Exogenously supplied compatible solutes rapidly ameliorate NaCl-induced potassium efflux from barley roots

It has been suggested that the role of compatible solutes in plant stress responses is not limited to conventional osmotic adjustment, but also includes some other regulatory or osmoprotective functions. In this study, we hypothesized that one such function is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, a feature that may confer salt tolerance in many species, particularly in barley. This hypothesis was investigated using the non-invasive microelectrode ion flux (MIFE) measuring technique. We show that low (0.5-5 mM) concentrations of exogenously supplied proline or betaine significantly reduced NaCl-induced K+ efflux from barley roots in a dose-response manner. This effect was instantaneous, implying that large intracellular concentrations of compatible solutes are not required for an amelioratory role. Exogenously supplied betaine also significantly enhanced NaCl-induced H+ efflux, but only in pre-incubated roots, implying some alternative mechanism of regulation. Sap K+ and Na+ analysis and membrane potential measurements are also consistent with the model that one function of compatible solutes is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, possibly through the enhanced activity of H+-ATPase, controlling voltage-dependent outward-rectifying K+ channels and creating the electrochemical gradient necessary for secondary ion transport processes. These data provide the first direct evidence for regulation of ion fluxes across the plasma membrane by physiologically relevant low concentrations of compatible solutes.     

Characterization of neutralizing monoclonal antibodies recognizing a 15-residues epitope on the spike protein HR2 region of severe acute respiratory syndrome coronavirus (SARS-CoV)

Summary The spike (S) glycoprotein is thought to play a complex and central role in the biology and pathogenesis of SARS coronavirus infection. In this study, a recombinant protein (rS268, corresponding to residues 268–1255 of SARS-CoV S protein) was expressed in Escherichia coli and was purified to near homogeneity. After immunization with rS268, S protein-specific BALB/c antisera and mAbs were induced and confirmed using ELISA, Western blot and IFA. Several BALB/c mAbs were found to be effectively to neutralize the infection of Vero E6 cells by SARS-CoV in a dose-dependent manner. Systematic epitope mapping showed that all these neutralizing mAbs recognized a 15-residues peptide (CB-119) corresponding to residues 1143–1157 (SPDVDLGDISGINAS) that was located to the second heptad repeat (HR2) region of the SARS-CoV spike protein. The peptide CB-119 could specifically inhibit the interaction of neutralizing mAbs and spike protein in a dose-dependent manner. Further, neutralizing mAbs, but not control mAbs, could specifically interact with CB-119 in a dose-dependent manner. Results implicated that the second heptad repeat region of spike protein could be a good target for vaccine development against SARS-CoV.     

International Union of Physiological Sciences teaching workshop, April 7-10, 2005, Pali Mountain, California

Fifty-six physiologists from fourteen countries participated in the sixth International Union of Physiological Sciences Teaching Workshop. The four-day program included a poster session, a debate on integrative versus disciplinary curricula, and interactive lectures on evidence-based education, inquiry laboratories, and student preparation for physiology courses. Participants worked in small groups on one of four topics: developing free web-based laboratory resources, information technology, curriculum planning and design, and other issues in classroom teaching.     

Phylogenetic analyses of mtDNA sequences corroborate taxonomic designations based on cuticular hydrocarbons in subterranean termites

Cuticular hydrocarbons (CHCs) are valuable characters for the analysis of cryptic insect species with few discernible morphological characters. Yet, their use in insect systematics, specifically in subterranean termites in the genus Reticulitermes (Isoptera: Rhinotermitidae), remains controversial. In this paper, we show that taxonomic designations in Reticulitermes from California (USA) suggested in light of differences among CHC phenotypes are corroborated by phylogenetic analyses using mtDNA sequences. Analyses based on CHC phenotypes and supported, in part, by behavioral and ecological differences have suggested the presence of more species than the two currently recognized: R. hesperus Banks and R. tibialis Banks. We analyze a 680 base pair fragment of the mitochondrial DNA cytochrome oxidase (COII) gene from 45 new (21 collection localities) and two previously recorded samples of Reticulitermes from California using parsimony and maximum likelihood methods. Both methods result in trees with highly similar topologies. Bootstrapping indicates support for six clades of Reticulitermes, and corroborates groupings based on cuticular hydrocarbons. One of the clades, R. hesperus, is already recognized in California, while four clades appear to be previously undescribed taxa. Although identification of the final clade is inconclusive, it includes a sample putatively identified as R. tibialis. Therefore, using phylogenetic analyses we corroborate chemical characters used to identify taxa, associate a chemical phenotype with a previously described species, and provide additional support for undescribed taxa of Reticulitermes.     

