Nano-scale dynamic recognition imaging on vascular endothelial cells

Combination of high-resolution atomic force microscope topography imaging with single molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Here simultaneous topography and recognition imaging (TREC) was applied to gently fixed microvascular endothelial cells from mouse myocardium (MyEnd) to identify binding sites of vascular endothelial (VE)-cadherin, known to play a crucial role in calcium-dependent, homophilic cell-to-cell adhesion. TREC images were acquired with magnetically oscillating atomic-force microscope tips functionalized with a recombinant VE-cadherin-Fc cis-dimer. The recognition images revealed single molecular binding sites and prominent, irregularly shaped dark spots (domains) with sizes ranging from 10 to 100 nm. These domains arose from a decrease of the oscillation amplitude during specific binding between active VE-cadherin cis-dimers. The VE-cadherin clusters were subsequently assigned to topography features. TREC represents an exquisite method to quickly obtain the local distribution of receptors on cellular surface with an unprecedented lateral resolution of 5 nm.     

Chromosome Separation and Exit from Mitosis in Budding Yeast: Dependence on Growth Revealed by cAMP-Mediated Inhibition

Cell cycle progression of somatic cells depends on net mass accumulation. In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis. By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth. In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed. In S. cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p. The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells. Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells. Thus, the cAMP pathway and APC functionally interact in S. cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation. Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase. PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest. Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells. We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system. A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed.     

Chitin synthase genes in the arbuscular mycorrhizal fungus Glomus versiforme: full sequence of a gene encoding a class IV chitin synthase

Chitin synthase genes of the arbuscular mycorrhizal fungus Glomus versiforme were sought in an investigation of the molecular basis of fungal growth. Three DNA fragments (Gvchs1, Gvchs2 and Gvchs3) corresponding to the conserved regions of distinct chitin synthase (chs) genes were amplified by means of the polymerase chain reaction (PCR) with two sets of degenerate primers. Gvchs1 and Gvchs2 encode two class I chitin synthases, whereas Gvchs3 encodes a class IV chitin synthase. A genomic library was used to obtain the Gvchs3 complete gene (1194 amino acids), which shows a very close similarity to the class IV chitin synthase from Neurospora crassa.     

Fluctuating asymmetry and morphometric variation of hand bones

The major aim of this study was to test three hypotheses: 1) more complex traits of the hand are less prone to developmental insults and therefore show lower fluctuating asymmetry (FA) as compared with simple traits; 2) the manifestation of FA correlates with the variability of the trait (i.e., CV); and 3) FA is an organ-wide property, and therefore a concordance exists between the FA measures of different traits in hand bones. Seventy-two bilateral measurements of hand bones, were made from plain-film radiographs of 365 cadavers. A complex trait was considered as the total length of the three phalanges of a finger and their contiguous metacarpals. Simple traits were considered to be the lengths of individual bone that made up the complex trait. The following results were obtained: 1) on the average simple traits, composing the complex trait, show much higher FA than the corresponding complex trait, but this result is expected if there is no correlation (or low correlation) between FA of simple traits within the complex trait, due to random direction of right-left differences; 2) strong and highly significant correlation was observed between FA and CV of studied traits, regardless of sex and age of individuals; and 3) the majority of FA measurements of hand bones showed no correlation. However, correlations between some sets of FA traits were highly significant. They were interpreted, although not specifically tested, as the result of a tight relationship between traits related not only developmentally but also by active performance of the same function. Am J Phys Anthropol 107:125–136, 1998. © 1998 Wiley-Liss, Inc.     

Mosaic cri-du-chat syndrome in a girl with a mild phenotype

We report on the clinical observation of a girl patient with few signs of cri-du-chat syndrome. The chromosomal analysis in lymphocyte culture showed 46,XX,del(5)(p15.3) in 38% of cells. Psychological tests revealed motor, perceptive and visual-spatial problems, as well as immaturity and emotional dependence. The phoniatric evaluation showed poor vocabulary, difficulty with repeating words or numbers in sequence, and better receptive than expressive language. The spectrographic measurements showed disturbance of fundamental frequency (F₀) in vocal pronunciation. The anatomic findings of the laryngoscopic evaluation were normal, indicating that the voice and speech problems were functional disorders. The present case revealed moderate clinical signs and vocal disturbance associated with a low percentage of 5p-cells and the breakpoint at 5p15.3. The short terminal deletion with a possible loss of the critical region for cat-like cry and the presence of a normal cell line, explain the cry not so typical at birth (weak but not high-pitched), the intermediate values of F₀, and the moderate mental retardation. This case is compared with other mosaic 5p-patients reported in the literature.     

