Towards the genetic architecture of seed lipid biosynthesis and accumulation in Arabidopsis thaliana.

We report the quantitative genetic analysis of seed oil quality and quantity in six Arabidopsis thaliana recombinant inbred populations, in which the parent accessions were from diverse geographical origins, and were selected on the basis of variation for seed oil content and lipid composition. Although most of the biochemical steps involved in lipid biosynthesis are known and the key genes have been identified, the regulation of the processes that results in the final oil composition and total amount is not understood. By using physically anchored markers it was possible to compare results across populations. A total of 219 quantitative trait loci (QTLs) were identified, of which 81 were significant at P<0.001. Some of these colocalise with QTLs identified previously, but many novel QTLs were also identified. The results highlight the importance of studying traits in multiple populations, which will lead to a better understanding of the contribution that natural variation makes to the genetic architecture of a phenotype.

Comparative physical mapping of segments of the genome of Brassica oleracea var. alboglabra that are homoeologous to sequenced regions of chromosomes 4 and 5 of Arabidopsis thaliana.

Due to their relatedness to Arabidopsis thaliana (Arabidopsis), the cultivated Brassica species represent the first group of crops with which to evaluate comparative genomics approaches to understanding biological processes and manipulating traits. We have constructed a high-quality binary BAC library (JBo) from genomic DNA of Brassica oleracea var. alboglabra, in order to underpin such investigations. Using the Arabidopsis genome sequence and clones from the JBo library, we have analysed aspects of gene conservation and microsynteny between a 222 kb region of the genome of Arabidopsis and homoeologous segments of the genome of B. oleracea. All 19 predicted genes tested were found to hybridize to clones in the JBo library, indicating a high level of gene conservation. Further analyses and physical mapping with the BAC clones identified allowed us to construct clone contig maps and analyse in detail the gene content and organization in the set of paralogous segments identified in the genome of B. oleracea. Extensive divergence of gene content was observed, both between the B. oleracea paralogous segments and between them and their homoeologous segment within the genome of Arabidopsis. However, the genes present show highly conserved collinearity with their orthologues in the genome of Arabidopsis. We have identified one example of a Brassica gene in a non-collinear position and one rearrangement. Some of the genes not present in the discernible homoeologous regions appear to be located elsewhere in the B. oleracea genome. The implications of our findings for comparative map-based cloning of genes from crop species are discussed.

Human leucocyte antigen class II associations in chronic idiopathic urticaria.

The major histocompatibility complex (MHC) acts as a marker for self during T-cell ontogeny and is associated with the pathogenesis of many autoimmune diseases. Recent investigations have shown about 30% of patients with chronic idiopathic urticaria (CIU) have IgG autoantibodies against the high-affinity IgE receptor, FcepsilonRI, or IgE. A link between MHC class II alleles and CIU has not been reported previously. DNA was extracted from blood of 100 Caucasian patients with CIU, and the MHC class II type determined using the polymerase chain reaction with sequence-specific primers, testing for DRB and DQB1 alleles. The frequency of alleles in CIU patients was compared with that found in 603 controls. Further human leucocyte antigen (HLA) typing on patient subsets, classified by the patients' responses to intradermal injection of autologous serum and their serum-induced histamine release from basophil leucocytes of healthy donors, was undertaken. HLA DRB1*04 (DR4) and its associated allele, DQB1*0302 (DQ8), are raised in CIU patients compared with a control population (P = 2 x 10-5 and P = 2 x 10-4, respectively). HLA DRB1*15 (DR15) and its associated allele, DQB1*06 (DQ6), are significantly less frequently associated with CIU. The HLA DRB1*04 association is particularly strong (corrected P = 3.6 x 10-6) for patients whose serum has in vivo and in vitro histamine-releasing activity. HLA class II typing is consistent with the concept that CIU is a heterogeneous disease, and supports an autoimmune pathogenesis in a subset of patients.

An interdisciplinary home healthcare program for patients with Parkinson's disease.

The long-term treatment of Parkinson's disease presents a special challenge in the home healthcare setting. This article describes a successful interdisciplinary home healthcare program that addresses the special needs of patients with Parkinson's disease and associated disorders. The program, which combines skilled assessment, professional healthcare services, patient and family education, and case management to optimize patient outcomes, has become a national model for agencies hoping to start or expand Parkinson's disease programs.

