Genetic analysis of temperature-sensitive male sterilty in rice

The present study of genetic analysis is an attempt to precisely characterize diverse temperature-sensitive genic male-sterile (TGMS) lines so as to explore the possibilities of utilizing the most promising in large-scale hybrid seed production. Genetical studies revealed that the TGMS segregants derived from crosses involving TGMS lines ID24 and SA2 expressed differential fertility levels at low-temperature conditions. A majority of these progenies expressed transgressive segregation towards either sterility of fertility, causing instability of sterility and low reversibilty of fertility which may be due to large numbers of single-locus QTLs and their epistatic interactions. We identified two putative genes imparting temperature-sensitive male sterility after observing crosses involving diverse TGMS sources. To identify suitable molecular markers closely linked to the trait we used RAPD, AFLP and microsatellites which generated polymorphism through bulked segregant analysis. AFLP analysis using a smaller genome kit resulted in enormous polymorphism, out of which the combination EAA/MCAG amplified a 330-bp fragment, which closely segregated with the gene at a distance of 5.3 cM. This fragment was eluted for cloning and from the sequence a STS primer (TS200) was developed which produced a dominant polymorphism specific to TGMS. The microsatellite RM257, located earlier on chromosome 9, was linked with the TGMS trait in SA2 at a distance of 6.2 cM. RM257 produced a codominant polymorphism with 145-bp (sterile) and 132-bp (fertile) products. Both individually and collectively, the markers TS200 and RM257 located on either side of the TGMS locus are very useful for marker-assisted selection. Received: 10 April 1999 / Accepted: 29 July 1999     

Glycine reduces platelet aggregation

It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl^? influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1–10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor.     

TRAF6 is a critical mediator of signal transduction by the viral oncogene latent membrane protein 1

The oncogenic latent membrane protein 1 (LMP1) of the Epstein-Barr virus recruits tumor necrosis factor-receptor (TNFR)-associated factors (TRAFs), the TNFR-associated death domain protein (TRADD) and JAK3 to induce intracellular signaling pathways. LMP1 serves as the prototype of a TRADD-binding receptor that transforms cells but does not induce apoptosis. Here we show that TRAF6 critically mediates LMP1 signaling to p38 mitogen-activated protein kinase (MAPK) via a MAPK kinase 6-dependent pathway. In addition, NF-kappaB but not c-Jun N-terminal kinase 1 (JNK1) induction by LMP1 involves TRAF6. The PxQxT motif of the LMP1 C-terminal activator region 1 (CTAR1) and tyrosine 384 of CTAR2 together are essential for full p38 MAPK activation and for TRAF6 recruitment to the LMP1 signaling complex. Dominant-negative TRADD blocks p38 MAPK activation by LMP1. The data suggest that entry of TRAF6 into the LMP1 complex is mediated by TRADD and TRAF2. In TRAF6-knockout fibroblasts, significant induction of p38 MAPK by LMP1 is dependent on the ectopic expression of TRAF6. We describe a novel role of TRAF6 as an essential signaling mediator of a transforming oncogene, downstream of TRADD and TRAF2.     

Circadian variations in influx and efflux of the S phase in a partially synchronized cell system Double-labelling with [3H]thymidine in the epithelium of the hamster cheek pouch

Abstract. By means of a double-labelling experiment, circadian variations in the kinetic parameters of the S phase of the hamster cheek pouch epithelium were studied. The evaluation of the experiment included a recently developed correction for deviations from the strict pulse interpretation of the labelling technique.
Pronounced circadian variations were found in S phase influx and efflux; the diurnal mean of both was estimated as 0.5%/hr, when based on measurements of all nucleated epithelial cells. Variations in S phase influx seem mainly responsuble for the diurnal variation in cell proliferation, although diurnal variation in DNA synthesis rate, and thus in mean transit time, was also found. The increases in LI and influx were closely correlated and related to the beginning of the dark period.
A circadian variation in cell number was also observed.     

The eIF1A solution structure reveals a large RNA-binding surface important for scanning function

The translation initiation factor eIF1A is necessary for directing the 43S preinitiation complex from the 5' end of the mRNA to the initiation codon in a process termed scanning. We have determined the solution structure of human eIF1A, which reveals an oligonucleotide-binding (OB) fold and an additional domain. NMR titration experiments showed that eIF1A binds single-stranded RNA oligonucleotides in a site-specific, but non-sequence-specific manner, hinting at an mRNA interaction rather than specific rRNA or tRNA binding. The RNA binding surface extends over a large area covering the canonical OB fold binding site as well as a groove leading to the second domain. Site-directed mutations at multiple positions along the RNA-binding surface were defective in the ability to properly assemble preinitiation complexes at the AUG codon in vitro.     