Reactive Oxygen Species Production by Potato Tuber Mitochondria Is Modulated by Mitochondrially Bound Hexokinase Activity

Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of mitochondrial HK activity in PTM reactive oxygen species generation. Mitochondrial HK has a 10-fold higher affinity for glucose (Glc) than for fructose (K_MGlc = 140 μm versus K_MFrc = 1,375 μm). Activation of PTM respiration by succinate led to an increase in hydrogen peroxide (H₂O₂) release that was abrogated by mitochondrial HK activation. Mitochondrial HK activity caused a decrease in the mitochondrial membrane potential and an increase in oxygen consumption by PTM. Inhibition of Glc phosphorylation by mannoheptulose or GlcNAc induced a rapid increase in H₂O₂ release. The blockage of H₂O₂ release sustained by Glc was reverted by oligomycin and atractyloside, indicating that ADP recycles through the adenine nucleotide translocator and F₀F₁ATP synthase is operative during the mitochondrial HK reaction. Inhibition of mitochondrial HK activity by 60% to 70% caused an increase of 50% in the maximal rate of H₂O₂ release. Inhibition in H₂O₂ release by mitochondrial HK activity was comparable to, or even more potent, than that observed for StUCP (S. tuberosum uncoupling protein) activity. The inhibition of H₂O₂ release in PTM was two orders of magnitude more selective for the ADP produced from the mitochondrial HK reaction than for that derived from soluble yeast (Saccharomyces cerevisiae) HK. Modulation of H₂O₂ release and oxygen consumption by Glc and mitochondrial HK inhibitors in potato tuber slices shows that hexoses and mitochondrial HK may act as a potent preventive antioxidant mechanism in potato tubers.Production of reactive oxygen species (ROS) is an unavoidable consequence of aerobic respiration (Chance et al., 1979). The mitochondrial electron transport system (ETS) is the major site of ROS production in mammalian and nonphotosynthesizing plant cells (Puntarulo et al., 1991; Halliwell and Gutteridge, 2007). Depending on the mitochondrial respiratory states, a small portion of the consumable oxygen is partially reduced to generate ROS (Skulachev, 1996; Liu, 1997; Turrens, 1997; Møller, 2001; Considine et al., 2003; Smith et al., 2004). In plants, the monoelectronic reduction of oxygen by ETS leads to the production of superoxide radicals (O₂^·−) that can be dismutated by SOD, producing hydrogen peroxide (H₂O₂), and further decomposed by catalase and/or ascorbate-glutathione peroxidase cycles (Møller, 2001). An imbalance between the ROS production and antioxidant defenses can lead to an oxidative stress condition. Increased levels of ROS may be a consequence of the action of plant hormones, environmental stress, pathogens, or high levels of sugars and fatty acids (Bolwell et al., 2002; Couée et al., 2006; Gechev et al., 2006; Liu et al., 2007; Rhoads and Subbaiah, 2007). These conditions may lead to storage deterioration or impairment of seedling growth decreasing on crop yield. To avoid the harmful accumulation of ROS or to fine tune the steady-state levels of ROS, various enzymatic systems control the rate of ROS production in mitochondria (Schreck and Baeuerle, 1991; Møller, 2001).Mitochondrial ROS production is highly dependent on the membrane potential (ΔΨ_m) generated by the proton gradient formed across the inner mitochondrial membrane. High ΔΨ_m was shown to stimulate ROS production when the ETS is predominantly in a reduced state (i.e. when NADH, FADH₂, and O₂ are present in abundance but ADP or Pi levels are low). This condition is reached in resting metabolic states after a full oxidation of Glc or fatty acids. Stimulating electron flow by decreasing ΔΨ_m, either by the use of uncouplers or by coupling respiration to ATP synthesis, slows the ROS generation rate (Boveris and Chance, 1973; Korshunov et al., 1997). It has been observed that in isolated potato tuber (Solanum tuberosum) mitochondria (PTM) the uncoupling protein (referred to as PUMP in plants, or UCP in animals) causes a small decrease in ΔΨ_m when this proton carrier protein is activated by the presence of anionic fatty acids, a condition that blocks ROS generation (Vercesi et al., 1995, 2006). Nucleotides, such as ATP, antagonize this effect (Considine et al., 2003; Vercesi et al., 2006). On the other hand, fluctuations in free hexose levels due to environmental or developmental conditions (Morrell and ap Rees, 1986; Geigenberger and Stitt, 1993; Renz and Stitt, 1993) lead to variations in the oxygen consumption rate in heterotrophic tissues of plant (Brouquisse et al., 1991; Dieuaide et al., 1992). As a result, ROS-producing pathways may be either stimulated or repressed (Couée et al., 2006). Unlike PUMP activity, which is activated by an excess of free fatty acids, a specific mechanism for mitochondrial ROS production caused by an excess of hexose remains elusive.The metabolism of free hexoses begins by their phosphorylation in a reaction catalyzed by the hexokinase (HK):HK is a ubiquitous enzyme found in many organisms. In plants, the binding mechanism of HK to the outer mitochondrial membrane is not fully established, but some reports indicate that it may differ considerably from those properties described for mammal cells (Dry et al., 1983; Miernyk and Dennis, 1983; Rezende et al., 2006). It has been shown that in several mature and developing plant tissues, multiple HK isoforms are expressed with different kinetic properties and subcellular localizations. The HKs are found in cytosol, bound to the mitochondrial membrane, or in stroma of plastids in plant cells (Miernyk and Dennis, 1983; Galina et al., 1995; Damari-Weissler et al., 2007). Beyond its obvious role in glycolysis regulation, HK activity may also function as a sugar sensor, triggering a signal transduction pathway in plants (Rolland et al., 2006).In mammals, HK types I and II are associated with the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT). These associations were found in tissues with a high energy demand, such as heart, brain, and tumor cells (Arora and Pedersen, 1988; BeltrandelRio and Wilson, 1992; Wilson, 2003). In addition, recent evidence in mammalian cells has shown that binding of HK to VDAC located at the outer mitochondrial membrane is somehow involved in the protection against proapoptotic stimuli (Nakashima et al., 1986; Gottlob et al., 2001; Vander Heiden et al., 2001; Pastorino et al., 2002; Cesar and Wilson, 2004). Similar observations were reported for tobacco (Nicotiana tabacum) plant mitochondrial HK (mt-HK; Kim et al., 2006). However, it has been shown that drugs such as the fungicide clotrimazole and the anesthetic thiopental, which promptly disrupt the association between mt-HK and VDAC in mammalian mitochondria, are unable to promote this effect in maize (Zea mays) root mitochondria (Rezende et al., 2006). These observations suggest a different type of association of mt-HK with plant mitochondria. The binding of mt-HK with mitochondria in many plants involves a common N-terminal hydrophobic membrane anchor domain of about 24 amino acids that is related to the membrane targeting, but the exact mechanism of association is unknown (Damari-Weissler et al., 2007).Recently, our group demonstrated that mt-HK activity plays a key preventive antioxidant role by reducing mitochondrial ROS generation through a steady-state ADP recycling mechanism in rat brain neurons. The mitochondrial ADP recycling leads to a decrease in the ΔΨ_m coupled to the synthesis of ATP by oxidative phosphorylation (da-Silva et al., 2004; Meyer et al., 2006).Although plant HK is recognized to fulfill a catalytic function, the role of mt-HK activity in the regulation of both mitochondrial respiration and ROS production in plants is unknown. Recently, an authentic HK activity was detected in PTM (Graham et al., 2007) and its involvement in potato tuber glycolysis suggested, but its involvement in PTM ROS generation was not explored. We then raise the hypothesis that HK bound to PTM would contribute to produce a steady-state ADP recycling that regulates ROS formation. However, whether this association is capable of controlling the rate of ROS generation in plant mitochondria is unknown. Here, we aim to investigate the role of mt-HK activity in PTM physiology. The data indicate that mt-HK activity plays a key role as a regulator of ROS levels in respiring plant tissues exposed to high hexose levels.     