HLA-C genes and susceptibility to type 1 autoimmune hepatitis.

Susceptibility to autoimmune hepatitis (AIH) is associated with the HLA A1-B8-DR3 haplotype, DR4 antigen, and, more specifically, the HLA DRB3*0101, DRB1*0301, and DRB1*0401 alleles. Few investigators, however, have examined the HLA C locus in AIH, which warrants detailed study in view of its recently described roles in immunoregulation. Eighty-seven adult, white patients with well-characterized type 1 AIH and 100 controls were studied. HLA C and HLA DRB1 alleles were assigned by polymerase chain reaction (PCR)-based genotyping. HLA A and B antigens were determined by standard microlymphocytotoxicity assay. Extended haplotypes were constructed according to known patterns of linkage disequilibrium. Only one HLA C locus allele, Cw*0701, which was present in 54% of patients versus 34% of controls (P = .006; relative risk [RR] = 1.54) was associated with AIH. The overall increase in the frequency of the Cw*07 gene (70.1% of patients vs. 54% of controls; P = .024; RR = 1.3) was due entirely to inheritance of the Cw*0701 allele rather than the other Cw*07 alleles, Cw*0702, *0703, and *0704. The RR for Cw*0701 (RR = 1.54) is greater than that for HLA A1 (RR = 1.33) and DRB1*0301 (RR = 1.49), but less than that for HLA-B8 (RR = 1.75). The present findings suggest that the gene or genes conferring susceptibility to AIH lie in the region centromeric to the HLA A locus between HLA C and DRB1. Although linkage disequilibrium with both B8 and DRB1*0301 may account for our finding of an increased frequency of Cw*0701, it is also possible that this allele contributes to disease susceptibility, perhaps by interaction with natural killer cells or cytotoxic T lymphocytes.

Somatic embryogenesis and plant regeneration from stem explants of Moricandia arvensis.

Efficient and reproducible plant regeneration has been established from stem internode explants of Moricandia arvensis, a crucifer of special interest due to its C3-C4 intermediate photosynthetic activity. Somatic embryogenesis was induced in one-third of explants cultured on Murashige and Skoog based medium containing 9 mM 2,4-dichlorophenoxyacetic acid. High frequencies of plant regeneration (>90%) resulted when somatic embryos were germinated on medium lacking growth regulators. Regenerated plants were diploid, fertile and morphologically similar to seed-derived plants of M. arvensis. This is the first report of somatic embryogenesis in M. arvensis. This plant regeneration system should facilitate gene identification and localisation studies of C3-C4 physiology by insertional mutagenesis, a prerequisite for the isolation and transfer of genes involved in C3-C4 metabolism from Moricandia to cultivated brassicas.

Expression of the C3-C 4 intermediate character in somatic hybrids between Brassica napus and the C3-C 4 species Moricandia arvensis.

The wild crucifer Moricandia arvensis is a potential source of alien genes for the genetic improvement of related Brassica crops. In particular M. arvensis has a C3-C4 intermediate photosynthetic mechanism which results in enhanced recapture of photorespired CO2 and may increase plant water-use efficiency. In order to transfer this trait into Brassica napus, somatic hybridisations were made between leaf mesophyll protoplasts from cultured M. arvensis shoot tips and hypocotyl protoplasts from three Brassica napus cultivars, 'Ariana', 'Cobra' and 'Westar'. A total of 23 plants were recovered from fusion experiments and established in the greenhouse. A wide range of chromosome numbers were observed among the regenerated plants, including some apparent mixoploids. Thirteen of the regenerated plants were identified as nuclear hybrids between B. napus and M. arvensis on the basis of isozyme analysis. The phenotypes of these hybrids were typically rather B. napus-like, but much variability was observed, including variation in flower colour, leaf shape and colour, leaf waxiness, fertility and plant vigour. CO2 compensation point measurements on the regenerated plants demonstrated that 3 of the hybrids express the M. arvensis C3-C4 intermediate character at the physiological level. Semi-thin sections through leaf tissues of these 3 plants revealed the presence of a Kranz-like leaf anatomy characteristic of M. arvensis but not found in B. napus. This is the first report of the expression of this potentially important agronomic trait, transferred from Moricandia, in M. arvensis x B. napus hybrids.

