Fast neutron mutagenesis of soybean (Glycine soja L.) produces a supernodulating mutant containing a large deletion in linkage group H

We describe for the first time the application of fast neutron mutagenesis to the genetic dissection of root nodulation in legumes. We demonstrate the utility of chromosomal deletion mutations through production of a soybean supernodulation mutant FN37 that lacks the internal autoregulation of nodulation mechanism. After inoculation with microsymbiont Bradyrhizobium japonicum, FN37 forms at least 10 times more nodules than the wild type G. soja parent and has a phenotype identical to that of chemically induced allelic mutants nts382 and nts1007 (NTS-1 locus). Reciprocal grafting of shoots and roots confirmed systemic shoot control of the FN37 nodulation phenotype. RFLP/PCR marker pUTG132a and AFLP marker UQC-IS1 which are tightly linked to NTS-1 allowed the isolation of BAC contigs delineating both ends of the deletion. The genetic/physical distance ratio in the NTS-1 region is 279 kb/cM. The deletion is estimated to be about 460 kb based on the absence of markers and bacterial artificial chromosomes (BAC) ends as well as genetic and physical mapping. Deletion break points were determined physically and placed within flanking BAC contigs.

Ion transport induced by proteinase-activated receptors (PAR2) in colon and airways

Protease-activated receptors type 2 (PAR2) are activated by serine proteases like trypsin and mast cell tryptase. The function and physiological significance of PAR2 receptors is poorly understood, but recent studies suggest a role during inflammatory processes in both airways and intestine. PAR2 receptors are also likely to participate in the control of ion transport in these tissues. We demonstrate that stimulation of PAR2 in airways and intestine significantly enhanced ion transport. Trypsin induced CI- secretion in both airways and intestine when added to the basolateral but not to the luminal side of these tissues. In both airways and intestine, stimulation of ion transport was largely dependent on the increase in intracellular Ca2+. Effects of trypsin were largely reduced by basolateral bumetanide and barium and by trypsin inhibitor. Thrombin, an activator of proteinase-activated receptors types 1, 3, and 4 had no effects on equivalent short-circuit current in either airways or intestine. Expression of PAR2 in colon and airways was further confirmed by reverse transcription-polymerase chain reaction. We postulate that these receptors play a significant role in the regulation of electrolyte transport, which might be important during inflammatory diseases of airways and intestine.

Genes induced by growth hormone in a model of adipogenic differentiation

A substantial number of GH regulated genes have been reported in mature hepatocytes. but genes involved in GH-initiated cell differentiation have not yet been identified. Here we have studied a, ell-characterised model of GH-dependent differentiation, adipogenesis of 3T3-F442A preadipocytes, to identify genes rapidly induced by GH. Using the suppression subtractive hybridisation technique, we have identified eight genes induced within 60 min of GH treatment, and verified these by northern analysis. Six were identifiable as Stat 2. Stat 3, thrombospondin-1. oncostatin M receptor beta chain. a DEAD box RNA helicase. and muscleblind. a developmental transcription factor. Bioinformatic approaches assigned one of the two remaining unknown genes as a novel 436 residue serine,threonine kinase. As each of the identified genes hake important developmental roles. they may be important in initiating GH-induced adipogenesis. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Hypoalgesia induced by elbow manipulation in lateral epicondylalgia does not exhibit tolerance

Previous studies have demonstrated that the initial hypoalgesic effect of spinal manipulative therapy was not antagonized by naloxone and did not exhibit tolerance with repeated applications. The implication is that endogenous opioid mechanisms of pain relief are probably not at play in spinal manipulative therapy. The role of endogenous opioid peptides in manipulation of the peripheral joints has not been investigated. The aim of this study was to evaluate whether the initial hypoalgesic effect of a peripheral manipulative technique (mobilization-with-movement treatment for the elbow) demonstrated a tolerance to repeated applications (ie, reduction in magnitude of effect over repeated applications). Twenty-four participants with unilateral chronic lateral epicondylalgia participated in the study. A repeated measures study was conducted to examine the effect of repeated applications of the mobilization-with-movement treatment for the elbow on 6 separate treatment occasions at least 2 days apart. Pain-free grip strength and pressure pain threshold were chosen as the pain-related outcome measures. Changes in the percent maximum possible effect scores of measures of hypoalgesia were evaluated across the 6 treatment sessions by using linear trend analysis. The results showed no significant difference for the hypoalgesic effect of the treatment technique between sessions (P >.05). This peripheral manipulative therapy treatment technique appeared to have a similar effect profile to previously studied spinal manipulative therapy techniques, thereby contributing to the body of knowledge that indicates that manipulative therapy most likely induces a predominant non-opioid form of analgesia.

