Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs.

A series of novel hGRF(1-29)-NH2 analogs were synthesized and biotinylated. The immunological and biological activities of these analogs were then characterized. To distance the biotin moiety from the putative bioactive core, a C-terminal spacer arm consisting of -Gly-Gly-Cys-NH2 (-GGC) was added to hGRF(1-29)-NH2 (hGRF29) and analogs, with subsequent biotinylation performed at the cysteine residue. Neither addition of the C-terminal spacer arm nor biotinylation affected affinity of these analogs for GRF antibody. Relative to hGRF(1-44)-NH2 (hGRF44: potency = 1.0), the biotinylated analogs were equipotent in vitro to their nonbiotinylated, parent compounds: [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2 (4.7) = [Ala15]hGRF29-GGC-(tpBiocytin)-NH2 (3.9) greater than hGRF29-GGC-(tpBiocytin)-NH2 (0.8). Based upon cumulative GH release data in vivo (0-60 min postinjection), [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2, [Ala15]hGRF29-GGC-(tpBiocytin)-NH2, and hGRF29-GGC-(tpBiocytin)-NH2 displayed 8.6, 5.5, and 0.8 times, respectively, the potency of hGRF44. These in vivo potency values were not significantly different from the corresponding parent compounds (i.e., with or without the C-terminal spacer arm). In summary, biotinylated hGRF analogs have been developed that retain full immunoreactivity and potent bioactivity (in vitro and in vivo), thus permitting their use in GRF receptor isolation, ELISA, and histochemical procedures.     

Oxygen activation during the interaction between MPTP metabolites and synthetic neuromelanin--an ESR-spin trapping, optical, and oxidase electrode study

Oxidase electrode measurements as well as optical and electron spin resonance spectroscopic data have shown that synthetic neuromelanin oxidizes the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium in a dose-dependent manner forming 1-methyl-4-phenylpyridinium and hydrogen peroxide. Hydroxyl radicals are formed in this reaction which is promoted by iron chelates. In contrast, neither 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine nor 1-methyl-4-phenylpyridinium reacts with synthetic neuromelanin in a similar fashion. The mechanism of selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in pigmented neuronal cells is discussed in the light of these findings.     

A line-balancing strategy for designing flexible assembly systems

We present a rough-cut analysis tool that quickly determines a few potential cost-effective designs at the initial design stage of flexible assembly systems (FASs) prior to a detailed analysis such as simulation. It uses quantitative methods for selecting and configuring the components of an FAS suitable for medium to high volumes of several similar products. The system is organized as a series of assembly stations linked with an automated material-handling system moving parts in a unidirectional flow. Each station consists of a single machine or of identical parallel machines. The methods exploit the ability of flexible hardware to switch almost instantaneously from product to product. Our approach is particularly suitable where the product mix is expected to be stable, since we combine the hardware-configuration phase with the task-allocation phase. For the required volume of products, we use integer programming to select the number of stations and the number of machines at each station and to allocate tasks to stations. We use queueing network analysis, which takes into account the mean and variance of processing times among different products to determine the necessary capacity of the material-handling system. We iterate between the two analyses to find the combined solution with the lowest costs. Work-in-process costs are also included in the analysis. Computational results are presented.     

Characterization of a new and highly distinguishable HLA-A allele in a Spanish family

Correspondence to: J. L. Vicario.     

Activation of Retinal Tyrosine Hydroxylase In Vitro by Cyclic AMP-Dependent Protein Kinase: Characterization and Comparison to Activation In Vivo by Photic Stimulation

