Endothelin-1 response to mental stress in early ischemic lesions of the extremities due to systemic sclerosis

We studied circulating levels of endothelin-1, catecholamines and nitric oxide after a mental arithmetic test in 14 patients with early ischemic lesions of the extremities due to systemic sclerosis and slightly impaired peripheral vascular flow. The test induced an increase (P < 0.01) in blood pressure, heart rate, endothelin-1 and catecholamine levels, whereas it did not change the low basal levels of nitric oxide. In healthy subjects (n = 20) the test significantly (P < 0.01) decreased endothelin-1 without affecting nitric oxide. The low basal levels of nitric oxide and the high plasma concentration of endothelin-1 after psychological stress cannot be explained by an impaired release from the limited ischemic lesions alone. This suggests a diffuse microvascular derangement that aggravates the course of peripheral microvascular ischemic lesions.     

Structural analysis of the sheath of a sheathed bacterium, Leptothrix cholodnii

Leptothrix cholodnii is an aerobic sheath-forming bacterium often found in oligotrophic and metal-rich aquatic environments. The sheath of this bacterium was isolated by selectively lysing the cells. Glycine and cysteine were the major amino acids of the sheath. The sheath was readily dissolved in hydrazine, and a polysaccharide substituted with cysteine was recovered from the solution. Galactosamine, glucosamine and galacturonic acid were detected in the hydrazinolysate by gas liquid chromatography analysis. FAB-MS analysis of the hydrazinolysate suggested a sugar sequence of HexN-GalA-HexN-HexN. Methylation linkage analysis revealed the presence of 4-linked GalA, 3-linked HexN and 4-linked HexN. The sulfhydryl groups of the sheath were used for labeling with the fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The labeled sheath (ABD-sheath) was partially hydrolyzed and three fluorescent fragments were purified by HPLC. One of them was identified as ABD-cysteine. The second one was found to be the ABD-cysteine tetramer. Another fragment was indicated to be a pentasaccharide substituted with ABD-cysteine by nuclear magnetic resonance (NMR) analysis. It can be assumed that the polysaccharide and peptide moieties of the sheath are connected by a cysteine residue. NMR analysis of the hydrazinolysate revealed that the polysaccharide moiety of the sheath was constructed from a pentasaccharide repeating unit containing 2-amino-2-deoxygalacturonic acid (GalNA), as shown below. -->4)-alpha-GalNA-(1-->4)-alpha-D-GalN(p)-(1-->4)-alpha-D-GalA(p)-(1-->4)-beta-D-GlcN(p)-(1-->3)-beta-D-GalN(p)-(1-->.     

Changes in responses of UVB irradiated skin of brownish guinea pigs with aging

It is known that skin often shows irregular pigmentation during aging, which is frequently associated with hyperpigmentation. Many studies have utilized brownish A1 guinea pigs to investigate the pathogenesis of ultraviolet B (UVB)-induced skin pigmentation, however, responses associated with aging following UVB irradiation have not been elucidated. To characterize those responses, dorsal skin of A1 guinea pigs from 14-weeks to 5-yr old were investigated. The minimal erythema dose was found to increase with aging. Further, in pigmentation induced by UVB radiation, skin brightness (DeltaL*-value) decreased equally in both the 14-week old (young) group and in the 3-yr old (old) group of guinea pigs. The DeltaL*-value recovered in the young group from 21 d after UVB irradiation, whereas no such recovery was seen in the old group. In addition, the amount of melanin and the number of melanocytes returned near pre-irradiation levels in the young group, while they remained high in the old group. Our results therefore demonstrate for the first time that skin responses following UVB irradiation change with aging in A1 guinea pigs.     

Phospholipase Cdelta4 associates with glutamate receptor interacting protein 1 in testis

We reported previously that phospholipase C (PLC) delta4 is required for calcium mobilization in the zona pellucida-induced acrosome reaction in sperm. Here we focused on the function of the C2 domain of PLCdelta4 and report that glutamate receptor-interacting protein1 (GRIP1) was identified as a binding protein of the PLCdelta4-C2 domain on yeast two-hybrid screening. Physiological interaction of GRIP1 with PLCdelta4 in mouse testis was confirmed by immunoprecipitation with anti-PLCdelta4 antibodies and the association seemed to correlate with the maturation stage of sperm. We also determined that a PDZ-binding motif at the C-terminus of the PLCdelta4-C2 domain is responsible for GRIP1 binding, whereas the sixth or seventh PDZ domain of GRIP1 is essential and sufficient for association with the PLCdelta4-C2 domain. These results indicate that PLCdelta4 binds via its C2 domain to the PDZ6 or PDZ7 domain of GRIP1, and that this association may play a role in spermatogenesis.     

A novel phospholipase C, PLC(eta)2, is a neuron-specific isozyme

Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, beta-, gamma-, delta-, epsilon-, zeta-, and eta-type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLC(eta)2. PLC(eta)2 is composed of conserved domains including pleckstrin homology, EF-hand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLC(eta)2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLC(eta)2 was more sensitive to calcium concentration than the PLC activity of the PLCdelta-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLC(eta)2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLC(eta)2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLC(eta)2 in pyramidal cells of the hippocampus. Finally, we found that PLC(eta)2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLC(eta)2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.     

