Alpha-helical small molecular size analogues of neuropeptide Y: structure-activity relationships.

C-terminal analogues of neuropeptide Y (NPY) of small molecular size have been synthesized. The influence of chain length, single or multiple amino acid substitution, and segment substitutions on receptor binding, pre- and postsynaptic biological activity, and conformational properties have been investigated. Receptor binding and in vivo assays revealed biological activity for NPY Ac-25-36 that increased with increasing alpha-helicity. In attempts to stabilize the alpha-helical content, three independent types of modified NPY Ac-25-36 analogues were synthesized. Strong agonistic activities could be detected in a series of discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was NPY 1-4-Aca-25-36 (Aca, epsilon-aminocaproic acid). For the first time conformational properties of a series of small NPY analogues have been investigated by CD, and correlated with biological activity and receptor binding. A C-terminal dodecapeptide segment of NPY with an amount of 50% substitution to the native C-terminal sequence of NPY was found to exhibit significant receptor binding.     

Biological activity of the antitumor protein neocarzinostatin coupled to a monoclonal antibody by N-succinimidyl 3-(2-pyridyldithio)-propionate

The chromophore free apoprotein of neocarzinostatin was coupled to monoclonal IgG₁ antibody using N-Succinimidyl 3-(2-pyridyldithio)-propionate as heterobifunctional reagent. After coupling active chromophore was reassociated with the apoprotein. We present here experimental evidence that the hybrid protein retains biological activity as measured by the degradation of T2-DNA and bacteriostatic action.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

Manganese oxidation by an intracellular protein of a Pseudomonas species.

Cultures of a Pseudomonas sp. strain MnB 1 produce an intracellular, manganese oxidizing protein (abbrev. as Mn ox. protein) during the stationary phase of growth. This protein is heat labile, can be inactivated by protease and has a pH-optimum for manganese oxidation at pH 7.0. Mn2+ is oxidized only at concentrations below 3-10(-5) M. The occurrence of the protein is not dependent on the presence of Mn2+, but is clearly related to the cessation of growth after the end of the exponential growth phase. Oxygen, coenzymes, and low molecular weight components of the cell extract seem not to be involved in the reaction as electron acceptors for the oxidation of Mn2+. Continued manganese oxidation by Mn ox. protein results in a progressive decrease in activity which corresponds to the amount of formed manganese oxide.     

Role of catecholamines in hypotensive response to dieting.

The effect of dieting on blood pressure and catecholamine metabolism was assessed in 11 normotensive obese women by providing first a weight-maintenance regimen high in carbohydrate and then a low-energy diet. All dietary constituents other than carbohydrate were maintained constant throughout the 18-day study. The low-carbohydrate diet led within 48 hours to a 41% fall in the urinary output of 4-hydroxy-3-methoxy mandelate and a significant fall in systolic and diastolic blood pressure. Plasma noradrenaline concentrations also fell and the hypotensive effect of the diet continued despite a maintained total body sodium. Thus the fall in blood pressure appeared to be mediated by changes in catecholamine metabolism independent of sodium intake. This may explain both the usefulness of weight reduction in hypertensive patients and the fainting that occurs in some normotensive obese subjects taking slimming regimens low in carbohydrate.     

Structural and membrane modifying porperties of suzukacillin, a peptide antibiotic related to alamethicin. Part B. Pore formation in black lipid films.

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete condustance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger alpha-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of "inactivation".     

Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part B. Pore formation in black lipid films

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete conductance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation tion starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger α-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of “inactivation”.     

Electrophoretic studies of several hydrolytic enzymes in relation to the cold tolerance of alfalfa.

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to determine the characteristics and relationships of several hydrolytic enzymes to cold tolerance.Soluble enzymatic proteins, extracted from lyophilized crown and root tissues with three different solvents, were separated by polyacrylamide disc-gel electrophoresis and evaluated on the basis of equal dry weights of tissue and equal quantities of protein.Gels assayed for amylases, acid phosphatases, esterases, leucine aminopeptidases, and adenosine triphosphatases exhibited mainly quantitative differences in isoenzymes depending upon extractant, cultivar, and environmental differences. The qualitative differences detected were generally due to differential solubilities of isoenzymes in the three extractants and, to a lesser extent, were related to environmental, cultivar, or stability differences.While activities of esterases, acid phosphatases, and leucine aminopeptidases increased in winter samples, as soluble protein increased, only slight decreases in these enzymes occurred during dehardening. Conversely, activities of amylases were slightly lower in winter samples than in the other samples, and adenosine triphosphatase activity decreased in the most coldtolerant sample.The measured levels of total nonstructural carbohydrate, total soluble sugar, and starch indicated differences between cultivars in starch-sugar conversion. Further, the differential heat stabilities of the isoamylases also provided some information as to the nature of “protected activity” of diastatic enzymes.Differential cryostabilities of peptidases and adenosine triphosphatases detected between cultivars and environments also demonstrated the influence of the extraction medium in maintaining enzyme activity, and these observations may be important to an understanding of cold tolerance in alfalfa. The obvious speculations regarding enzyme stability and the factors involved as related to the cold tolerance of alfalfa require further examination.     

Sterically hindered disulfide bridges in cystine diketopiperazine, cysteinyl-cysteine disulfide and derivatives.

L-Cystine diketopiperazine (1), L-cysteinyl-cysteine disulfide -HCl (2), L-cysteinyl-cysteine disulfide methyl ester -HCl (3), and t-butyloxycarbonyl-L-cysteinyl-cysteine disulfide methyl ester (4) are investigated by CD, ultraviolet, 13C NMR, infrared and laser Raman spectroscopy. The temperature dependence of the 13C NMR signals of 1 reveals an exceptionally high energy barrier of deltaGNo. = 15.8 +/- 0.2 kcal/mol for the reversible change in helicity of the inherently dissymmetric disulfide bridge of 1. The P-helical diastereomer predominates in dimethyl-sulfoxide at 25 degrees C, with 80-85% of the molecules having this configuration. The Cotton effects of 1 are larger and show smaller temperature coefficients than the conformationally more mobile cystine compounds 2 and 3. After dissolving crystals of 1 in 95% ethanol there is a time-dependent decrease of the ellipticity of the negative Cotton effect at 225 nm, indicating a conformational change in going from crystal to solution. Besides 1, 2 and 3 are at present the only known examples of cystine derivatives with C-S-S-C torsional angles around 90 degrees, which do not exhibit optical activity in the long wavelength disulfide absorption, as is predicted for 1 from the Linderberg-Michl model. At 305 nm a new weak Cotton effect was discovered for 1. The solvent dependence of the CD spectra is discussed and the infrared and Raman spectra are assigned.     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.