A radiation hybrid map of the rat genome containing 5,255 markers.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

Convergent evolution to an aptamer observed in small populations on DNA microarrays.

The development of aptamers on custom synthesized DNA microarrays, which has been demonstrated in recent publications, can facilitate detailed analyses of sequence and fitness relationships. Here we use the technique to observe the paths taken through sequence-fitness space by three different evolutionary regimes: asexual reproduction, recombination and model-based evolution. The different evolutionary runs are made on the same array chip in triplicate, each one starting from a small population initialized independently at random. When evolving to a common target protein, glucose-6-phosphate dehydrogenase (G6PD), these nine distinct evolutionary runs are observed to develop aptamers with high affinity and to converge on the same motif not present in any of the starting populations. Regime specific differences in the evolutions, such as speed of convergence, could also be observed.

A gene map of the human genome.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

L-ß-N-methylamino-l-alanine (BMAA) nitrosation generates a cytotoxic DNA damaging alkylating agent: An unexplored mechanism for neurodegenerative disease.

BACKGROUND: L-ß-N-methylamino-l-alanine (BMAA) is a non-proteinic amino acid, that is neurotoxic in vitro and in animals, and is implicated in the causation of amyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS-PDC) on Guam. Given that natural amino acids can be N-nitrosated to form toxic alkylating agents and the structural similarity of BMAA to other amino acids, our hypothesis was that N-nitrosation of BMAA might result in a toxic alkylating agent, providing a novel mechanistic hypothesis for BMAA action. FINDINGS: We have chemically nitrosated BMAA with sodium nitrite to produce nitrosated BMAA (N-BMAA) which was shown to react with the alkyl-trapping agent, 4-(p-nitrobenzyl)pyridine, cause DNA strand breaks in vitro and was toxic to the human neuroblastoma cell line SH-SY5Y under conditions in which BMAA itself was minimally toxic. CONCLUSIONS: Our results indicate that N-BMAA is an alkylating agent and toxin suggesting a plausible and previously unrecognised mechanism for the neurotoxic effects of BMAA.

Steroid recognition by chloramphenicol acetyltransferase: engineering and structural analysis of a high affinity fusidic acid binding site.

The antibiotic fusidic acid and certain closely related steroidal compounds are potent competitive inhibitors of the type I variant of chloramphenicol acetyltransferase (CATI). In the absence of crystallographic data for CATI, the structural determinants of steroid binding were identified by (1) construction in vitro of genes encoding chimaeric enzymes containing segments of CATI and the related type III variant (CATIII) and (2) site-directed mutagenesis of the gene encoding CATIII, followed by kinetic characterisation of the substituted variants. Replacement of four residues of CATIII (Gln92, Asn146, Tyr168 and Ile172) by their equivalents from CATI yields an enzyme variant that is susceptible to competitive inhibition by fusidate with respect to chloramphenicol (Ki = 5.4 microM). The structure of the complex of fusidate and the Q92C/N146F/Y168F/I172V variant, determined at 2.2 A resolution by X-ray crystallography, reveals the inhibitor bound deep within the chloramphenicol binding site and in close proximity to the side-chain of His195, an essential catalytic residue. The aromatic side-chain of Phe146 provides a critical hydrophobic surface which interacts with non-polar substituents of the steroid. The remaining three substitutions act in concert both to maintain the appropriate orientation of Phe 146 and via additional interactions with the bound inhibitor. The substitution of Gln92 by Cys eliminates a critical hydrogen bond interaction which constrains a surface loop (residues 137 to 142) of wild-type CATIII which must move in order for fusidate to bind to the enzyme. Only two hydrogen bonds are observed in the CAT-fusidate complex, involving the 3-alpha-hydroxyl of the A-ring and both hydroxyl of Tyr25 and NE2 of His195, both of which are also involved in hydrogen bonds with substrate in the CATIII-chloramphenicol complex. In the acetyl transfer reaction catalysed by CAT, NE2, of His195 serves as a general base in the abstraction of a proton from the 3-hydroxyl of chloramphenicol as the first chemical step in catalysis. The structure of the CAT-inhibitor complex suggests that deprotonation of the 3-alpha-hydroxyl of bound fusidate by this mechanism could produce an oxyanion nucleophile analogous to that seen with chloramphenicol, but one which is incorrectly positioned to attack the thioester carbonyl of acetyl-CoA, accounting for the observed failure of CAT to acetylate fusidate.

Miniaturised nucleic acid analysis.

The application of micro total analysis systems has grown exponentially over the past few years, particularly diversifying in disciplines related to bioassays. The primary focus of this review is to detail recent new approaches to sample preparation, nucleic acid amplification and detection within microfluidic devices or at the microscale level. We also introduce some applications that have as yet to be explored in a miniaturised environment, but should benefit from improvements in analytical efficiency and functionality when transferred to planar-chip formats. The studies described in this review were published in commonly available journals as well as in the proceedings of three major conferences relevant to microfluidics (Micro Total Analysis Systems, Transducers and The Nanotechnology Conference and Trade Show). Although an emphasis has been placed on papers published since 2002, pertinent articles preceding this publication year have also been included.

Relation of level of total serum cholesterol to amount of calcific deposits in operatively excised stenotic mitral valves: analysis of 155 cases.