Helicobacter Pylori's Plasticity Zones Are Novel Transposable Elements

Background

Genes present in only certain strains of a bacterial species can strongly affect cellular phenotypes and evolutionary potentials. One segment that seemed particularly rich in strain-specific genes was found by comparing the first two sequenced Helicobacter pylori genomes (strains 26695 and J99) and was named a “plasticity zone”.

Principal Findings

We studied the nature and evolution of plasticity zones by sequencing them in five more Helicobacter strains, determining their locations in additional strains, and identifying them in recently released genome sequences. They occurred as discrete units, inserted at numerous chromosomal sites, and were usually flanked by direct repeats of 5′AAGAATG, a sequence generally also present in one copy at unoccupied sites in other strains. This showed that plasticity zones are transposable elements, to be called TnPZs. Each full length TnPZ contained a cluster of type IV protein secretion genes (tfs3), a tyrosine recombinase family gene (“xerT”), and a large (≥2800 codon) orf encoding a protein with helicase and DNA methylase domains, plus additional orfs with no homology to genes of known function. Several TnPZ types were found that differed in gene arrangement or DNA sequence. Our analysis also indicated that the first-identified plasticity zones (in strains 26695 and J99) are complex mosaics of TnPZ remnants, formed by multiple TnPZ insertions, and spontaneous and transposable element mediated deletions. Tests using laboratory-generated deletions showed that TnPZs are not essential for viability, but identified one TnPZ that contributed quantitatively to bacterial growth during mouse infection and another that affected synthesis of proinflammatory cytokines in cell culture.

Conclusions

We propose that plasticity zone genes are contained in conjugative transposons (TnPZs) or remnants of them, that TnPZ insertion is mediated by XerT recombinase, and that some TnPZ genes affect bacterial phenotypes and fitness.     

In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol.