-mediated coupling. Both esters and amides were investigated for their in vitro inhibitory potency and selectivity against acetylcholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from equine serum using Ellman’s spectrophotometric method. The derivatives exhibited mostly a moderate activity against both cholinesterases. IC₅₀ values for AChE were in a closer concentration range of 24.05–86.85 μM when compared to BChE inhibition (7.92–227.19 μM). The esters caused the more efficient inhibition of AChE than amides and parent acid. The esterification and amidation of the rhodanine-3-acetic acid increased inhibition of BChE, even up to 26 times. Derivatives of 4-nitroaniline/phenol showed the activity superior to other substituents (H, Cl, CH₃, OCH₃, CF₃). Rhodanines produced a balanced inhibition of both cholinesterases. Seven derivatives produced the more potent inhibition of AChE than rivastigmine, a clinically used drug; additional three compounds were comparable. Two amides exceeded inhibitory potency of rivastigmine towards BChE. Importantly, this is the first evidence that rhodanine-based compounds are able to inhibit BChE.     

Internalization of insulin and its receptor in the isolated rat adipose cell: Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37°C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [¹²⁵I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [¹²⁵I]insulin to these same membrane fractions. At 37°C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20–30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16°C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.     

Renal protective effects of arjunolic acid in a cisplatin-induced nephrotoxicity model

Cisplatin is the first platinum-containing anti-cancer drugs. Cisplatin notable side effect of nephrotoxicity limits its use in clinic. Meanwhile, arjunolic acid possesses anti-inflammatory properties and plays protective roles against chemically induced organ pathophysiology. This study was conducted to find out whether arjunolic acid could attenuate kidney damage in rats, and to elucidate its possible mechanism of action. Fifty rats were treated with cisplatin (10 mg/kg) in the presence/absence of 100 or 250 mg/kg arjunolic acid. Arjunolic acid is given 1 h after cisplatin. Morphological changes were assessed in kidney sections stained with Hematoxylin/Eosin and Masson Trichrome. Kidney samples were used for measurements of transforming growth factor (TGF)-β1 and its type 1 receptor (TGF-βR1), tumor necrosis factor (TNF)-α and interleukin (IL)-1β by ELISA. Gene expression NFκB was determined by real time-PCR. Kidney tissue apoptosis was assessed by measuring the activities of caspase-3/8/9. The renal protective effect of arjunolic acid was confirmed by approximately normal appearance of renal tissue and the relatively unaffected serum creatinine and urea levels. Furthermore, arjunolic acid showed dose dependent reduction in cisplatin-induced elevation in renal levels of TGF-βR1, TGF-β1, TNF-α, IL-1β and caspases. These findings demonstrated that arjunolic acid attenuates cisplatin nephrotoxicity either indirectly by enhancing body antioxidant activity or directly through several mechanisms, including inhibition of pro-inflammatory cytokines, blocking activation of TGF-β1, and anti-apoptotic effects.     

Purification and partial characterization of rat kidney histamine-N-methyltransferase

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromotography and S-adenosylhomocysteine affinity chromotography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35 000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 μM parahydroxymercuric benzoate and in 10 μM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The K_m values for histamine and S-adenosylmethionine were 6.0 and 7.1 μM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 μM S-adenosylhomocysteine and 100 μM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 μM caused substrate inhibition.     

Regulation of human leukocyte microsomal hydroxymethylglutaryl-CoA reductase activity by a phosphorylation and dephosphorylation mechanism

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50°C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.