An EST-enriched comparative map of Brassica oleracea and Arabidopsis thaliana

A detailed comparative map of Brassica oleracea and Arabidopsis thaliana has been established based largely on mapping of Arabidopsis ESTs in two Arabidopsis and four Brassica populations. Based on conservative criteria for inferring synteny, "one to one correspondence" between Brassica and Arabidopsis chromosomes accounted for 57% of comparative loci. Based on 186 corresponding loci detected in B. oleracea and A. thaliana, at least 19 chromosome structural rearrangements differentiate B. oleracea and A. thaliana orthologs. Chromosomal duplication in the B. oleracea genome was strongly suggested by parallel arrangements of duplicated loci on different chromosomes, which accounted for 41% of loci mapped in Brassica. Based on 367 loci mapped, at least 22 chromosomal rearrangements differentiate B. oleracea homologs from one another. Triplication of some Brassica chromatin and duplication of some Arabidopsis chromatin were suggested by data that could not be accounted for by the one-to-one and duplication models, respectively. Twenty-seven probes detected three or more loci in Brassica, which represent 25.3% of the 367 loci mapped in Brassica. Thirty-one probes detected two or more loci in Arabidopsis, which represent 23.7% of the 262 loci mapped in Arabidopsis. Application of an EST-based, cross-species genomic framework to isolation of alleles conferring phenotypes unique to Brassica, as well as the challenges and opportunities in extrapolating genetic information from Arabidopsis to Brassica and to more distantly related crops, are discussed.     

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Expression of the macrophage scavenger receptor, a multifunctional lipoprotein receptor, in microglia associated with senile plaques in Alzheimer's disease.

The macrophage scavenger receptor is a multifunctional receptor whose ligands include oxidized low density lipoprotein (LDL), as well as several other polyanionic macromolecules. Although the capacity of the receptor to bind modified LDL has implicated it in the process of atherosclerosis, its physiological role remains uncertain. We have examined human brain for expression of macrophage scavenger receptor as part of ongoing studies of lipoprotein receptors in the central nervous system. The receptor is expressed on microglia, but not on astrocytes, neurons, or vessel-associated structures. In Alzheimer disease, there is strong expression of the scavenger receptor in association with senile plaques.     

Hormone therapy and affect

This study investigated the influence of hormone therapy on affect in a double blind crossover trial. The sample consisted of 49 women who had previously undergone hysterectomy and bilateral oophorectomy. Therapy consisted of 3 mth each of ethinyl oestradiol — 50 μg/day, levonorgestrel — 250 μg/day, “Nordial” — a combination of these two substances, and placebo. Affect was measured by the Hamilton Depression Rating Scale, verbal reports by women and self-ratings on visual analogue scales. Ethinyl oestradiol was found to have a beneficial influence on aspects of affect such as Hamilton scores, anxiety, irritability and insomnia.

The influence of hormones on Hamilton scores could be partly but not fully explained by the alleviation of hot flushes. Norgestrel showed less favourable changes initially but these tended to diminish by the third therapy month. Most of the women studied were not clinically depressed. Anxiety symptoms were the major features exhibited in the group of women investigated. The results of this study suggest that visual analogue rating scales are of questionable validity in assessing affect in patients without any appreciable psychiatric morbidity.     

Reduction of adhesion formation in rabbits by intraperitoneal administration of lazaroid formulations

Adhesion formation is a major source of postoperative morbidity and mortality. In this study, the ability of a variety of lazaroid formulations [the antioxidant 21-aminosteroid PNU74006F (tirilazad) and the non-steroidal 2-methylaminochroman derivative PNU83,836E] to reduce i.p. adhesion formation in three rabbit models was examined. In initial studies, PNU83836E was administered via Alzet miniosmotic pump to the site of injury. In the sidewall and double uterine horn models, PNU83,836E was administered via Alzet miniosmotic pump for the entire postoperative interval. In the sidewall model, there was a dose- dependent reduction in the area of the sidewall injury that was involved in adhesions. In the double uterine horn model, PNU83,836E was administered via Alzet miniosmotic pump to the area of injury for 1, 2, 3 or 7 days. Administration for as little as 24 h after surgery significantly reduced the extent of adhesion formation and the reduction was increased if it was administered for longer. Further studies were conducted in which various lazaroid formulations were administered as a bolus at the end of surgery. In both the sidewall and double uterine horn models, administration of either PNU83,386E (in citrate buffer) or PNU74006F (in cyclodextrin or lipid emulsion vehicles) at the end of surgery reduced adhesion formation. Administration of a bolus of PNU74006F 10 min prior to initiation of surgery with or without additional treatment at the end of surgery further increased its efficacy in the reduction of adhesion formation. Administration of a minimum of 1.5 mg before and after surgery (3 mg total) was required for maximal efficacy. These studies demonstrate that pre- and postoperative administration of either a steroidal (PNU74006F) or non-steroidal (PNU83,836E) lazaroid intraperitoneally reduced the formation and reformation of postoperative adhesions in three animal models.      

Alteration in the pattern of nerve terminal protein immunoreactivity in the perforant pathway in Alzheimer's disease and in rats after entorhinal lesions.

Neurons in layer II of the entorhinal cortex consistently develop neurofibrillary tangles in Alzheimer's disease (AD). Experimental neuroanatomical studies have shown that these neurons give rise to the perforant pathway, a major excitatory projection to the hippocampal formation, which terminates in a discrete pattern in the outer portion of the molecular layer of the dentate gyrus. The distribution of two nerve terminal associated proteins, synaptophysin and NT75, was studied in the molecular layer of the dentate gyrus in AD and control cases to determine whether Alzheimer neuronal pathology is associated with loss of synaptic markers. In parallel studies, the effect of ablation of the entorhinal cortex in rats was evaluated. In AD as compared to controls, a decrease in synaptophysin immunostaining was evident in the terminal zone of the perforant pathway. NT75 nerve terminal immunostaining was too weak to interpret in the human hippocampal formation. Both synaptophysin and NT75 immunoreactivity were found in association with some neuritic plaques. In rats, entorhinal lesions resulted in diminished immunoreactivity for both synaptophysin and NT75 in the perforant pathway terminal zone. These results suggest that nerve terminal protein loss is a concomitant feature of neuronal pathology in AD.     

Genome location of polyadenylated transcripts of herpes simplex virus type 1 and type 2 DNA.

The genome location of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) polyadenylated RNA has been studied by hybridization of herpesvirus-specific nuclear and cytoplasmic polyadenylated RNA to the Eco R, restriction enzyme fragments of herpesvirus DNA, which have been separated by agarose gel electrophoresis and transferred to nitrocellulose paper. For both HSV-1 and HSV-2 DNA, the nuclear and the cytoplasmic polyadenylated RNA are distributed on all the Eco R_I fragments of the homologous DNA. In a heterologous system, total polyadenylated RNA from HSV-1-infected cells was annealed to the Eco R_I fragments of HSV-2 DNA and vice versa. HSV-2 polyadenylated RNA hybridized to all fragments of HSV-1 DNA, and HSV-1 polyadenylated RNA hybridized to all but three of the fragments of HSV-2 DNA. These data indicate that the DNA base sequence homology between the two herpesvirus strains is dispersed throughout the genome and is not located in a single contiguous block.