The many roles of small RNAs in leaf development

Leaf development involves many complex genetic interactions,signals between adjacent cells or between more distant tissues and consequent changes in cell fate.This review describes three stages in leaf development where regulation by small RNAs have been used to modulate gene expression patterns.     

Effect of experimental glaucoma on the non-image forming visual system

Glaucoma is a leading cause of blindness worldwide, characterized by retinal ganglion cell degeneration and damage to the optic nerve. We investigated the non-image forming visual system in an experimental model of glaucoma in rats induced by weekly injections of chondroitin sulphate (CS) in the eye anterior chamber. Animals were unilaterally or bilaterally injected with CS or vehicle for 6 or 10 weeks. In the retinas from eyes injected with CS, a similar decrease in melanopsin and Thy-1 levels was observed. CS injections induced a similar decrease in the number of melanopsin-containing cells and superior collicular retinal ganglion cells. Experimental glaucoma induced a significant decrease in the afferent pupil light reflex. White light significantly decreased nocturnal pineal melatonin content in control and glaucomatous animals, whereas blue light decreased this parameter in vehicle- but not in CS-injected animals. A significant decrease in light-induced c-Fos expression in the suprachiasmatic nuclei was observed in glaucomatous animals. General rhythmicity and gross entrainment appear to be conserved, but glaucomatous animals exhibited a delayed phase angle with respect to lights off and a significant increase in the percentage of diurnal activity. These results indicate the glaucoma induced significant alterations in the non-image forming visual system.     

LcrV Mutants That Abolish Yersinia Type III Injectisome Function

LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR⁺] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV_*327). The addition of at least four amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR⁻ phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, LcrV_*327 mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with either injectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR⁺, LCR⁻, or dominant negative LCR⁻ phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor.     

Factors affecting the accuracy of urine-based biomarkers of BSE

Background  

Transmissible spongiform encephalopathy diseases are untreatable, uniformly fatal degenerative syndromes of the central nervous system that can be transmitted both within as well as between species. The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob disease (vCJD), have profoundly influenced beef production processes as well as blood donation and surgical procedures. Simple, robust and cost effective diagnostic screening and surveillance tools are needed for both the preclinical and clinical stages of TSE disease in order to minimize both the economic costs and zoonotic risk of BSE and to further reduce the risk of secondary vCJD.     

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles

Background  

This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles.     

An entry to curcuphenol/elvirol core structures via a retro-Aldol reaction

Analogs of curcuphenol/elvirol, naturally occurring bisabolane sesquiterpenes, were prepared in six steps from alkyl-α-tetralones employing an aromatization reaction of cyclic dienone precursors and olefination of the key aldehyde intermediates. The in vitro antifungal activities of 6a, 6b, 6d, and 6g are also reported.     

Rational engineering of the TOL meta-cleavage pathway

The meta-cleavage pathway of Pseudomonas putida mt-2 was simulated using a biochemical systems simulation developed by Regan (1996). A non-competitive inhibition term for catechol-2,3-dioxygenase (C23O) by 2-OH-pent-2,4-dienoate (Ki = 150 μM) was incorporated into the model. The simulation predicted steady state accumulation levels in the μM range for metabolites pre-meta-cleavage, and in the mM range for metabolites post-meta-cleavage. The logarithmic gains L[V-i, Xj] and L[X-i, Xj] clearly indicated that the pathway was most sensitive to the concentration of the starting substrate, benzoate, and the first enzyme of the pathway, toluate-1, 2-dioxygenase (TO). The simulation was validated experimentally; it was found that the amplification of TO increased the steady state flux from 0.024 to 0.091 (mmol/g cell dwt)/h. This resulted in an increased accumulation of a number of the pathway metabolites (intra- and extracellularly), especially cis-diol, 4-OH-2-oxovalerate, and 4-oxalocrotonate. Metabolic control analysis indicated that C23O was, in fact, the major controling enzymic step of the pathway with a scaled control coefficient of 0.83. The amplification of TO resulted in a shift of some of the control away from C23O. Catechol-2,3-dioxygenase, however, remained as the major controling element of the pathway. Copyright 1998 John Wiley & Sons, Inc.     

