The granin (chromogranin/secretogranin) family

The chromogranins/secretogranins, referred to in abbreviated form as granins, are a family of acidic secretory proteins that are found in the secretory granules of a wide variety of endocrine cells and neurons, being stored together with many different peptide hormones and neuropeptides. The recent elucidation of their primary structure has provided insights into possible functions of these proteins. Moreover, the granins have been successfully used as markers for normal and neoplastic endocrine and neuronal cells, as well as model proteins to understand the sorting mechanism involved in the formation of secretory granules.     

Approach to the analysis of diuretics and masking agents by high-performance liquid chromatography—mass spectrometry in doping control

The application of thermospray and plasmaspray high-performance liquid chromatography—mass spectrometry to the analysis of diuretics and probenecid has been investigated. The latter method gave better ionization efficiency than the former, and its response was optimized by altering the solvent composition: best results were obtained with water—methanol—acetonitrile—trifluoroacetic acid. Using different proportions of these solvents, three isocratic systems were developed to separate the compounds under study. The principal characteristic of plasmaspray positive-ion mass spectra was a protonated molecular ion and very little fragmentation was evident. In the negative ionization mode, the plasmaspray method gave mass spectra showing more fragmentation, which resulted in additional structural information. The ability of trifluoroacetic acid to form negative cluster ions precluded its use as a mobile phase component. The minimum detectable amounts determined by the analysis in the positive-ion mode was compound-dependent, but generally ca. 10–150 ng. In many cases the compounds could be detected in urine extracts.     

Redescription of Branchinecta granulosa Daday, 1902 from Argentina (Crustacea: Branchiopoda)

The species Branchinecta granulosa Daday 1902, is redescribed on the basis of adult material from near Facundo (Chubut Province, Argentina). Its relationship to its regional congeners is outlined.     

Biological characterization of Trichinella isolates from various host species and geographical regions.

Forty isolates of Trichinella collected from 5 continents were compared for 7 biological characters: newborn larvae produced per female worm cultured in vitro at the seventh, eighth, and ninth day postinfection, host muscle nurse cell development time, reproductive capacity index in rats and chickens, and resistance of muscle larvae to freezing. The isolates also were compared by analyses of an environmental character of the location from which they were isolated: the isotherms for January and July. By factorial analysis of correspondence of the biological and environmental data, the 40 isolates were grouped into 8 gene pools (T1-T8). The environmental temperature-related distribution was more evident for the sylvatic isolates (T2, T3, T5, T6, T7, T8), than for T1, which was isolated from domestic pigs, and for T4, a bird-adapted, nonencapsulating genetic type. The 8 biological groups correlated closely with the 8 gene pools previously identified on the basis of allozyme analysis. These results support the concept that the genus Trichinella is composed of at least 5 distinct gene pools or sibling species: Trichinella spiralis sensu stricto (T1), Trichinella nativa (T2), Trichinella sp. (T3), Trichinella pseudospiralis (T4), and Trichinella nelsoni (T7), and 3 other groups of uncertain taxonomic status (i.e., T5, T6, and T8).     

Distance measurements using paramagnetic ion-induced relaxation in the saturation transfer electron spin resonance of spin-labeled biomolecules: Application to phospholipid bilayers and interdigitated gel phases

The saturation transfer electron spin resonance (STESR) spectra of spin-labeled phosphatidylcholines in gel phase lipid bilayers are shown to be sensitive to dipolar spin-spin interactions with paramagnetic ions in the aqueous phase. The reciprocal integrated intensity of the STESR spectrum is linearly dependent on aqueous Ni²⁺ ion concentration, hence, confirming the expectation that the STESR intensity is directly proportional to the spin-lattice relaxation time of the spin label. The gradient of the relaxation rate with respect to Ni²⁺ ion concentration decreases strongly with the position of the nitroxide group down the sn-2 chain of the spin-labeled lipid and is consistent with a 1/R³ dependence on the distance, R, from the bilayer surface. The values derived for the dimensions of the bilayer and lipid molecules in the case of dipalmitoyl phosphatidylcholine (DPPC) are in good agreement with those available from x-ray diffraction studies. Allowance for the multibilayer nature of the DPPC dispersions gives an estimate of the water layer thickness that is also consistent with results from x-ray diffraction. The profile of the paramagnetic ion-induced relaxation is drastically changed with DPPC dispersions in glycerol for which the lipid chains are known to be interdigitated in the gel phase. The terminal methyl groups of the lipid chains are located approximately in register with the C-3 atoms of the sn-2 chain of the oppositely oriented lipid molecules in the interdigitated phase. The thickness of the lipid layer and the effective thickness of the lipid polar group are reduced by ~40% in the interdigitated phase as compared with the bilayer phase. The calibrations of the distance dependence established by use of spin labels at defined chain positions should be applicable to STESR measurements on other biological systems.     

Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network.

The effects of brefeldin A (BFA) on membrane traffic between the trans-Golgi network (TGN) and the plasma membrane were investigated in intact PC12 cells and in a cell-free system derived from PC12 cells. In intact cells, BFA caused a virtually complete block of constitutive secretion, as indicated by the lack of release from, and accumulation in, the cells of a [35S]sulfate-labeled heparan sulfate proteoglycan (hsPG). Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that this block was due to the inhibition of formation of constitutive secretory vesicles (CSVs) from the TGN. BFA did not block the depolarization-induced release of [35S]sulfate-labeled chromogranin B (CgB) and secretogranin II (SgII) from secretory granules formed prior to the addition of the drug, showing that BFA does not block secretory granule fusion with the plasma membrane. The presence of BFA did, however, prevent the appearance of [35S]sulfate-labeled CgB and SgII in secretory granules, indicating that the drug inhibits the formation of secretory granules from the TGN. Evidence for a direct block of vesicle formation by BFA was obtained using a cell-free system derived from [35S]sulfate-labeled PC12 cells. In this system, low concentrations of BFA (5 micrograms/ml) inhibited the formation of the hsPG-containing CSVs and that of the SgII-containing secretory granules from the TGN to the same extent (50-60%) as, and in a non-additive manner with, the nonhydrolyzable GTP analogue GTP gamma S. Consistent with the inhibitory effects of BFA on vesicle formation from the TGN, BFA treatment of intact PC12 cells led to the hypersialylation of CgB, which presumably was due to the increased residence time of the protein in the TGN. In conclusion, our data are consistent with, and allow the generalization of, the concept that the BFA-induced block of anterograde membrane traffic results from the inhibition of vesicle formation from a donor compartment.     

Flavin-photosensitized oxidation of reduced c-type cytochromes. Reaction mechanism and comparison with photoreduction of oxidized cytochromes by flavin semiquinones

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin-cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.     

Asymmetric reduction of ketones with resting cells of Sulfolobus solfataricus

The method of resting cells has been of interest in the development of biocatalysts applied to organic reactions.This article deals with the use of resting cells of a thermophilic archaebacterium Sulfolobus solfataricus, in the asymmetric reduction of acyclic, cyclic, and aromatic ketones. The system allows the continuous regeneration of endogenous coenzyme with the coupled substrate approach. The results indicate that the direction of hydride attack was equatorial on the re face of the carbonyl group of substrates producing (S)-alcohols with a good optical yield. A convenient system for the reuse of resting cells has been set out to synthesize (S)-alcohols on a preparative scale.     

Archaebacterial elongation factor Tu insensitive to pulvomycin and kirromycin

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.