Population genetics of geographically restricted and widespread species of Myrica (Myricaceae)

Allozyme variation of 11 putative loci in five populations of the rare Myrica adenophora Hance, and four populations of its widespread congeneric species, M. rubra (Lour.) Sieb. & Zucc. was studied. Among the 21 alleles studied, no unique allele was detected for M. adenophora, whereas M. rubra had 3 alleles not found in the former species. In terms of genetic diversity, populations of the rare species contained fewer alleles per locus (1.5 versus 1.7), fewer effective number of alleles per locus (1.12 versus 1.20), fewer number of alleles per polymorphic locus (2.14 versus 2.46), lower percentage of polymorphic loci (30.9 versus 40.9), and lower expected heterozygosity (0.106 versus 0.163) than populations of the widespread species. Genetic distances within species average 0.043 for M. adenophora and 0.045 for M. rubra, and between species ranged from 0.052 to 0.177, with a mean of 0.103, which agrees with the very similar gross morphologies of these two species. Intrapopulation differentiation was similar in both species: G(ST) = 0.152 for M. adenophora, and 0.146 for M. rubra, whereas estimated gene flow based on G(ST) values were moderate in these two species (Nm = 1.39 versus 1.46). We inferred that M. rubra and M. adenophora are a progenitor-derivative species pair that emerged before migrating into Taiwan during the last glacial period. We consider the Hengchun population (Chiupeng, Hsuhai, and Chufengpi) and Taitung population (Tienkuan and Lanshan) of M. adenophora which probably arose from two subsets of the genome of M. rubra. Genetic drift was inferred to be one of the forces shaping the observed genetic structure in M. adenophora and M. rubra.     

Isolation and characterization of Corynebacterium ulcerans from cephalic implants in macaques

To determine the prevalence of colonization by Corynebacterium ulcerans, we cultured samples from the cephalic implant-skin margin and pharynx of 26 rhesus macaques and one pig-tailed macaque. All but one of the samples from the cephalic implants yielded a mixed population of bacteria. C. ulcerans grew from the cephalic implants in 56% and from the pharynx in 3% of the implanted animals. We screened nine of these isolates for diphtheria toxin (DT) and phospholipase D (PLD). Polymerase chain reactions (PCR) failed to identify DT in any of the tested isolates, which also lacked DT activity in Elek tests. However, all nine isolates tested had PLD toxin activity as determined by conjoint hemolysis on sheep blood agar plates in the presence of equi factor (Rhodococcus equi). In addition, PCR assays and Southern blot hybridization confirmed the presence of pld in the isolates. The role of the PLD toxin in promoting colonization of cephalic implants by C. ulcerans is unknown. We found C. ulcerans to be a frequent contaminant of the cephalic implant-skin margin. Further studies are necessary to investigate the relative clinical importance of this organism and the efficacy of various implant maintenance protocols in preventing infection.     

Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-a-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of ¹⁴C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.     

Inhibition of testosterone secretion by S-petasin in rat testicular interstitial cells

S-petasin, a kind of sesquiterpene ester, is the anti-inflammatory ann analgesic component of the butterbur (Petasites hybridus). The clinical benefit of S-petasin is the spasmolytic activity, but its side effects on the reproductive endocrinology are not clear yet. The present study was to explore the effects of S-petasin on the secretion of testosterone in vivo and in vitro. We found that single intravenous injection of S-petasin (1 microg/kg) decreased basal plasma testosterone concentration in adult male rats. The enzymatically dispersed rat testicular interstitial cells were incubated with S-petasin (0-4.3 x 10(-5)M) in the presence or absence of human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (adenylyl cyclase activator, 10(-5) M), and androstenedione (testosterone biosynthesis precursor, 10(-9) M) at 34 degrees C for 1 h. The concentrations of testosterone in the incubation medium were measured by radioimmunoassay. S-petasin at 4.3 x 10(-7) M was effective to reduce the basal and hCG-stimulated release of testosterone in rat testicular interstitial cells. The stimulatory effects of testosterone secretion induced by forskolin and androstenedione were significantly reduced by S-petasin at 4.3 x 10(-5) M and 4.3 x 10(-6) M, respectively. These results suggest that S-petasin inhibits the production of testosterone in rat testicular interstitial cells in part through diminishing the activities of adenylyl cyclase and 17-ketosteroid reductase.     

