Spatial regulation of DELTA expression mediates NOTCH signalling for segmentation of Drosophila legs.

The Notch (N) signalling pathway is recruited for segregation of cell fates in a number of Drosophila tissue types. We show here that N dependent segmentation of Drosophila legs is regulated by a dynamic pattern of expression of its ligand, DELTA (DL). During third larval instar and early stages of pupation, high levels of DL expression is seen in stripes of cells in the leg imaginal discs which later form the proximal borders of leg joints. These domains also displayed heightened Dl enhancer activity. During subsequent stages of pupation, following segmentation of the leg primordium, DL expression becomes uniform throughout these segments barring the joints. We further show that regulatory Dl mutations or mis-expression of DL abolish leg segmentation. Domains of N signalling for segmentation of legs of flies are thus set up by a stringent spatial regulation of expression of its ligand at the segment border. Further, a comparable role of DL in antennal development reveals a common paradigm of DL-N signalling for segmentation of appendages in flies.     

Multiple Functions of Human Papillomavirus Type 16 E6 Contribute to the Immortalization of Mammary Epithelial Cells

The E6 proteins from cervical cancer-associated human papillomavirus (HPV) types such as HPV type 16 (HPV-16) induce proteolysis of the p53 tumor suppressor protein through interaction with E6-AP. We have previously shown that human mammary epithelial cells (MECs) immortalized by HPV-16 E6 display low levels of p53. HPV-16 E6 as well as other cancer-related papillomavirus E6 proteins also binds the cellular protein E6BP (ERC-55). To explore the potential functional significance of these interactions, we created and analyzed a series of E6 mutants for their ability to interact with E6-AP, p53, and E6BP in vitro. While there was a similar pattern of binding among these E6 targets, a subset of mutants differentiated E6-AP binding, p53 binding, and p53 degradation activities. These results demonstrated that E6 binding to E6-AP is not sufficient for binding to p53 and that E6 binding to p53 is not sufficient for inducing p53 degradation. The in vivo activity of these HPV-16 E6 mutants was tested in MECs. In agreement with the in vitro results, most of these p53 degradation-defective E6 mutants were unable to reduce the p53 level in early-passage MECs. Interestingly, several mutants that showed severely reduced ability for interacting with E6-AP, p53, and E6BP in vitro efficiently immortalized MECs. These immortalized cells exhibited low p53 levels at late passage. Furthermore, mutants defective for p53 degradation but able to immortalize MECs were also identified, and the immortal cells retained normal levels of p53 protein. These results imply that multiple functions of HPV-16 E6 contribute to MEC immortalization.     

On the mechanism of the chromate reduction by glutathione: ESR evidence for the glutathionyl radical and an isolable Cr(V) intermediate

With a view of elucidating the role of glutathione (GSH) in the biochemical pathways of the chromate-exposure related carcinogenesis, we carried out electron spin resonance (ESR) spectroscopic investigations of the chromate-GSH redox reactions. The ESR measurements, employing spin-traps, provide evidence for the involvement of the glutathione (GS) radical, as well as an isolable Cr(V)-glutathione intermediate. These results indicate a new mechanism for the reduction of chromate by GSH in in vitro cellular environment and help understand the (unexpected) increase in Cr(VI)-induced DNA strand breaks at elevated GSH levels.     

Molecular cloning and characterization of five annexin genes from Indian mustard (Brassica juncea L. Czern and Coss)

Plant annexins constitute a multigene family having suggested roles in a variety of cellular processes including stress responses. We have isolated and characterized five different cDNAs of mustard, Brassica juncea (AnnBj1, AnnBj2, AnnBj3, AnnBj6 and AnnBj7) encoding annexin proteins using a RT-PCR/RACE-PCR based strategy. The predicted molecular masses of these annexins are ～36.0 kDa with acidic pIs. At the amino acid level, they share high sequence similarity with each other and with annexins from higher plants. Phylogenetic analysis revealed their evolutionary relationship with corresponding orthologous sequences in Arabidopsis and deduced proteins in various plant species. Expression analysis by semi-quantitative RT-PCR revealed that these genes are differentially expressed in various tissues. The expression patterns of these genes also showed regulation by various stress conditions such as exposure to signaling molecules, salinity and oxidative stress and wounding. Additionally, the in silico promoter analysis (of AnnBj1, AnnBj2 and AnnBj3) showed the presence of different cis-responsive elements that could respond to various stress conditions. These results indicate that AnnBj genes may play important roles in adaptation of plants to various environmental stresses.     

High-resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe grisea

In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ^h ) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F₂ individuals, we mapped the Pi-k ^h gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ^h gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ^h gene revealed that its expression is pathogen inducible.     

