Application of Bruchin B to pea pods results in the up-regulation of CYP93C18, a putative isoflavone synthase gene, and an increase in the level of pisatin, an isoflavone phytoalexin.

Bruchins, mono and bis (3-hydroxypropanoate) esters of long chain alpha,omega-diols, are a recently discovered class of insect elicitors that stimulate cell division and neoplasm formation when applied to pods of peas and certain other legumes. Differential display analysis resulted in the identification of an mRNA whose level was increased by the application of Bruchin B to pea pods. The corresponding amplification product was cloned and sequenced and a full length cDNA sequence was obtained. This cDNA and the gene from which it was derived were assigned the name CYP93C18 based upon sequence similarities to the cytochrome P450 mono-oxygenase CYP93C subfamily, which contains isoflavone synthase genes from legumes. RNA gel blots and quantitative RT-PCR demonstrated that expression of CYP93C18 increased within 8 h of bruchin treatment to a maximum of 100-200-fold of the level in untreated pods, and then declined. The up-regulation of CYP93C18 was followed by an increase in the level of the isoflavone phytoalexin, pisatin. Pisatin was detectable in the bruchin-treated pods after 16 h and reached a maximum between 32 h and 64 h. This, the first report of induction of phytoalexin biosynthesis by an insect elicitor, suggests that Bruchin B not only stimulates neoplasm formation, but also activates other plant defence responses.

Mapping Ds insertions in barley using a sequence-based approach.

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgaresubsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions. The principal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.

Gene structure and expression of the targeting subunit, RGL, of the muscle-specific glycogen-associated type 1 protein phosphatase, PP1G.

The type I phosphatase associated with glycogen, PP1G, plays an important role in glycogen metabolism. PP1G is targeted to glycogen by the R(GL) subunit, which regulates the function of the enzyme. We report the cloning and characterization of the gene as well as the pattern of expression of the R(GL) subunit from mouse. The gene covers more than 37 kb, is composed of four exons and three introns, and codes for a 1089 residue polypeptide with a calculated molecular weight of 121,000. The amino acid sequence has 60% identity with the human and rabbit R(GL). The 5' flanking region of the gene contains a TATA box, c-Myc sites, and a potential cAMP-responsive element. Muscle specific motifs, such as MyoD and MEF-2, were also found. The A-T rich 3'-UTR contained several polyadenylation signals, two associated with poly(A) down-stream consensus motifs. ARE elements, which regulate mRNA stability, were dispersed throughout the 3'-UTR. Northern analysis of poly(A) mRNA from various murine tissues indicates a major transcript of 7.5 kb in skeletal muscle and heart. Western analysis demonstrates that R(GL) protein is present in skeletal and cardiac muscle from mouse, rat, and rabbit but not in L6 myoblasts, L6 myotubes, 3T3 L1 fibroblasts, 3T3 L1 or rat primary adipocytes, confirming that expression of the gene is specific to striated muscle. Analysis of skeletal muscle from rats made diabetic by streptozotocin treatment reveals that the level of R(GL) protein is the same as in control animals, indicating that expression is not regulated by insulin.

Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL.

The regulatory-targeting subunit (RGL), also called GM) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/RGL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of RGL. To investigate the function of the phosphatase, RGL knockout mice were generated. Animals lacking RGL show no obvious defects. The RGL protein is absent from the skeletal and cardiac muscle of null mutants and present at approximately 50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by approximately 50% in the RGL-deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant RGL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by approximately 90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and RGL null mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that RGL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/RGL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.

A high-resolution radiation hybrid map of the proximal region of rat chromosome 4.

Radiation hybrid (RH) mapping has been used to produce genome maps in the human and mouse, but as yet the technique has been applied little to other species. We describe the use of RH mapping in the rat, using a newly available rat/hamster RH panel, to construct an RH map of the proximal part of rat Chromosome (Chr) 4. This region is of interest because quantitative trait loci (QTLs) for defective insulin and catecholamine action, hypertension, and dyslipidemia map to this region. The RH map includes 23 rat genes or microsatellites previously mapped to this part of Chr 4, one rat gene not previously mapped in the rat, and markers for four new genes, homologs of which map to the syntenic region of the mouse genome. The RH map integrates genetic markers previously mapped on several rat crosses, increases the resolution of existing maps, and may provide a suitable basis for physical map construction and gene identification in this chromosomal region. Our results demonstrate the utility of RH mapping in the rat genome and show that RH mapping can be used to localize, in the rat genome, the homologs of genes from other species such as the mouse. This will facilitate identification of candidate genes underlying QTLs on this chromosomal segment.