A diphenyldiselenide derivative induces autophagy via JNK in HTB‐54 lung cancer cells

Symmetric aromatic diselenides are potential anticancer agents with strong cytotoxic activity. In this study, the in vitro anticancer activities of a novel series of diarylseleno derivatives from the diphenyldiselenide (DPDS) scaffold were evaluated. Most of the compounds exhibited high efficacy for inducing cytotoxicity against different human cancer cell lines. DPDS 2 , the compound with the lowest mean GI₅₀ value, induced both caspase‐dependent apoptosis and arrest at the G₀/G₁ phase in acute lymphoblastic leucemia CCRF‐CEM cells. Consistent with this, PARP cleavage; enhanced caspase‐2, ‐3, ‐8 and ‐9 activity; reduced CDK4 expression and increased levels of p53 were detected in these cells upon DPDS 2 treatment. Mutated p53 expressed in CCRF‐CEM cells retains its transactivating activity. Therefore, increased levels of p21^CIP1 and BAX proteins were also detected. On the other hand, DPDS 6 , the compound with the highest selectivity index for cancer cells, resulted in G₂/M cell cycle arrest and caspase‐independent cell death in p53 deficient HTB‐54 lung cancer cells. Autophagy inhibitors 3‐methyladenine, wortmannin and chloroquine inhibited DPDS 6 ‐induced cell death. Consistent with autophagy, increased LC3‐II and decreased SQSTM1/p62 levels were detected in HTB‐54 cells in response to DPDS 6 . Induction of JNK phosphorylation and a reduction in phospho‐p38 MAPK were also detected. Moreover, the JNK inhibitor SP600125‐protected HTB‐54 cells from DPDS 6 ‐induced cell death indicating that JNK activation is involved in DPDS 6 ‐induced autophagy. These results highlight the anticancer effects of these derivatives and warrant future studies examining their clinical potential.     

Mutations in yscC, yscD, and yscG prevent high-level expression and secretion of V antigen and Yops in Yersinia pestis.

The Yersinia pestis low-Ca2+ response stimulon is responsible for the temperature- and Ca(2+)-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37 degrees C in the absence of Ca2+. In this study, we constructed and characterized mutants with in-frame deletions in yscC, yscD, and yscG of the ysc operon that contains yscA through yscM. All three mutants lost the Ca2+ requirement for growth at 37 degrees c, expressed only basal levels of V antigen and YopM in the presence or absence of Ca2+, and failed to secrete these proteins to the culture supernatant. Overproduction of YopM in these mutants failed to restore YopM export, showing that the mutations had a direct effect on secretion. The protein products of yscC, yscD, and yscG were identified and localized by immunoblot analysis. YscC was localized to the outer membrane of Y. pestis, while YscD was found in the inner membrane. YscG was distributed equally between the soluble and total membrane fractions. Double mutants were characterized to assess where YscC and YscD act in low-Ca2+ response (LCR) regulation. lcrH::cat-yscC and lcrH::cat-yscD double mutants were constitutively induced for expression of V antigen and YopM; however, these proteins were not exported. This finding showed that the ysc mutations did not directly decrease induction of LCR stimulon genes. In contrast, lcrE-yscC, lcrG-yscC, lcrE-yscD, and lcrG-yscD double mutants as well as an lcrE-lcrD double mutant expressed only basal levels of V antigen and YopM and also failed to secrete these proteins to the culture supernatant. These results indicated that a functional LCR secretion system was necessary for high-level expression of LCR stimulon proteins in the lcrE and lcrG mutants but not in an lcrH::cat mutant. Possible models of regulation which incorporate these results are discussed.