Intracellular measurement of ammonium in Chara corallina using ion-selective microelectrodes

The study of ammonium (NH₄ ⁺) transport across plant cell membranes requires accurate measurement of NH₄ ⁺ gradients across subcellular gradients. We have developed an ammonium-selective microelectrode based on the ionophore nonactin. This electrode can detect NH₄ ⁺ activities (a_NH4) in vivo in the millimolar range in the presence of cytosolic levels of potassium, the main interfering ion. The electrode was used to measure intracellular a_NH4 in internodal cells of the giant alga Chara corallina. Results from cells incubated in media supplemented with 1 mM NH₄ ⁺ produced two populations, with means of 7.3 and 30.8 mM, respectively. HPLC analysis of vacuolar sap suggests the higher population represents vacuolar impalements, and the lower population can thus be assumed to be cytosolic. These results suggest a four-fold accumulation of NH₄ ⁺ in the vacuolar compartment of Chara. This revised version was published online in June 2006 with corrections to the Cover Date.     

HCV全长NS3基因表达及在抗体检测中的应用

丙型肝炎病毒(HCV)是引起非甲非乙型肝炎的主要病原因子.被HCV感染的病例中,超过50%以上会引起持续性感染、慢性肝炎,最终可能引起肝硬化和肝细胞癌[1].HCV严重威胁人类健康,但目前对丙肝患者尚缺乏有效的治疗手段,因此,严格把好血源关,提高对丙肝患者检出的灵敏度,是阻止丙肝血源传播的有效手段.     

Alpha-tocopherol and gamma-tocopherol attenuate ethanol-induced changes in membrane fatty acid composition in embryonic chick brains

BACKGROUND: This project investigated whether or not EtOH-induced reductions in the levels of long-chain polyunsaturated membrane fatty acids could be attenuated by exogenous exposure to either alpha-tocopherol, gamma-tocopherol, or diallyl sulfide (DAS). METHODS: At 0 days of development, fertile chicken eggs were injected with a single dose of either saline supplemented with various concentrations of EtOH, alpha- or gamma-tocopherol and EtOH, or DAS and EtOH. At 18 days of development, brains were isolated and subjected to membrane analyses. RESULTS: When exposed to EtOH, concentrations ranging from 0-60.50 microm/Kg egg, dose-dependent decreases in the levels of brain 18:0, 18:1 (n-9), 18:2 (n-6), 18:3 (n-3), and 20:4 (n-6) were observed. These ethanol-induced changes in membrane fatty acid composition correlated with ethanol-induced reductions in brain mass, brain protein levels, acetylcholine esterase (AChE) activities and correlated with increased lipid hydroperoxide levels. Exposure to either 2.5 microm alpha-tocopherol/Kg egg and 6.050 mm EtOH/Kg egg, or 2.5 microm alpha-tocopherol/ Kg egg and 6.050 mm EtOH/Kg egg attenuated EtOH-induced changes in membrane fatty acid composition, brain mass, brain protein levels, AChE activities, and lipid hydroperoxide levels. Embryonic exposure to the cytochrome p450-2E1 inhibitor, diallyl sulfide (DAS), also attenuated EtOH-induced decreases in long-chain, unsaturated membrane fatty acids. However, embryonic exposure to DAS promoted abnormally low brain mass. CONCLUSION: EtOH-induced reductions in the levels of brain long-chain polyunsaturated fatty acid are caused by lipid peroxidation.     

Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase

We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller, J. Am. Chem. Soc. 120(3) (1998) 461–467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn²⁺, Fe³⁺ or Fe²⁺ are bound, based on EPR and NMR spectroscopy. However, the mutant’s Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD’s activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn²⁺, Fe³⁺ and ¹H in the presence of Fe²⁺ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe²⁺SOD and Fe²⁺(Mn)SOD by ¹⁵N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe²⁺ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower E_m in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH⁻ coordinated to the metal ion.     