The grant portfolio of the National Center for Medical Rehabilitation Research: the first five years.

OBJECTIVE: To relate grant funding activities of the National Center for Medical Rehabilitation Research (NCMRR) with the Center's mission, priorities, and terminology for disability classification. STUDY DESIGN: Retrospective review by the National Advisory Board on Medical Rehabilitation Research (NABMRR). DATA: Abstracts of 153 research proposals and one contract funded by the NCMRR from 1992 through 1996. METHOD: A six-member research group participated in the development of a rating form and related instructions used to evaluate each abstract. The form was piloted and revised, and interrater agreement was monitored. RESULTS: Funded proposals reflected each of the NCMRR priorities evaluated, with the highest proportion in the areas of assistive technology and whole body system, and the lowest in the area of behavioral adaptation. Although some proposals were funded in each of the domains of the disability classification system, proportionately fewer addressed the domains of disability and societal limitations. Findings also indicated that few funded proposals addressed more than one domain in the disability classification system and that most abstracts did not address consumers' perspectives on quality of life. RECOMMENDATIONS: The NABMRR recommended that the NCMRR (1) encourage more research in the areas of disability and societal limitations and in behavioral adaptation, (2) examine funded proposals in light of a recent Institute of Medicine report, and (3) explore quality-of-life measurements. Further, members of the rehabilitation community are encouraged to e-mail their responses to this review to NCMRR staff at (1q2n@nih.govA) and to suggest areas of research emphasis.

Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Medical, functional, and legal aspects of vocational rehabilitation for people with multiple sclerosis.

A majority of individuals with multiple sclerosis (MS) are not employed, even though most are at an age of prime employability and have been successfully employed in the past. Factors that have been shown to decrease employment in these individuals include mobility impairment, bladder and bowel dysfunction, impairment in activities of daily living, cognitive dysfunction, social and family issues, lack of physical and functional access to the workplace, and attitudes of the patient, family, and employer about chronicity and disability in MS. This paper reviews the functional, psychosocial, and legal barriers to employment in these individuals and discusses effective interventions to maintain or regain successful employment in this population.

Duration of signals for intertrial reinforcement and nonreinforcement in random control procedures.

In the random control procedure, responding to a conditioned stimulus (target CS) is prevented when the probability of unsignaled, unconditioned stimuli (USs) in the intertrial interval (ITI) is equal to the probability of the US in the presence of the target CS. Three experiments used an autoshaping procedure with White Carneaux pigeons to examine the effects of the temporal duration of signals for the ITI USs (cover CSs) and for concomitant periods of nonreinforcement. In Experiment 1, a short duration cover, but not a long duration cover, resulted in responding to the target CS. In Experiment 2, an explicit CS- cue during periods of nonreinforcement did not affect target acquisition. In Experiment 3, a long CS-, but not a short cover CS, was a sufficient condition for the acquisition of responding to the target CS. These results imply that the acquisition of responding to a target CS requires a discriminable period of nonreinforcement that is long relative to the target CS duration.

Verbal training to improve explanations of conservation with aphasic adults.

Recent research has indicated a need to study the relationship between the language of the adult aphasic and his attempts at cognitive processing. Nine aphasic adults who demonstrated a minimal ability to explain conservation (as defined by Piaget), a cognitive task which they understood, were given verbal model training to improve their explanations of weight and liquid conservation. Each subject was given a pretest, an experimental condition during which explanations for weight conservation only were trained, a control condition during which subjects named pictured common objects, and a posttest. Order of presentation of the experimental and control conditions was varied. As a result of training, a greater number of explanations (quantitative improvement) and a greater number of explanatory concepts (qualitative improvement) were expressed for both the trained and nontrained conservation tasks. It is suggested that the improvement in conservation explanations is the result of "response facilitation effects" as described by Bandura. Furthermore, the improvement in conservation explanations is supportive of Schuell's concept of impaired linguistic retrieval mechanisms in aphasia.