Distinct roles for the AAA ATPases NSF and p97 in the secretory pathway

NSF and p97 are related AAA proteins implicated in membrane trafficking and organelle biogenesis. p97 is also involved in pathways that lead to ubiquitin-dependent proteolysis, including ER-associated degradation (ERAD). In this study, we have used dominant interfering ATP-hydrolysis deficient mutants (NSF(E329Q) and p97(E578Q)) to compare the function of these AAA proteins in the secretory pathway of mammalian cells. Expressing NSF(E329Q) promotes disassembly of Golgi stacks into dispersed vesicular structures. It also rapidly inhibits glycosaminoglycan sulfation, reflecting disruption of intra-Golgi transport. In contrast, expressing p97(E578Q) does not affect Golgi structure or function; glycosaminoglycans are normally sulfated and secreted, as is the VSV-G ts045 protein. Instead, expression of p97(E578Q) causes ubiquitinated proteins to accumulate on ER membranes and slows degradation of the ERAD substrate cystic-fibrosis transmembrane-conductance regulator. In addition, expression of p97(E578Q) eventually causes the ER to swell. More specific assessment of effects of p97(E578Q) on organelle assembly shows that the Golgi apparatus disperses and reassembles normally after treatment with brefeldin A and during mitosis. These findings demonstrate that ATP-hydrolysis-dependent activities of NSF and p97 in the cell are not equivalent and suggest that only NSF is directly involved in regulating membrane fusion.     

Activation of adenosine A1 and A2A receptors modulates dopamine D2 receptor-induced responses in stably transfected human neuroblastoma cells

Adenosine can influence dopaminergic neurotransmission in the basal ganglia via postsynaptic interaction between adenosine A2A and dopamine D2 receptors. We have used a human neuroblastoma cell line (SH-SY5Y) that was found to express constitutively moderate levels of adenosine A1 and A2A receptors (approximately 100 fmol/mg of protein) to investigate the interactions of A2A/D2 receptors, at a cellular level. After transfection with human D2L receptor cDNA, SH-SY5Y cells expressed between 500 and 1,100 fmol of D2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A2A receptors decreased the affinity of dopamine D2 receptors for dopamine. In intact cells, the calcium concentration elevation induced by KCI treatment was moderate, and dopamine had no effect on either resting intracellular free Ca2+ concentration ([Ca2+]i) or KCI-induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca2+]i evoked by KCI, which then was totally reversed by dopamine. The effects induced by 48-h adenosine inactivation were mimicked by application of adenosine A1 antagonists and could not be further reversed by acute activation of either A1 or A2A receptors. Acute application of the selective A2 receptor agonist CGS-21680 counteracted the D2 receptor-induced [Ca2+]i responses. The present study shows that SH-SY5Y cells are endowed with functional adenosine A2A and A1 receptors and that A2A receptors exert an antagonistic acute effect on dopamine D2 receptor-mediated functions. In contrast, A1 receptors induce a tonic modulatory role on these dopamine functions.     

The hydrophobic hinge region of rat DNA polymerase beta is critical for substrate binding pocket geometry

The hydrophobic hinge of DNA polymerase beta facilitates closing and stabilization of the enzyme once the nucleotide substrate has bound. Alteration of the hydrophobic nature of the hinge by the introduction of a hydrophilic glutamine residue in place of isoleucine 260 results in an inaccurate polymerase. The kinetic basis of infidelity is lack of discrimination during the binding of substrate. The I260Q polymerase beta variant has lower affinity than wild type enzyme for the correct substrate and much higher affinity for the incorrect substrate. Our results demonstrate that the hinge is important for formation of the substrate binding pocket. Our results are also consistent with the interpretation that DNA polymerase beta discriminates the correct from incorrect substrate during the binding step.     

Centromeric chromatin: what makes it unique?

Centromeres represent the final frontier of eukaryotic genomes. Although they are defining features of chromosomes--the points at which spindle microtubules attach--the fundamental features that distinguish them from other parts of the chromosome remain mysterious. The function of centromeres is conserved throughout eukaryotic biology, but their DNA sequences are not. Rather, accumulating evidence favors chromatin-based centromeric identification. To understand how centromeric identity is maintained, researchers have studied DNA-protein interactions at native centromeres and ectopic "neocentromeres". Other studies have taken a comparative approach focusing on centromere-specific proteins, of which mammalian CENP-A and CENP-C are the prototypes. Elucidating the assembly and structure of chromatin at centromeres remain key challenges.     

Identification and quantitative analysis of beta-sitosterol oxides in vegetable oils by capillary gas chromatography-mass spectrometry

As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of beta-sitosterol oxides using capillary gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify beta-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time.