Efficiency of the use of pedigree and molecular marker information in conservation programs

The value of molecular markers and pedigree records, separately or in combination, to assist in the management of conserved populations has been tested. The general strategy for managing the population was to optimize contributions of parents to the next generation for minimizing the global weighted coancestry. Strategies differed in the type of information used to compute global coancestries, the number and type of evaluated individuals, and the system of mating. Genealogical information proved to be very useful (at least for 10 generations of management) to arrange individuals' contributions via the minimization of global coancestry. In fact, the level of expected heterozygosity after 10 generations yielded by this strategy was 88-100% of the maximum possible improvement obtained if the genotype for all loci was known. Marker information was of very limited value if used alone. The amount and degree of polymorphism of markers to be used to compute molecular coancestry had to be high to mimic the performance of the strategy relying on pedigree, especially in the short term (for example, >10 markers per chromosome with 10 alleles each were needed if only the parents' genotype was available). When both sources of information are combined to calculate the coancestry conditional on markers, clear increases in effective population size (Ne) were found, but observed diversity levels (either gene or allelic diversity) in the early generations were quite similar to the ones obtained with pedigree alone. The advantage of including molecular information is greater when information is available on a greater number of individuals (offspring and parents vs. parents only). However, for realistic situations (i.e., large genomes) the benefits of using information on offspring are small. The same conclusions were reached when comparing the use of the different types of information (genealogical or/and molecular) to perform minimum coancestry matings.     

Confocal and video imaging of cytoskeleton dynamics in the leech zygote

Ooplasmic segregation in the late interphase zygote of the leech Theromyzon trizonare is accomplished by reorganization of an ectoplasmic cytoskeleton formed by polar rings and meridional bands. The dynamic properties of this cytoskeleton were explored by time-lapse confocal and video microscopy. Cytoskeleton assembly was investigated in zygotes pulse-labeled with microinjected fluorophore-tagged or biotin-tagged dimeric tubulin and G-actin. Cytoskeleton disassembly was studied by comparing the linear dimensions of the cytoskeleton at different time points during late interphase. The relative distributions of F- and-G-actin were determined after microinjection of rhodamine-labeled actin and fluorescein-labeled DNase I. Results showed that labeled precursors were readily incorporated into a network of microtubules or actin filaments. Bipolar translocation of the rings and meridional bands was accompanied by the rapid assembly and disassembly of microtubules and actin filaments. Because labeled microtubules and microfilaments gradually decreased, the rate of cytoskeleton disassembly was greater than the rate of cytoskeleton assembly. Hence, ooplasmic segregation was accompanied by the rapid turnover of cytoskeletal components. Co-distribution of F- and-G-actin during mid and late interphase may favor polymer-monomer interchange. We conclude that cytoskeleton reorganization during foundation of cytoplasmic domains can be conveniently studied in the live leech zygote after microinjection of labeled precursors.     

NMR study on the binding of neuropeptide achatin-I to phospholipid bilayer: the equilibrium, location, and peptide conformation

Molecular mechanism of the binding of neuropeptide achatin-I (Gly-D-Phe-Ala-Asp) to large unilamellar vesicles of zwitterionic egg-yolk phosphatidylcholine (EPC) was investigated by means of natural-abundance (13)C and high-resolution (of 0.01 Hz order) (1)H NMR spectroscopy. The binding equilibrium was found to be sensitive to the ionization state of the N-terminal NH(3)(+) group in achatin-I; the de-ionization of NH(3)(+) decreases the bound fraction of the peptide from approximately 15% to nearly none. The electrostatic attraction between the N-terminal positive NH(3)(+) group and the negative PO(4)(-) group in the EPC headgroup plays an important role in controlling the equilibrium. Analysis of the (13)C chemical shifts (delta) of EPC showed that the binding location of the peptide within the bilayer is the polar region between the glycerol and ester groups. The binding caused upfield changes Delta delta of the (13)C resonance for almost all the carbon sites in achatin-I. The changes Delta delta for the ionic Asp at the C-terminus are more than five times as large as those for the other residues. The drastic changes for Asp result from the dehydration of the ionic CO(2)(-) groups, which are strongly hydrated by electrostatic interactions in bulk water. The side-chain conformational equilibria of the aromatic d-Phe and ionic Asp residues were both affected by the binding, and the induced changes in the equilibria appear to reflect the peptide-lipid hydrophobic interactions.     

Sphingosine 1-phosphate breakdown in platelets

We examined the formation of sphingolipid mediators in platelets, which abundantly store, and release extracellularly, sphingosine 1-phosphate (Sph-1-P). Challenging [(3)H]Sph-labeled platelet suspensions with thrombin or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a decrease in Sph-1-P formation and an increase in sphingosine (Sph), ceramide (Cer), and sphingomyelin formation. Sph conversion into Cer, and Cer conversion into sphingomyelin were not affected upon activation, suggesting that Sph-1-P dephosphorylation may initiate the formation of sphingolipid signaling molecules. In fact, Sph-1-P phosphatase (but not lyase) activity was detected in platelets, but this activity was not enhanced by thrombin or TPA. When quantified with [(3)H]acetic anhydride acetylation, followed by HPLC separation, the amounts of Sph-1-P and Sph decreased and increased, respectively, upon stimulation with thrombin or TPA, and these changes were attenuated by staurosporine. Under these TPA treatment conditions, over half of the [(3)H]Sph-1-P (formed in platelets incubated with [(3)H]Sph) was detected extracellularly, possibly due to its release from platelets, which was completely inhibited by staurosporine pretreatment. Furthermore, when TPA-induced Sph-1-P release was blocked by staurosporine after the stimulation, the extracellular [(3)H]Sph-1-P radioactivity decreased, suggesting that the Sph-1-P released may undergo dephosphorylation extracellularly. To support this, [(32)P]Sph-1-P, when added extracellularly to platelet suspensions, was rapidly degraded, possibly due to the ecto-phosphatase activity. Our results suggest the presence in anucleate platelets of a transmembrane cycling pathway starting with Sph-1-P dephosphorylation and leading to the formation of other sphingolipid mediators.