Phase Variation in Xenorhabdus nematophilus

Xenorhabdus nematophilus is a symbiotic bacterium that inhabits the intestine of entomopathogenic nematodes. The bacterium-nematode symbiotic pair is pathogenic for larval-stage insects. The phase I cell type is the form of the bacterium normally associated with the nematode. A variant cell type, referred to as phase II, can form spontaneously under stationary-phase conditions. Phase II cells do not elaborate products normally associated with the phase I cell type. To better define phase variation in X. nematophilus, several strains (19061, AN6, F1, N2-4) of this bacterium were analyzed for new phenotypic traits. An analysis of pathogenicity in Manduca sexta larvae revealed that the phase II form of AN6 (AN6/II) was significantly less virulent than the phase I form (AN6/I). The variant form of N2-4 was also avirulent. On the other hand, F1/II and 19061/II were as virulent as the respective phase I cells. Strain 19061/II was found to be motile, and AN6/II regained motility when the bacteria were grown in low-osmolarity medium. In contrast, F1/II remained nonmotile. The phase II cells did not produce the outer membrane protein, OpnB, that is normally induced during the stationary phase. Both phase I and phase II cells were able to support nematode growth and development. These findings indicate that while certain phenotypic traits are common to all phase II cells, other characteristics, such as virulence and motility, are variable and can be influenced by environmental conditions.     

Different Desensitization Patterns for Sensory and Vascular TRPV1 Populations in the Rat: Expression,Localization and Functional Consequences

Background and purpose

TRPV1 is expressed in sensory neurons and vascular smooth muscle cells, contributing to both pain perception and tissue blood distribution. Local desensitization of TRPV1 in sensory neurons by prolonged, high dose stimulation is re-engaged in clinical practice to achieve analgesia, but the effects of such treatments on the vascular TRPV1 are not known.

Experimental approach

Newborn rats were injected with capsaicin for five days. Sensory activation was measured by eye wiping tests and plasma extravasation. Isolated, pressurized skeletal muscle arterioles were used to characterize TRPV1 mediated vascular responses, while expression of TRPV1 was detected by immunohistochemistry.

Key results

Capsaicin evoked sensory responses, such as eye wiping (3.6±2.5 versus 15.5±1.4 wipes, p<0.01) or plasma extravasation (evans blue accumulation 10±3 versus 33±7 µg/g, p<0.05) were reduced in desensitized rats. In accordance, the number of TRPV1 positive sensory neurons in the dorsal root ganglia was also decreased. However, TRPV1 expression in smooth muscle cells was not affected by the treatment. There were no differences in the diameter (192±27 versus 194±8 µm), endothelium mediated dilations (evoked by acetylcholine), norepinephrine mediated constrictions, myogenic response and in the capsaicin evoked constrictions of arterioles isolated from skeletal muscle.

Conclusion and implications

Systemic capsaicin treatment of juvenile rats evokes anatomical and functional disappearance of the TRPV1-expressing neuronal cells but does not affect the TRPV1-expressing cells of the arterioles, implicating different effects of TRPV1 stimulation on the viability of these cell types.     

Carbamoyloximes as novel non-competitive mGlu5 receptor antagonists

Hit-to-lead optimization of a HTS hit led to new carbamoyloxime derivatives. After identification of an advanced hit (8d) the CYP enzyme inhibitory activity of this class of compounds was successfully eliminated. Systematic exploration of different parts of the advanced hit led us to some promising lead compounds with mGluR5 affinities comparable to that of MPEP.     

The intracellular dissipation of cytosolic calcium following glucose re-addition to carbohydrate depleted Saccharomyces cerevisiae

Glucose re-addition to carbohydrate starved yeast cells leads to a transient elevation of eytosolic calcium (TECC). Concomitantly, a cytosolic proton extrusion occurs through the activation of the vacuolar H(+)-ATPase and the plasma membrane H(+)-ATPases. This study addressed the dissipation of the TECC through intracellular compartmentalization and the possible affects of the H(+)-ATPases on this process. Both the vacuole and the Golgi-ER apparatus were found to play important roles in distributing calcium to internal stores. Additionally, the inhibition of cytosolic proton extrusion augmented cytosolic calcium responses. A model where pH dependent cytosolic calcium buffering plays an important role in the dissipation of the TECC in Saccharomyces cerevisiae is proposed.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Assembly of Slx4 signaling complexes behind DNA replication forks

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.     

Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to environ-mental stimuli such as an exposure to pheromone or to hexoses after carbon source limitation with a transient elevation of cytosolic calcium (TECC) response. In this study, we examined whether hexose transport and phosphorylation are necessary for the TECC response. We found that a mutant strain lacking most of the known hexose transporters was unable to carry out the TECC response when exposed to glucose. A mutant strain that lacked the ability to phosphorylate glucose was unable to respond to glucose addition, but displayed a normal TECC response after the addition of galactose. These results indicate that hexose uptake and phosphorylation are required to trigger the hexose-induced TECC response. We also found that the TECC response was significantly smaller than normal when the level of environmental calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high-affinity calcium channel of the yeast plasma membrane. These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space. Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc-1-P (or a related glucose metabolite) in yeast.     