Abstract: Tyrosine hydroxylase in rat retina is activated in vivo as a consequence of photic stimulation. Tyrosine hydroxylase in crude extracts of dark-adapted retinas is activated in vitro by incubation under conditions that stimulate protein phosphorylation by cyclic AMP-dependent protein kinase. Comparison of the activations of the enzyme by photic stimulation in vivo and protein phosphorylation in vitro demonstrated several similarities. Both treatments decreased the apparent K _m of the enzyme for the synthetic pterin cofactor 6MPH₄. Both treatments also produced the same change in the relationships of tyrosine hydroxylase activity to assay pH. When retinal extracts containing tyrosine hydroxylase activated either in vivo by photic stimulation or in vitro by protein phosphorylation were incubated at 25°C, the enzyme was inactivated in a time-dependent manner. The inactivation of the enzyme following both activation in vivo and activation in vitro was partially inhibited by sodium pyrophosphate, an inhibitor of phosphoprotein phosphatase. In addition to these similarities, the activation of tyrosine hydroxylase in vivo by photic stimulation was not additive to the activation in vitro by protein phosphorylation. These data indicate that the mechanism for the activation of tyrosine hydroxylase that occurs as a consequence of light-induced increases of neuronal activity is similar to the mechanism for activation of the enzyme in vitro by protein phosphorylation. This observation suggests that the activation of retinal tyrosine hydroxylase in vivo may be mediated by phosphorylation of tyrosine hydroxylase or some effector molecule associated with the enzyme.     

Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids

Experimental details for the "Fmoc solid phase peptide synthesis" of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. N alpha-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55-60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91%) yield. Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.     

The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes.

Diphosphonates are known to inhibit bone resorption in tissue culture and in experimental animals. This effect may be due to their ability to inhibit the dissolution of hydroxyapatite crystals, but other mechanisms may be important. Since lysosomal enzymes have implicated in the process of bone resorption, we have examined the effect of several phosphonates and of a polyphosphate (P20,2) on lysosomal hydrolases derived from rat liver and rat bone. Dichloromethylene diphosphonate strongly inhibited acid beta-glycerophosphatase (EC 3.1.3.2) and acid p-nitrophenyl phosphatase (EC 3.1.3.2) and to a lesser degree (in descending order) acid pyrophosphatase (EC 3.1.3.-), arylsulfatase A (EC 3.1.6.1), deoxyribonuclease II(EC 3.1.4.6) and phosphoprotein phosphatase (EC 3.1.3.16) of rat liver. Inhibition of acid p-nitrophenyl phosphatase and arylsulfatase A was competitive. Ethane-1-hydroxy-1, 1-diphosphonate did not inhibit any of these enzymes, except at high concentrations. Neither dichloromethylene diphosphonate nor ethane-1-hydroxy-1, 1-diphosphonate had any effect on beta-glucuronidase (EC 3.2.1.31), arylesterase (EC 3.1.1.2) and cathepsin D (EC 3.4.23.5). Of several other phosphonates tested only undec-10-ene-1-hydroxy-1, 1-diphosphonic acid inhibited acid p-nitrophenyl phosphatase strongly, the polyphosphate (P20, I) had little effect. Acid p-nitrophenyl phosphatase in rat calvaria extract behaved in the same way as the liver enzyme and was also strongly inhibited by dichloromethylene diphosphonate, but not by ethane-1-hydroxy-1, 1-diphosphonate. It is suggested that the inhibition of bone resorption by dichloromethylene diphosphonate might be due in part to a direct effect of this diphosphonate on lysosomal hydrolases.     

Using genetic variation to disentangle the complex relationship between food intake and health outcomes

Diet is considered as one of the most important modifiable factors influencing human health, but efforts to identify foods or dietary patterns associated with health outcomes often suffer from biases, confounding, and reverse causation. Applying Mendelian randomization in this context may provide evidence to strengthen causality in nutrition research. To this end, we first identified 283 genetic markers associated with dietary intake in 445,779 UK Biobank participants. We then converted these associations into direct genetic effects on food exposures by adjusting them for effects mediated via other traits. The SNPs which did not show evidence of mediation were then used for MR, assessing the association between genetically predicted food choices and other risk factors, health outcomes. We show that using all associated SNPs without omitting those which show evidence of mediation, leads to biases in downstream analyses (genetic correlations, causal inference), similar to those present in observational studies. However, MR analyses using SNPs which have only a direct effect on the exposure on food exposures provided unequivocal evidence of causal associations between specific eating patterns and obesity, blood lipid status, and several other risk factors and health outcomes.