Evidence for intracellular spatial separation of hexokinases and fructokinases in tomato plants

Four hexokinase (LeHXK1–4) and four fructokinase (LeFRK1–4) genes were identified in tomato plants. Previous GFP fusion studies indicate that the gene product of LeHXK3 is associated with the mitochondria while that of LeHXK4 is located within plastids. In this study we found that the enzyme encoded by the fructokinase gene LeFRK3 is also located within plastids. The presence of LeFrk3 enzyme in plastids raises the question of the origin of fructose in these organelles. The other three FRKs enzymes, LeFrk1&2&4, are located in the cytosol. Unlike LeFrk1&2&4, the two additional HXKs, LeHxk1&2, share a common membrane anchor domain and are associated with the mitochondria similar to LeHxk3. The difference in the locations of the cytoplasmic FRK and HXK isozymes suggests that glucose phosphorylation is confined to defined special intracellular localizations while fructose phosphorylation is less confined.Contribution from the Agriculture Research Organization, The Volcani Center, Bet Dagan, Israel, No. 126/2006 series.     

Alteration of the menstrual cycle in baboons placed on tethering devices and moved to individual housing--a stress model for a follicular phase defect

BACKGROUND: During an attempt to identify endocrine characteristics in the baboon that would more precisely predict ovulatory status for assisted reproductive techniques, we observed severe alterations in the menstrual cycle length upon introducing an environmental stress. This environmental stress involved moving animals from their baseline gang cage environment to individual indoor caging and placing them on a tethering apparatus. METHODS: Five adult female baboons were followed for changes in sex skin indicative of menstrual cycle timing and move from outdoor gang gages to individual indoor cages during the early follicular phase of their cycle. A tether device including a surgically implanted cannula was then installed to facilitate daily blood draws without sedation. Radioimmuonoassays were performed to monitor serum estradiol levels and lapraroscopic surveillance was used to confirm time of ovulation. RESULTS: Complete data sets were collected from four of the female baboons. In each case, a prolongation of the menstrual cycle was noted either during the cycle during which the females were moved to indoor caging or during the cycle immediately following the move. This prolongation was isolated to the follicular phase of the affected cycle. CONCLUSIONS: We conclude that otherwise normal handling procedures, including movement to new caging, and/or installation of a tether device, can impart a stress effect on reproductively cycling adult female baboons, such that folliculogenesis is delayed.     

Pyridazines. Part XXIX: synthesis and platelet aggregation inhibition activity of 5-substituted-6-phenyl-3(2H)-pyridazinones. Novel aspects of their biological actions

A series of 6-phenyl-3(2H)-pyridazinones with a diverse range of substituents in the 5-position have been prepared and evaluated in the search for new antiplatelet agents. A significant dependence of the substituent on the inhibitory effect has been observed. The pharmacological study of these compounds confirms that modification of the chemical group at position 5 of the 6-phenyl-3(2H)-pyridazinone system influences both variations in the antiplatelet activity and the mechanism of action.     

Crystal structure of a human rhinovirus that displays part of the HIV-1 V3 loop and induces neutralizing antibodies against HIV-1

We report the 2.7 A resolution structure of a chimeric rhinovirus, MN-III-2, that displays part of the HIV-1 gp120 V3 loop and elicits HIV-neutralizing antibodies. The V3 loop insert is dominated by two type I beta turns. The structures of two adjacent tripeptides resemble those of analogous segments in three Fab/V3 loop peptide complexes. Although two of the three corresponding antibodies bind and neutralize MN-III-2 well, only one of the three can bind without significant rearrangement. These results suggest that the V3 loop insert: (1) can share some local conformational similarity to V3 loop sequences presented on different structural frameworks; (2) must be able to adopt multiple conformations, even in a relatively constrained environment; and (3) may mimic the conformational variability of the epitope on HIV-1, increasing the likelihood of eliciting appropriate neutralizing immune responses.     

Developmental endocrinology of the reproductive axis in the chicken embryo

In mammals, the phenotype of the homogametic sex develops in the (relative) absence of steroids and the phenotype of the heterogametic sex is imposed by the early action of steroids. In contrast, the heterogametic sex in avian species is the female and the presence of estrogens and their receptors plays a crucial role in female sexual differentiation. The time- and sex-dependent expression of enzymes involved in steroidogenesis which determine the ratio of androgens/estrogens produced by the gonads has been extensively investigated during the last 5-6 years. These results all show that the lack of estrogen synthesis in the male appears to be due to the extremely low levels of 17beta-hydroxysteroid dehydrogenase and P450aromatase expression. In females, extensive expression of the aromatase gene (around day 5-6 of incubation), leading to estrogen synthesis, and specific expression of the estrogen receptor-mRNA in the left gonad results in the development of a functional left ovary. Other sex differences can be found in the expression of the inhibin subunit genes in gonads of chicken embryos and in circulating concentrations of inhibin, follicle stimulating hormone (FSH) and steroids. Sex reversal attempts have been made by varying incubation temperatures, by using anti-estrogens, androgens, aromatase inhibitors and synthetic steroids. In ovo administration of a sex steroid hormone or an inhibitor of endogenous sex steroid synthesis can cause phenotypical sex reversal. All these experiments show that the development of gonads in birds is very sensitive to changes in the embryonic hormonal environment, sometimes resulting in changes of postnatal reproduction and even growth.     