Longevity pathways converge on autophagy genes to regulate life span in Caenorhabditis elegans

Aging is a multifactorial process with many mechanisms contributing to the decline. Mutations decreasing insulin/IGF-1 (insulin-like growth factor-1) or TOR (target of rapamycin) kinase-mediated signaling, mitochondrial activity and food intake each extend life span in divergent animal phyla. Understanding how these genetically distinct mechanisms interact to control longevity is a fundamental and fascinating problem in biology. Here we show that mutational inactivation of autophagy genes, which are involved in the degradation of aberrant, damaged cytoplasmic constituents accumulating in all aging cells, accelerates the rate at which the tissues age in the nematode Caenorhabditis elegans. According to our results Drosophila flies deficient in autophagy are also short-lived. We further demonstrate that reduced activity of autophagy genes suppresses life span extension in mutant nematodes with inherent dietary restriction, aberrant insulin/IGF-1 or TOR signaling, and lowered mitochondrial respiration. These findings suggest that the autophagy gene cascade functions downstream of and is inhibited by different longevity pathways in C. elegans, therefore, their effects converge on autophagy genes to slow down aging and lengthen life span. Thus, autophagy may act as a central regulatory mechanism of animal aging.     

Polyunsaturated fatty acid pattern in liver and erythrocyte phospholipids from obese patients

Objective: Our aim was to study the fatty acid (FA) composition of liver phospholipids and its relation to that in erythrocyte membranes from patients with obese nonalcoholic fatty liver disease (NAFLD), as an indication of lipid metabolism alterations leading to steatosis. Research Methods and Procedures: Eight control subjects who underwent antireflux surgery and 12 obese patients with NAFLD who underwent subtotal gastrectomy with a gastro‐jejunal anastomosis in Roux‐en‐Y were studied. The oxidative stress status of patients was assessed by serum F₂‐isoprostanes levels (gas chromatography/negative ion chemical ionization tandem mass spectrometry). Analysis of FA composition of liver and erythrocyte phospholipids was carried out by gas‐liquid chromatography. Results: Patients with NAFLD showed serum F₂‐isoprostanes levels 84% higher than controls. Compared with controls, liver phospholipids from obese patients exhibited significantly 1) lower levels of 20:4n‐6, 22:5n‐3, 22:6n‐3 [docosahexaenoic acid (DHA)], total long‐chain polyunsaturated FA (LCPUFA), and total n‐3 LCPUFA, 2) higher 22:5n‐6 [docosapentaenoic acid (DPAn‐6)] levels and n‐6/n‐3 LCPUFA ratios, and 3) comparable levels of n‐6 LCPUFA. Levels of DHA and DPAn‐6 in liver were positively correlated with those in erythrocytes (r = 0.77 and r = 0.90, respectively; p < 0.0001), whereas DHA and DPAn‐6 showed a negative association in both tissues (r = ?0.79, p < 0.0001 and r = ?0.58, p < 0.01, respectively), associated with lower DHA/DPAn‐6 ratios. Discussion: Obese patients with NAFLD showed marked alterations in the polyunsaturated fatty acid pattern of the liver. These changes are significantly correlated with those found in erythrocytes, thus suggesting that erythrocyte FA composition could be a reliable indicator of derangements in liver lipid metabolism in obese patients.     

Structural cooperativity in histone H3 tail modifications

Post-translational modifications of histone H3 tails have crucial roles in regulation of cellular processes. There is cross-regulation between the modifications of K4, K9, and K14 residues. The modifications on these residues drastically promote or inhibit each other. In this work, we studied the structural changes of the histone H3 tail originating from the three most important modifications; tri-methylation of K4 and K9, and acetylation of K14. We performed extensive molecular dynamics simulations of four types of H3 tails: (i) the unmodified H3 tail having no chemical modification on the residues, (ii) the tri-methylated lysine 4 and lysine 9 H3 tail (K4me3K9me3), (iii) the tri-methylated lysine 4 and acetylated lysine 14 H3 tail (K4me3K14ace), and (iv) tri-methylated lysine 9 and acetylated lysine 14 H3 tail (K9me3K14ace). Here, we report the effects of K4, K9, and K14 modifications on the backbone torsion angles and relate these changes to the recognition and binding of histone modifying enzymes. According to the Ramachandran plot analysis; (i) the dihedral angles of K4 residue are significantly affected by the addition of three methyl groups on this residue regardless of the second modification, (ii) the dihedral angle values of K9 residue are similarly altered majorly by the tri-methylation of K4 residue, (iii) different combinations of modifications (tri-methylation of K4 and K9, and acetylation of K14) have different influences on phi and psi values of K14 residue. Finally, we discuss the consequences of these results on the binding modes and specificity of the histone modifying enzymes such as DIM-5, GCN5, and JMJD2A.