Stomatal sensitivity to vapour pressure deficit of temperate and tropical evergreen rainforest trees of Australia

Although rainforests of eastern Australia grow in regions of high precipitation, there is a shift from a summer dry season in the temperate south to a winter dry season in the tropical north. Therefore, rainforest trees of eastern Australia provide an opportunity to investigate stomatal sensitivity of mesic trees to vapour pressure deficit (VPD) along a gradient in seasonality of precipitation. Eight rainforest canopy tree species were selected to cover the latitudinal range of rainforests in eastern Australia. Seedlings of these species were grown for a year in glasshouses under ambient conditions or at low VPD and water vapour exchange of leaves was measured during summer. Tropical species, which experience summer-dominant precipitation, showed higher stomatal sensitivities to VPD than temperate species, which experience winter-dominant precipitation. Growing plants under a low VPD increased stomatal sensitivity to increasing VPD in most species. The high stomatal sensitivity to VPD of the tropical species is consistent with the infrequent water stress experienced during their growing season and suggests a high susceptibility to water deficits. In contrast, temperate species may use other mechanisms to maintain photosynthesis under the relatively drier conditions of the temperate growing season.     

Author Index Volume 71 2004

Authors Index

Author Index Volume 71 2004     

Applications of proteomic methodologies to human pregnancy research: a growing gestation approaching delivery?

Maternal and perinatal morbidity and mortality rates are significantly higher in pregnancies complicated by preterm labor, pre-eclampsia and fetal growth restriction. Decades of research have not translated into a clear understanding of the underlying pathophysiologies or effective identification of women who are at high risk of developing these complications. Often the severity of these diseases does not correlate with the clinical symptoms, and current diagnostic methods are unable to accurately predict the conditions prior to clinical presentation. Though several potential markers have been proposed for each of these disorders, to date none have proven clinical utility. Emerging proteomic technology is only beginning to be employed in pregnancy research. A comprehensive analysis of gestational tissues can be expected to contribute to the elucidation of the complex molecular mechanisms of pregnancy and related complications. Comparison of the expression profiles of normal and pathogenic tissues and biofluids may also highlight novel candidate marker proteins that have so far remained undetected. More interestingly, rapidly evolving technologies using sophisticated bioinformatic tools are demonstrating their potential in disease diagnostics by using overall protein profiles to detect diseases. The clinical significance of these methodological advances is enormous. Early diagnosis together with improved understanding of underlying molecular mechanisms can enhance outcomes and increase effective management and therapeutic options.     

Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes

The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.     

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.     

Evidence that acute serotonergic activation potentiates the locomotor-stimulating effects of corticotropin-releasing hormone in juvenile chinook salmon (Oncorhynchus tshawytscha)

The present study investigated whether the serotonergic system is involved in mediating the behavioral effects of corticotropin-releasing hormone (CRH) in juvenile spring chinook salmon, Oncorhynchus tshawytscha. An intracerebroventricular (ICV) injection of CRH induced hyperactivity. The effect of CRH was potentiated in a dose-dependent manner by the concurrent administration of the serotonin (5-HT) selective reuptake inhibitor fluoxetine. However, administration of fluoxetine alone had no effect on locomotor activity, suggesting that the locomotor-stimulating effect of CRH is mediated by the activation of the serotonergic system. Conversely, ICV injections of the 5-HT(1A) receptor antagonist NAN-190 attenuated the effect of CRH on locomotor activity when given in combination with CRH but had no effect when administered alone. These results provide the first evidence to support the hypothesis that the effect of CRH on locomotor activity in teleosts is mediated by activating the serotonergic system.     

Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure

Desmosomes are intercellular junctions in which cadherin cell adhesion molecules are linked to the intermediate filament (IF) system. Desmoplakin is a member of the plakin family of IF-binding proteins. The C-terminal domain of desmoplakin (DPCT) mediates binding to IFs in desmosomes. The DPCT sequence contains three regions, termed A, B and C, consisting of 4.5 copies of a 38-amino acid repeat motif. We demonstrate that these regions form discrete subdomains that bind to IFs and report the crystal structures of domains B and C. In contrast to the elongated structures formed by other kinds of repeat motifs, the plakin repeats form a globular structure with a unique fold. A conserved basic groove found on the domain may represent an IF-binding site.     

Hormone trajectories leading to human birth

The mechanisms regulating human parturition and labor remain unknown. This ignorance is expensive as preterm birth is responsible for 70% of neonatal mortality and 50% of cerebral palsy. Methods for the prediction of preterm birth and treatments for women in preterm labor have poor efficacy reflecting our limited knowledge of the mechanisms involved. Recent research has supported the view that parturition is a cascade of events that commences early in pregnancy and involves the mother, fetus, placenta, membranes, cervix and myometrium. Although a number of the key hormones and proteins involved have been identified, the relationships between these factors in time and tissues remain unclear. Placental production of Corticotropin-releasing hormone (CRH) is proposed as an early event regulating the cascade of events. Central to the onset of parturition will be a mechanism for progesterone withdrawal and estrogen activation in human. Two forms of progesterone receptor with opposing actions exist in the human myometrium. Progesterone receptor A (PR-A) is a dominant negative repressor of progesterone receptor B (PR-B). Preliminary studies strongly support the hypothesis that the onset of human parturition is initiated by rising concentrations of PR-A in the myometrium.