The effects of proline, thioproline and methylglyoxal-bis-(guanylhydrazone) on shoot regeneration frequencies from stem explants of B. napus.

Internode segments from aseptic shoot cultures are the most prolific explants for the regeneration of Brassica shoots in vitro. These explants also have the advantage of not being subject to the genotypic variations in regeneration response observed in hypocotyl and cotyledon explants. Despite reports of 80-100% shoot regeneration from stem explants, observed frequencies are typically 50-60%. Three media additives, proline, thioproline and methylglyoxal-bis-(guanylhydrazone) (MGBG), were tested for their efficacy in promoting shoot regeneration from stem explants of two B. napus varieties, Westar and Cobra. The effects of proline and thioproline on both varieties were neutral or deleterious. In Cobra the MGBG treatments caused a uniform reduction in explant regeneration. However, at low concentrations (0.35µM) MGBG resulted in a 50% increase, to 92%, in regeneration from Westar. The potential of MGBG in promoting explant regeneration in B. napus is discussed in the light of its interaction with the explant genotype.

Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP).

We have developed a single DNA typing method which uses 144 sequence-specific primer (SSP) reactions to simultaneously detect all known HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as "Phototyping". Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA-B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem-free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.

Central venous oxygen saturation, arterial base deficit, and lactate concentration in trauma patients.

Our object was to explore the usefulness of central venous oxygen saturation, arterial base deficit, and lactate concentration in the evaluation of trauma patients. In busy urban trauma centers, limited operating room availability may necessitate that certain hemodynamically stable patients experience some delay between diagnosis of injury and surgery. Because hemodynamic compromise may occur before operation is undertaken, some means of identifying those patients who have the most severe injuries or who are at greatest risk for hemodynamic instability would be useful. We prospectively studied 40 patients with operative truncal injuries admitted to the Cook County Trauma Unit, Chicago, to examine the usefulness of postresuscitation central venous oxygen saturation (ScvO2), arterial lactate concentration, and arterial base deficit in this regard. Preoperative hypotension occurred in 12.5 per cent of these initially stable patients. ScvO2 did not significantly correlate with any of the parameters of blood loss and severity of injury examined. However, both base deficit and lactate concentration correlated with transfusion requirements; in addition, base deficit correlated with trauma score, and lactate correlated with peritoneal shed blood volume. Our data suggest that, after resuscitation, arterial base deficit and lactate concentration may be better indicators of blood loss than is ScvO2.

Effects of ischaemic conditions on uptake of glutamate, aspartate, and noradrenaline by cell lines derived from the human nervous system.

The effect of hypoglycaemic, hypoxic, and ischaemic conditions on high-affinity neurotransmitter transport was studied in the human astrocytoma clone D384 and the human neuroblastoma clone SH-SY5Y. Both cell lines expressed a sodium-dependent glutamate/aspartate transporter. Km values for D-[3H]aspartate uptake were 6.1 +/- 0.9 microM for D384 cells and 5.3 +/- 0.3 microM for SH-SY5Y cells (mean +/- SEM of three experiments). In addition, SH-SY5Y, but not D384, expressed a sodium-dependent noradrenaline transporter with Km = 0.6 +/- 0.1 microM (mean +/- SEM of three experiments). Up to 3 h of hypoglycaemic conditions had no effect on neurotransmitter uptake or on ATP levels of each cell line. In sharp contrast, during hypoxic conditions, the uptake of D-[3H]-aspartate and [3H]noradrenaline declined by 43-56% within 5 min. These reduced rates of neurotransmitter uptake were maintained over 30 min of hypoxic conditions. Five minutes of ischaemic conditions caused similar reductions in neurotransmitter uptake rates. A correlation between reductions in rates of neurotransmitter uptake and in ATP levels was observed for each cell line. Results are discussed in relation to other brain preparations, which are used as models of the nervous system to study the effects of ischaemic conditions on neurotransmitter and energy metabolism.

Grafting in vitro.

Graft formation in plants involves the severing of the vascular system with consequent loss of water and solute transport throughout the plant. This transport must be restored to prevent death resulting from nutrient starvation or dessication.