Valproic acid induces extracellular signal-regulated kinase 1/2 activation and inhibits apoptosis in endothelial cells

The histone deacetylase (HDAC) inhibitor valproic acid (VPA) was recently shown to inhibit angiogenesis, but displays no toxicity in endothelial cells. Here, we demonstrate that VPA increases extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation in human umbilical vein endothelial cells (HUVEC). The investigation of structurally modified VPA derivatives revealed that the induction of ERK 1/2 phosphorylation is not correlated to HDAC inhibition. PD98059, a pharmacological inhibitor of the mitogen-activated protein kinase kinase 1/2, prevented the VPA-induced ERK 1/2 phosphorylation. In endothelial cells, ERK 1/2 phosphorylation is known to promote cell survival and angiogenesis. Our results showed that VPA-induced ERK 1/2 phosphorylation in turn causes phosphorylation of the antiapoptotic protein Bcl-2 and inhibits serum starvation-induced HUVEC apoptosis and cytochrome c release from the mitochondria. Moreover, the combination of VPA with PD98059 synergistically inhibited angiogenesis in vitro and in vivo.     

Colloidal gold labeling of intracellular ligands in dorsal root sensory neurons, visualized by scanning electron microscopy

We report a novel technique that combines high-resolution scanning electron microscopy (SEM) of intracellular structures with backscattered electron imaging (BEI) of colloidal gold-labeled intracellular ligands. Murine dorsal root ganglia were immersion-fixed, freeze-cleaved, labeled with gold complexes, and critical point-dried. Specimens were carbon-coated and viewed by BEI. They were then minimally sputter-coated with gold and previously identified cells relocated by secondary electron imaging (SEI). This permitted increased resolution of intracellular detail while gold particles remained detectable by BEI. Incubation with RNAse-gold and DNAse-gold complexes resulted in specific labeling of cytoplasm and nucleus, respectively. Immunolabeling of neurofilament (NF) and small nuclear ribonucleoproteins (snRNP) resulted in selective labeling of intracellular antigens. Nonspecific binding was abolished by use of 1% skin milk. Specifically, incubation with monoclonal anti-NF68 resulted in labeling of cytoplasm in 66% of neurons, notably of the large cells known to contain large amounts of NF. Satellite cells, which lack NF, showed low levels of background label. Human autoimmune anti-Sm serum recognizes snRNP particles, with the exception of the nucleolar U3 snRNP. Labeling with this serum resulted in specific labeling of 92% of nuclei, with only background labeling over nucleoli and cytoplasm. The results show that it is feasible to employ high-resolution SEM in conjunction with colloidal gold labeling to localize intracellular ligands in situ.     

Induced expression of the class II chitinase gene during cold acclimation and dehydration of bermudagrass (Cynodon sp.)

Bermudagrass cultivars vary greatly in their ability to survive freezing temperatures as a result of a differential ability to cold acclimate (CA) at temperatures slightly above 0°C. Little information exists on the genetic and physiological mechanisms associated with the cold acclimation process in bermudagrass. Experiments were conducted to study the changes in chitinase gene expression during cold acclimation of freeze-tolerant bermudagrass cultivars. A chitinase gene (CynCHT1) was isolated from ’Midiron’ bermudagrass. Because the hydrophilic protein putatively encoded by the gene lacked an N-terminal cysteine-rich domain and a hydrophobic C-terminal extension, it was classified a class II chitinase. The expression patterns of this and related chitinase genes in response to CA, drought, and ABA were investigated in freeze-tolerant ’MSU’ (LT₅₀=?11°C), Midiron (LT₅₀=?10°C) and ’Uganda’ (LT₅₀=?8°C) bermudagrasses. Northern-blot analysis indicated expression in the crown tissues induced by CA at 8°C/2°C day/night temperature cycles. Induction of gene expression was evident in tissues sampled at 2 and 28 days after initiating CA. Expression after 2-days de-acclimation at 28°C/24°C was similar to control levels. Significantly higher levels of CA-induced chitinase gene expression were observed in MSU and Midiron, compared to Uganda. Similar expression patterns were observed among the cultivars in responses to drought and ABA. These results suggest that chitinases have important roles in bermudagrass response to low temperature and dehydration stresses.