Nm23H2 facilitates coat protein complex II assembly and endoplasmic reticulum export in mammalian cells

The cytosolic coat protein complex II (COPII) mediates vesicle formation from the endoplasmic reticulum (ER) and is essential for ER-to-Golgi trafficking. The minimal machinery for COPII assembly is well established. However, additional factors may regulate the process in mammalian cells. Here, a morphological COPII assembly assay using purified COPII proteins and digitonin-permeabilized cells has been applied to demonstrate a role for a novel component of the COPII assembly pathway. The factor was purified and identified by mass spectrometry as Nm23H2, one of eight isoforms of nucleoside diphosphate kinase in mammalian cells. Importantly, recombinant Nm23H2, as well as a catalytically inactive version, promoted COPII assembly in vitro, suggesting a noncatalytic role for Nm23H2. Consistent with a function for Nm23H2 in ER export, Nm23H2 localized to a reticular network that also stained for the ER marker calnexin. Finally, an in vivo role for Nm23H2 in COPII assembly was confirmed by isoform-specific knockdown of Nm23H2 by using short interfering RNA. Knockdown of Nm23H2, but not its most closely related isoform Nm23H1, resulted in diminished COPII assembly at steady state and reduced kinetics of ER export. These results strongly suggest a previously unappreciated role for Nm23H2 in mammalian ER export.     

Leishmania donovani amastigotes mobilize organic and inorganic osmolytes during regulatory volume decrease

The protozoan parasite Leishmania donovani encounters large fluctuations in osmolality as it cycles between its insect vector and human host. The flagellated promastigote exhibits regulatory volume responses involving organic and inorganic osmolytes, but little is known about volume regulation in the clinically relevant amastigote that multiplies within the parasitophorous vacuoles of mammalian host cells. Using a combination of morphological, X-ray microanalytical, and biochemical approaches we determined that non-motile amastigotes respond to hypotonic stress with (1) an amino acid and l-alanine-mediated regulatory volume decrease, and (2) a parallel release of Na+, K+, P (presumably as negatively charged phosphates), and subsequently Cl- from cytoplasm and the cell as a whole. In addition P, Zn2+, and subsequently Ca2+ increase in acidocalcisomes as Cl- content declines in this compartment. This evidence is the first to document subcellular translocation of, and thus a potential role for, zinc in volume regulatory responses. These coordinated changes in organic and inorganic osmolytes demonstrate that amastigote subcellular compartments, particularly acidocalcisomes, function in maintaining ionic homeostasis in the response of Leishmania amastigotes to hypo-osmotic stress.     

Ethics guidelines for research with the recently dead

The objective of the multidisciplinary expert Consensus Panel on Research with the Recently Dead (CPRRD) was to craft ethics guidelines for research with the recently dead. The CPRRD recommends that research with the recently dead: (i) receive scientific and ethical review and oversight; (ii) involve the community of potential research subjects; (iii) be coordinated with organ procurement organizations; (iv) not conflict with organ donation or required autopsy; (v) use procedures respectful of the dead; (vi) be restricted to one procedure per day; (vii) preferably be authorized by first-person consent, though both general advance research directives and surrogate consent are acceptable; (viii) protect confidentiality; (ix) not impose costs on subjects' estates or next of kin and not involve payment; (x) clearly explain ultimate disposition of the body.     

A miniaturized nebulization catheter for improved gene delivery to the mouse lung

BACKGROUND: The available methods for administration of gene delivery systems to the lungs of small animals via nebulization have several drawbacks. These include lack of control over the delivered dose and a negative impact on the stability of the formulation. This paper describes a new nebulization catheter device for the administration of plasmid-based gene delivery systems (polyplexes) as aerosols to the mouse lung in vivo. METHODS: The physical stability of naked pDNA and polyplexes formulated with chitosan oligomers and PEI was examined following nebulization with the catheter device. We also examined the in vitro transfection efficiency of the polyplexes recovered after nebulization. Lung distribution and gene expression after administration of the selected gene delivery systems to the mouse lung were also investigated. RESULTS: In contrast to previously described nebulization methods, the structural integrity of the unprotected naked pDNA was maintained following nebulization by the catheter device, which indicates relatively mild nebulization conditions. In addition, the nebulization procedure did not affect the physical stability of the formulated polyplexes. Small volumes of the pDNA aerosol (10-20 microl) were delivered in a highly controlled and reproducible manner. The aerosol droplet size varied with the molecular weight of the polycations. Aerosol delivery via this method resulted in improved lung distribution of pDNA polyplexes and a six-fold increase in the efficiency of gene delivery in vivo over that seen with the commonly used intratracheal instillation method. CONCLUSION: The use of the nebulization catheter device provides a promising alternative for aerosol gene delivery to the mouse lung.     

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post-messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock-induced gene expression, leading to abundant HSP induction in vitro or in vivo.