Ethylene enhances water transport in hypoxic aspen

Water transport was examined in solution culture grown seedlings of aspen (Populus tremuloides) after short-term exposures of roots to exogenous ethylene. Ethylene significantly increased stomatal conductance, root hydraulic conductivity (L(p)), and root oxygen uptake in hypoxic seedlings. Aerated roots that were exposed to ethylene also showed enhanced L(p). An ethylene action inhibitor, silver thiosulphate, significantly reversed the enhancement of L(p) by ethylene. A short-term exposure of excised roots to ethylene significantly enhanced the root water flow (Q(v)), measured by pressurizing the roots at 0.3 MPa. The Q(v) values in ethylene-treated roots declined significantly when 50 microM HgCl(2) was added to the root medium and this decline was reversed by the addition of 20 mM 2-mercaptoethanol. The results suggest that the response of Q(v) to ethylene involves mercury-sensitive water channels and that root-absorbed ethylene enhanced water permeation through roots, resulting in an increase in root water transport and stomatal opening in hypoxic seedlings.     

Involvement of the fungal nuclear migration gene nudC human homolog in cell proliferation and mitotic spindle formation.

Essential genes which are required for normal nuclear migration and play a role in developmental processes have been isolated from model genetic organisms. One such gene is nudC (nuclear distribution C), which is required for positioning nuclei in the cytoplasm of the filamentous fungus Aspergillus nidulans and for normal colony growth. This gene is highly conserved, structurally and functionally, throughout evolution and the human homolog, HnudC, has been cloned. To study the function of nudC in higher eukaryotic cells, HnudC was downregulated by developing triple ribozyme constructs, consisting of two cis-acting ribozymes which liberate an internal trans-acting ribozyme targeted to HnudC. Efficient cleavage sites in HnudC mRNA were identified using a library selection technique and HnudC-targeted internal ribozymes were cloned into a triple ribozyme cassette. Triple ribozyme constructs were subcloned into an ecdysone-inducible expression vector and stably transfected into human embryonic 293 cells. Muristerone A induced expression of the HnudC ribozyme and produced specific reduction of HnudC mRNA. Downregulation of HnudC mRNA resulted in significant inhibition of cell proliferation in clones expressing the HnudC-targeted triple ribozyme, which was not observed in uninduced cells or cells transfected with vector alone. In induced cultures, many mitotic cells demonstrated defects in spindle architecture during mitosis. The most common defect observed was multiple mitotic spindle poles rather than the expected bipolar structure. These data demonstrate the fundamental importance of HnudC in eukaryotic cell proliferation and a functional role for HnudC in spindle formation at mitosis.     

Sequence-specific recognition of collagen triple helices by the collagen-specific molecular chaperone HSP47

HSP47 is a molecular chaperone that plays an unknown role during the assembly and transport of procollagen. Our previous studies showed that, unlike most chaperones, HSP47 interacts with a correctly folded substrate. We suggested that HSP47 either stabilizes the correctly folded collagen helix from heat denaturation or prevents lateral aggregation prior to its transport from the endoplasmic reticulum. In this study we have addressed the role of triple helix stability in the binding of HSP47 to procollagen by expressing procollagen molecules with differing thermal stabilities and analyzing their ability to interact with HSP47 within the endoplasmic reticulum. Our results show that HSP47 interacts with thermostable procollagen molecules, suggesting that helix stabilization is not the primary function of HSP47 and that the interaction of HSP47 with procollagen depends upon the presence of a minimum of one Gly-X-Arg triplet within the triple helical domain. Interestingly, procollagen chains containing high proportions of stabilizing triplets formed triple helices and interacted with HSP47 even in the absence of proline hydroxylation, demonstrating that recognition does not depend upon this modification. Our results support the view that HSP47 functions early in the secretory pathway by preventing the lateral aggregation of procollagen chains.     

Calmodulin-dependent cyclic nucleotide phosphodiesterase in an experimental rat model of cardiac ischemia-reperfusion

In the present study, we investigated the activity and expression of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) and the effects of calpains in rat heart after ischemia and reperfusion. Immunohistochemical studies indicated that CaMPDE in normal heart is localized in myocardial cells. Rat ischemic heart showed a decrease in CaMPDE activity in the presence of Ca2+ and calmodulin; however, in ischemic-reperfusion tissue a progressive increase in Ca2+ and calmodulin-independent cyclic nucleotide phosphodiesterase (CaM-independent PDE) activity was observed. Perfusion of hearts with cell-permeable calpain inhibitor suppressed the increase of Ca2+ and CaM-independent PDE activity. Protein expression of CaMPDE was uneffected by hypoxic injury to rat myocardium. The purified heart CaMPDE was proteolyzed by calpains into a 45 kDa immunoreactive fragment in vitro. Based on these results, we propose that hypoxic injury to rat myocardium results in the generation of CaM-independent PDE by calpain mediated proteolysis, allowing the maintenance of cAMP concentrations within the physiological range.     