The VCAM-1 gene that encodes the vascular cell adhesion molecule is a target of the Sry-related high mobility group box gene, Sox18

VCAM-1 (vascular cell adhesion molecule-1) and Sox18 are involved in vascular development. VCAM-1 is an important adhesion molecule that is expressed on endothelial cells and has a critical role in endothelial activation, inflammation, lymphatic pathophysiology, and atherogenesis. The Sry-related high mobility group box factor Sox18 has previously been implicated in endothelial pathologies. Mutations in human and mouse Sox18 leads to hypotrichosis and lymphedema. Furthermore, both Sox18 and VCAM-1 have very similar spatio-temporal patterns of expression, which is suggestive of crosstalk. We use biochemical techniques, cell culture systems, and the ragged opossum (RaOP) mouse model with a naturally occurring mutation in Sox18 to demonstrate that VCAM-1 is an important target of Sox18. Transfection, site-specific mutagenesis, and gel shift analyses demonstrated that Sox18 directly targeted and trans-activated VCAM-1 expression. Importantly, the naturally occurring Sox18 mutant attenuates the expression and activation of VCAM-1 in vitro. Furthermore, in vivo quantitation of VCAM-1 mRNA levels in wild type and RaOP mice demonstrates that RaOP animals show a dramatic and significant reduction in VCAM-1 mRNA expression in lung, skin, and skeletal muscle. Our observation that the VCAM-1 gene is an important target of SOX18 provides the first molecular insights into the vascular abnormalities in the mouse mutant ragged and the human hypotrichosis-lymphedematelangiectasia disorder.

Sox18 mutations in the ragged mouse alleles ragged-like and opossum

The ragged (Ra) spontaneous mouse mutant is characterised by abnormalities in its coat and cardiovascular system. Four alleles are known and we have previously described mutations in the transcription factor gene Sox18 in the Ra and Ra-J alleles. We report here Sox18 mutations in the remaining two ragged alleles, opossum (Ra-op) and ragged-like (Ragl). The single-base deletions cause a C-terminal frameshift, abolishing transcriptional trans-activation and impairing interaction with the partner protein MEF2C. The nature of these mutations, together with the near-normal phenotype of Sox18-null mice, suggests that the ragged mutant SOX18 proteins act in a dominant-negative fashion. The four ragged mutants represent an allelic series that reveal SOX18 structure-function relationships and implicate related SOX proteins in cardiovascular and hair follicle development. (C) 2003 Wiley-Liss, Inc.

High frequency of BRAF mutations in nevi

To evaluate the timing of mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) during melanocytic neoplasia, we carried out mutation analysis on microdissected melanoma and nevi samples. We observed mutations resulting in the V599E amino-acid substitution in 41 of 60 (68%) melanoma metastases, 4 of 5 (80%) primary melanomas and, unexpectedly, in 63 of 77 (82%) nevi. These data suggest that mutational activation of the RAS/RAF/MAPK pathway in nevi is a critical step in the initiation of melanocytic neoplasia but alone is insufficient for melanoma tumorigenesis.

Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A

Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A, and caused radioresistant DNA synthesis (RDS). The basal turnover of Cdc25A operating in unperturbed S phase required Chk1-dependent phosphorylation of serines 123, 178, 278, and 292. IR-induced acceleration of Cdc25A proteolysis correlated with increased phosphate incorporation into these residues generated by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms.

Systematic characterization of mutations in yeast acetohydroxyacid synthase: Interpretation of herbicide-resistance data

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides. However, the selectivity of these mutants towards various sulfonylureas and imidazolinones has not been determined systematically. Now that the structure of the yeast enzyme is known, both in the absence and presence of a bound herbicide, a detailed understanding of the molecular interactions between the enzyme and its inhibitors becomes possible. Here we construct 10 active mutants of yeast AHAS, purify the enzymes and determine their sensitivity to six sulfonylureas and three imidazolinones. An additional three active mutants were constructed with a view to increasing imidazolinone sensitivity. These three variants were purified and tested for their sensitivity to the imidazolinones only. Substantial differences are observed in the sensitivity of the 13 mutants to the various inhibitors and these differences are interpreted in terms of the structure of the herbicide-binding site on the enzyme.