Identification of the protein kinases that activate the E3 ubiquitin ligase Pellino 1 in the innate immune system

The E3 ubiquitin ligase Pellino 1 can be interconverted between inactive and active forms by a reversible phosphorylation mechanism. In vitro, phosphorylation and activation can be catalysed by either the IRAKs [IL (interleukin)-1-receptor-associated kinases] IRAK1 and IRAK4, or the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase}-related kinases [IKK? and TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1)]. In the present study we establish that IRAK1 is the major protein kinase that mediates the IL-1-stimulated activation of Pellino 1 in MEFs (mouse embryonic fibroblasts) or HEK (human embryonic kidney)-293 cells, whereas the IKK-related kinases activate Pellino 1 in TNFα-stimulated MEFs. The IKK-related kinases are also the major protein kinases that activate Pellino 1 in response to TLR (Toll-like receptor) ligands that signal via the adaptors MyD88 (myeloid differentiation primary response gene 88) and/or TRIF [TIR (Toll/IL-1 receptor) domain-containing adaptor protein inducing interferon β]. The present studies demonstrate that, surprisingly, the ligands that signal via MyD88 do not always employ the same protein kinase to activate Pellino 1. Our results also establish that neither the catalytic activity of IRAK1 nor the activation of Pellino 1 is required for the initial transient activation of NF-κB and MAPKs (mitogen-activated protein kinases) that is triggered by IL-1 or TNFα in MEFs, or by TLR ligands in macrophages. The activation of Pellino 1 provides the first direct readout for IRAK1 catalytic activity in cells.     

The Influence of Sequential Male Courtship Behaviors on Courtship Success and Duration in a Terrestrial Salamander,Plethodon shermani

Most organisms use multiple cues across many modalities to assess potential mates. Salamanders in the genus Plethodon have an elaborate courtship display, yet previous studies primarily have concentrated on the influence of olfactory cues on courtship success. In this study, we tested whether two different cues (one chemical and one visual) affected courtship success and duration in the red‐legged salamander, Plethodon shermani. By staging and observing over 180 courtship trials, we found that ‘foot‐dancing’, a male visual cue performed in the earliest stages of courtship, significantly increased the likelihood that a male and female would progress to subsequent stages of courtship and, thus, the likelihood of insemination. In contrast, delivery of biologically relevant levels of a non‐volatile pheromone (produced in a large gland on the male's chin) during courtship trials did not increase overall insemination rate success, but did significantly reduce the duration of one of the later stages of courtship, tail‐straddling walk. Thus, we propose that early use of visual cues in Plethodon courtship, may (1) increase the detectability of the male message by increasing the range at which the male can be perceived by the female and (2) indicate the context of the male's approach and subsequent behaviors. In short, this study draws attention to the need to examine behavioral traits affecting courtship success at multiple stages and across several sensory modalities.     

RHO1 and RHO2 share partially overlapping functions in the regulation of cell wall integrity and hyphal polarity in Neurospora crassa

Rho proteins are key regulators of cellular morphogenesis, but their function in filamentous fungi is poorly understood. By generating conditional rho‐1 mutants, we dissected the function of the essential GTPase RHO1 in cell polarization and maintenance of cell wall integrity in Neurospora crassa. We identified NCU00668/RGF1 as RHO1‐specific exchange factor, which controls actin organization and the cell wall integrity MAK1 MAP kinase pathway through the direct interaction of active RHO1 with the formin BNI1 and PKC1 respectively. The activity of RGF1 is controlled by an intramolecular interaction of its DEP and GEF domains that blocks the activation of the GTPase. Moreover, the N‐terminal region including the DEP domain of RGF1 interacts with the plasma membrane sensor NCU06910/WSC1, potentially to activate the cell wall integrity pathway. RHO1 also functions as regulatory subunit of the glucan synthase. N. crassa possesses a second GTPase, RHO2, that is highly homologous to RHO1. RHO2 is of minor importance for growth and does not interact with BNI1. Conditional rho‐1;rho‐2 double mutants display strong synthetic growth and cell polarity defects. We show that RHO2 does not regulate glucan synthase activity and the actin cytoskeleton, but physically interacts with PKC1 to regulate the cell wall integrity pathway.     

CCN3/NOV small interfering RNA enhances fibrogenic gene expression in primary hepatic stellate cells and cirrhotic fat storing cell line CFSC

Nephroblastoma overexpressed gene encodes a matricellular protein (CCN3/NOV) of the CCN family, comprising CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). CCN proteins are involved in the regulation of mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration in multiple cell types. Compared to CCN2/CTGF, known as a profibrotic protein, the biological role of CCN3/NOV in liver fibrosis remains obscure. In this study we showed ccn3/nov mRNA to increase dramatically following hepatic stellate cell activation, reaching peak levels in fully transdifferentiated myofibroblasts. In models of experimental hepatic fibrosis, CCN3/NOV increased significantly at the mRNA and protein levels. CCN3/NOV was found mainly in non-parenchymal cells along the areas of tissue damage and repair. In the bile-duct ligation model, CCN3/NOV was localized mainly along portal tracts, while the repeated application of carbon tetrachloride resulted in CCN3/NOV expression mainly in the centrilobular areas. In contrast to CCN2/CTGF, the profibrotic cytokines platelet-derived growth factor-B and -D as well as transforming growth factor-β suppressed CCN3/NOV expression. In vitro, CCN3/NOV siRNA attenuated migration in the cirrhotic fat storing cell line CFSC well in line with in vivo findings that various types of cells expressing CCN3/NOV migrate into the area of tissue damage and regeneration. The suppression of CCN3/NOV enhanced expression of profibrotic marker proteins, such as α-smooth muscle actin, collagen type I, fibronectin, CCN2/CTGF and TIMP-1 in primary rat hepatic stellate cells and in CFSC. We further found that adenoviral overexpression of CCN2/CTGF suppressed CCN3/NOV expression, while the overexpression of CCN3/NOV as well as the suppression of CCN3/NOV by targeting siRNAs both resulted in enhanced CCN2/CTGF expression. These results indicate the complexity of CCN actions that are far beyond the classic Yin/Yang interplay.