A database for the provisional identification of species using only genotypes: web-based genome profiling

Background  

For a long time one could not imagine being able to identify species on the basis of genotype only as there were no technological means to do so. But conventional phenotype-based identification requires much effort and a high level of skill, making it almost impossible to analyze a huge number of organisms, as, for example, in microbe-related biological disciplines. Comparative analysis of 16S rRNA has been changing the situation, however. We report here an approach that will allow rapid and accurate phylogenetic comparison of any unknown strain to all known type strains, enabling tentative assignments of strains to species. The approach is based on two main technologies: genome profiling and Internet-based databases.     

Elevated end-tidal carbon dioxide during thoracoscopy

We report a case of a patient with right pleural effusion who, during video-assisted thoracoscopy for biopsy and diagnosis, developed a sudden rise in end-tidal carbon dioxide (EtCO2) after a small tear of the lung tissue. The purpose of this case report is to highlight this rare complication and to discuss possible alternative differential diagnosis.     

Protective effects of nicaraven,a new hydroxyl radical scavenger,on the endothelial dysfunction after exposure of pig coronary artery to hydroxyl radicals

Recently, we have reported that a new synthetic compound, 1,2bis(nicotinamido)-propane (nicaraven), improved cardiac function following preservation and reperfusion. In this study, we investigated the efficacy of nicaraven as a radical scavenger by using an in vitro model of oxidative stress, to clarify mechanisms of the protective effect of this new compound on reperfusion injury in rat heart. Ring segments of epicardial right coronary arteries (RCA) of pig were suspended in organ chambers and exposed to hydroxyl radicals (·OH), generated (by two different systems ) by 0.28 mM FeSO₄/0.28 mM H₂O₂ and DHF/Fe³⁺-ADP (2.4 mM, 43 nM, and 1.56 uM, respectively) to the bathing solution for 60 min. Prior exposure of the coronary arteries to ·OH significantly produced right-ward shift of the dose-response curves of the bradykinin-induced endothelium-dependent relaxations (an increase in the ED₅₀ value for bradykinin by 4.37 and 1.98 times than control in two different ·OH generating systems, respectively), but did not affect the maximum relaxation responses. The presence of nicaraven (10^-4 and 10^-5 M) in the ·OH generating system, shifted the dose-response curves to bradykinin to the control level, suggesting a significant hydroxyl radical scavenging effect of the drug. These results indicate that nicaraven, a new hydroxyl radical scavenger, exhibits a protective effect on hydroxyl radicalinduced endothelial dysfunctions of pig coronary artery.     

Characterization of the Human Homologue of the Yeast Spc98p and Its Association with γ-Tubulin

A trimeric complex formed by Tub4p, the budding yeast γ-tubulin, and the two spindle pole body components, Spc98p and Spc97p, has recently been characterized in Saccharomyces cerevisiae. We reasoned that crucial functions, such as the control of microtubule nucleation, could be maintained among divergent species. SPC98-related sequences were searched in dbEST using the BLASTN program. Primers derived from the human expressed sequence tag matching SPC98 were used to clone the 5′ and 3′ cDNA ends by rapid amplification of cDNA ends (RACE)-PCR. The human Spc98 cDNA presents an alternative splicing at the 3′ end. The deduced protein possesses 22% identity and 45% similarity with the yeast homologue. We further report that the human Spc98p, like γ-tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic complexes. Sucrose gradient sedimentation of the cytosolic fraction and immunoprecipitation experiments demonstrate that both γ-tubulin and HsSpc98p are in the same complex. Interestingly, Xenopus sperm centrosomes, which are incompetent for microtubule nucleation before their activation in the egg cytoplasm, were found to contain similar amounts of both Spc98p and γ-tubulin to human somatic centrosomes, which are competent for microtubule nucleation. Finally, affinity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in microinjected cells, suggesting that this novel protein is indeed required for the nucleation reaction.     

Genome analysis technologies: towards species identification by genotype.

Traditional identification of species has been based on phenotypic traits, although it is clear that, theoretically, genotype-based classification is more accurate. This is especially the case for microorganisms which possess less identifiable traits and are more easily influenced by environment. Therefore, technology that allows identification of species based on genotype is highly desirable. Whole genome sequencing can provide a sufficient amount of information and can be determinative for this purpose but is very impractical for routine use. Thus, a competent technology is needed that allows a reproducible reduction in the amount of information required about a whole genome, while still providing sufficiently accurate identification. It is almost imperative for such a technology to be of a high cost-performance and of easy handling. Universality and portability are also strongly desired. Based on these criteria, the current state of genome analysis technologies are reviewed. Among various methodologies discussed here, amplified fragment length polymorphism (AFLP), genome profiling (GP) and microarrays are the subject of particular attention. As species identification is a base for most fields of biology including microbiology, ecology, epidemiology and for various biotechnologies, it is of paramount importance to establish a more efficient, easily handled and more objective methodology, in parallel with conventional phenotype-based methodologies. GP is currently considered to have the most optimal nature for identification of species since it can reproducibly reduce a huge amount of genome information to a manageable size by way of random polymerase chain reaction and can extract a sufficient amount of information for species identification from the DNA fragments thus profiled by temperature gradient gel electrophoresis. The potential ability of DNA microarrays for this purpose is also discussed and promises much for the future.