Protective impacts of household-based tuberculosis contact tracing are robust across endemic incidence levels and community contact patterns

There is an emerging consensus that achieving global tuberculosis control targets will require more proactive case finding approaches than are currently used in high-incidence settings. Household contact tracing (HHCT), for which households of newly diagnosed cases are actively screened for additional infected individuals is a potentially efficient approach to finding new cases of tuberculosis, however randomized trials assessing the population-level effects of such interventions in settings with sustained community transmission have shown mixed results. One potential explanation for this is that household transmission is responsible for a variable proportion of population-level tuberculosis burden between settings. For example, transmission is more likely to occur in households in settings with a lower tuberculosis burden and where individuals mix preferentially in local areas, compared with settings with higher disease burden and more dispersed mixing. To better understand the relationship between endemic incidence levels, social mixing, and the impact of HHCT, we developed a spatially explicit model of coupled household and community transmission. We found that the impact of HHCT was robust across settings of varied incidence and community contact patterns. In contrast, we found that the effects of community contact tracing interventions were sensitive to community contact patterns. Our results suggest that the protective benefits of HHCT are robust and the benefits of this intervention are likely to be maintained across epidemiological settings.     

Rational design and PCR-based synthesis of an artificial Schizophyllum commune xylanase gene.

A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure. Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage. The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E. coli genes. The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases.     

Expression of nerve growth factor and its receptors in the somatosensory-motor cortex of Macaca nemestrina

The present study was designed to examine the nerve growth factor (NGF) system (ligand and receptor-expressing neurons) in the somatosensory (areas 1, 3a, and 3b) and motor (area 4) cortices of the mature macaque. Light and electron microscope immunohistochemistry was used to assess the distribution and identity of NGF-, p75-, and trk-expressing elements. In each cortical area examined, NGF-positive neuronal somata were distributed through all laminae; most immunolabeled neurons were in layers II, III, and V. Based upon light microscope criteria (e.g., the morphology of proximal dendrites), both pyramidal and stellate neurons expressed NGF. Of the identifiable NGF- immunoreactive cells, 92% were pyramidal neurons and the remainder was stellate neurons. The electron microscope study showed that most (88%) NGF-positive somata formed symmetric synapses, whereas the others formed both symmetric and asymmetric synapses. As the somata of pyramidal neurons form only symmetric synapses and those of inhibitory stellate neurons form both symmetric and asymmetric somatic synapses, the ultrastructural data support the light microscopic analyses. In contrast, neurotrophin receptors, p75 and trk, were expressed chiefly by the cell bodies of layer V pyramidal neurons and the supragranular neuropil. At the ultrastructural level, receptor-positive profiles were post-synaptic elements (e.g., dendritic shafts and spines) and the concentration of immunoreactivity was greatest in the vicinity of post-synaptic densities. Thus, NGF regulatory systems parallel excitatory and inhibitory neurotransmitter systems. Cortex contains the morphological framework by which pyramidal and/or inhibitory stellate neurons can affect the activity of post-synaptic pyramidal neurons via anterograde and autocrine/paracrine NGF systems.     

Support for the microenvironment hypothesis for adaptive value of gall induction in the California gall wasp, Andricus quercuscalifornicus

Three major hypotheses have been advanced for the adaptive nature of plant galls: nutrition, enemy-avoidance, and microenvironment. Of these, the microenvironment hypothesis has been frequently invoked, but rarely tested directly. We tested this hypothesis in a population of Andricus quercuscalifornicus (Bassett) (Hymenoptera: Cynipidae) wasps inducing galls on Quercus lobata Née (Fagaceae) trees in Northern California, USA. Relative humidity and temperature data gathered from both immature and mature galls in the field indicated that A. quercuscalifornicus larvae modify their microenvironments significantly by raising and stabilizing relative humidity levels inside galls to near saturation. In addition, excised larvae maintained under experimental conditions survived significantly longer under levels of high relative humidity. These data support the hypothesis that through gall induction, A. quercuscalifornicus manipulates its environment adaptively.