The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis

We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin-enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell-cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.     

Bacterial lipopolysaccharide activates protein kinase C, but not intracellular calcium elevation, in human peripheral T cells

The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human peripheral T cells. Bacterial lipopolysaccharide (LPS) is nonmitogenic in human T cells. However, in the presence of monocytes, LPS becomes mitogenic to proliferate T cells. The aim of this study was to define the incompetency of LPS on two mitogenic signals in human peripheral T cells. T cells were isolated from human peripheral blood. [Ca(2+)](i) and pH(i) were determined by loading the cells with the fluorescent dyes, Fura-2 acetoxymethyl ester (Fura-2/AM) and 2',7'-bis(2-carboxyethyl)-5-(and 6)carboxyfluorescein acetoxymethyl ester (BCECF/AM). PKC activity was determined by protein kinase assay and cell proliferation was estimated from the incorporation of [(3)H]-thymidine. The results indicated that (1) LPS (10 microg/ml) stimulated PKC activity significantly within 5 min, reached a plateau at 30 min, and maintained that level for at least 2 h; and (2) LPS stimulated cytoplasmic alkalinization but did not affect the levels of [Ca(2+)](i) and [(3)H]-thymidine incorporation into T cells. Moreover, the combination of calcium ionophore A23187 with LPS significantly stimulated [(3)H]-thymidine incorporation into T cells. Thus, the results demonstrate that LPS failed to proliferate T cells, probably because of a lack of the machinery necessary to stimulate the mitogenic signal on [Ca(2+)](i) elevation.     

Temperature transitions of protein properties in human red blood cells.

Human red blood cells (RBC) undergo a sudden change from blocking to passing through 1.3 +/- 0.2-micrometer micropipettes at a transition temperature (Tc) of 36.4 degrees C. For resealed RBC ghosts this transition occurs at 28.3 degrees C (Tg). These findings are attributed to an elastomeric transition of hemoglobin from being gel-like to a fluid and to an elastomeric transition of membrane proteins such as spectrin. Spectrin shows a uniform distribution along the aspirated RBC tongue above Tg in contrast to the linear gradient below Tg.     

Post-Translational Modifications of β Subunits of Voltage-Dependent Calcium Channels

Different post-translational modifications of Ca channel subunits have been identified. Recent studies have characterized the palmitoylation of the Ca channel _2a subunit, as well as one effect of this modification on channel function. The potential importance of palmitoylation on other channel properties is discussed. Other studies have addressed the role of phosphorylation of subunits in the regulation of voltage-dependent Ca channels. Phosphorylation of subunits by second messenger-activated protein kinases, as well as by unidentified protein kinases, may affect interactions between channel subunits and other aspects of channel function. The differential modification of Ca channel subunit isoforms by post-translational events likely results in diversely regulated channels with unique properties.     

Charge Accumulation and Hysteresis in Perovskite‐Based Solar Cells: An Electro‐Optical Analysis

Organic–inorganic hybrid perovskite solar cells based on CH₃NH₃PbI₃ have achieved great success with efficiencies exceeding 20%. However, there are increasing concerns over some reported efficiencies as the cells are susceptible to current–voltage (I–V) hysteresis effects. It is therefore essential that the origins and mechanisms of the I–V hysteresis can clearly be understood to minimize or eradicate these hysteresis effects completely for reliable quantification. Here, a detailed electro‐optical study is presented that indicates the hysteresis originates from lingering processes persisting from sub‐second to tens of seconds. Photocurrent transients, photoluminescence, electroluminescence, quasi‐steady state photoinduced absorption processes, and X‐ray diffraction in the perovskite solar cell configuration have been monitored. The slow processes originate from the structural response of the CH₃NH₃PbI₃ upon E‐field application and/or charge accumulation, possibly involving methylammonium ions rotation/displacement and lattice distortion. The charge accumulation can arise from inefficient charge transfer at the perovskite interfaces, where it plays a pivotal role in the hysteresis. These findings underpin the significance of efficient charge transfer in reducing the hysteresis effects. Further improvements of CH₃NH₃PbI₃‐based perovskite solar cells are possible through careful surface engineering of existing TiO₂ or through a judicious choice of alternative interfacial layers.