This report analyzes 155 patients with rheumatic mitral stenosis in whom the operatively excised mitral valve was x-rayed to determine the presence of and extent of calcific deposits and the preoperative level of total serum cholesterol (TC). The amount of mitral calcium was graded 0 to 4+ and the average TC for each of the 5 groups was: 0 deposits--21 patients (14%) (TC = 188 mg/dl); 1+-50 patients (32%) (TC = 196 mg/dl); 2+-22 patients (14%) (TC = 198 mg/dl); 3+-37 patients (24%) (TC = 205 mg/dl), and 4+-25 patients (16%) (TC = 184 mg/dl). These average values of TC and the mean ages of the patients in each of the 5 groups of mitral calcium were not significantly different.

Electrocardiographic observations in clinically isolated, pure, chronic, severe aortic regurgitation: analysis of 30 necropsy patients aged 19 to 65 years.

Certain electrocardiographic findings are described in 30 necropsy patients with clinically isolated pure, chronic, severe aortic regurgitation. They were 19 to 65 years old (mean 45). The hearts of the 22 men ranged in weight from 430 to 1,110 g (mean 717) and of the 8 women, from 375 to 950 g (mean 638). Four had grossly visible left ventricular (LV) scars. All but 1 patient was in sinus rhythm. The PR interval was greater than 0.20 second in 8 patients (28%) and the QRS duration was greater than or equal to 0.12 second in 6 patients (20%). Only 5 patients (17%) had 1 or more ventricular premature complexes recorded on the resting electrocardiogram analyzed. The mean QRS amplitude for each of the 12 leads averaged 23 mm. The highest mean QRS voltage occurred in leads V2 and V3 (each 38 mm), and the lowest in lead aVR (11 mm). The mean QRS voltage in V5 was higher than in V6 (33 vs 28 mm) and in 22 patients (73%) the QRS voltage in V5 was higher than in V6. The sum of the S wave in V1 plus the larger of the R wave in V5 or V6 (Sokolow-Lyon index) averaged 51 mm and in only 22 patients (73%) was it greater than 35 mm. The Romhilt-Estes voltage criteria for LV hypertrophy was fulfilled even less frequently, despite the severe degrees of LV hypertrophy in the patients studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Amounts of coronary arterial narrowing by atherosclerotic plaques in clinically isolated, chronic, pure aortic regurgitation: analysis of 37 necropsy patients older than 30 years.

The degree of cross-sectional area (XSA) narrowing by atherosclerotic plaque in each of the 4 major epicardial coronary arteries (right, left main, left anterior descending and left circumflex) was determined at necropsy in 37 patients (30 men and 7 women) aged 34 to 77 years (mean 54) with severe, isolated, chronic, pure aortic regurgitation (AR). In 7 patients (19%), greater than or equal to 1 major coronary artery was narrowed 76 to 100% in XSA at some point. Of the 148 major coronary arteries examined in the 37 patients, 12 arteries (8%) were narrowed at some point 76 to 100% in XSA. Each of the 148 major coronary arteries were divided into 5-mm-long segments (average 53 per patient) and a histologic section from each segment was examined. Of the 1,977 segments, 1,087 were narrowed 0 to 25%, 669 (34%) 26 to 50%, 170 (9%) 51 to 75%, 48 (2%) 76 to 95% and 3 (0.001%) 96 to 100%. The average amount of XSA narrowing by atherosclerotic plaque per segment was about 28%. Of the 37 patients, 9 had had angina pectoris, 2 of whom had significant (greater than 75% XSA reduction) coronary narrowing; 2 other patients had had acute myocardial infarction clinically, 1 of whom had significant coronary narrowing at necropsy. Thus, in general, the amount of coronary narrowing in our 37 adults with severe, pure, isolated, chronic AR was relatively mild.

An improved 191Os/191mIr generator using a hybrid anion exchanger.

Iridium-191m is an attractive radionuclide for first-pass radionuclide angiography, but development of an 191Os/191mIr generator with high 191mIr yield has proven difficult. The use of trans-dioxobisoxalatoosmate(VI) as a parent species results in a higher initial yield (20 to 25%/mL), but the yield decreases over time. Using an anion-exchange column of tridodecylmethylammonium chloride-treated silica gel and AG MP-1 resin, we have developed an improved 191Os/191mIr generator with higher initial yield (25 to 30%/mL) and a slower rate of decrease than the previous design.

Synthesis and biodistribution of 64Cu-labeled monocationic diiminedioxime copper(II) complexes.

Monocationic copper(II) complexes of three derivatives of the diiminedioxime ligand 2,10-dioximino-3,9-dimethyl-4,8-diazaundeca-3,8-diene were labeled with 64Cu in high radiochemical yield, and the biodistribution of the complexes was measured in mice. The concentration of the complexes in the heart was low, but the partition coefficients of the complexes are less than optimal for a myocardial perfusion agent. All three complexes are resistant to reduction by glutathione in vitro. This suggests that more lipophilic derivatives of these compounds merit further investigation as possible myocardial perfusion agents.

Comparison of uptake of 99mTc-alkylisonitriles in the rat 9L gliosarcoma tumor model.

The accumulation of three 99mTc(I) alkyl isonitriles was compared in vitro and in vivo using 9L gliosarcoma cells. In vitro, the uptake of 99mTc-EIBI and 99mTc-EPI was higher than that of 99mTc-MIBI. In vivo, however, there was no difference in the tumor concentration at 15 or 60 min postinjection and only a small difference at 24 h. The differences in uptake observed in vitro are apparently offset in vivo by differences in delivery of the tracers to the tumor.

Evans' syndrome, myelofibrosis and systemic lupus erythematosus: role of procollagens in myelofibrosis.

We describe an 18 yr old female with systemic lupus erythematosus presenting with Evans' syndrome (autoimmune hemolytic anemia and immune thrombocytopenia) and dense myelofibrosis. Her clinical course and response to treatment were monitored with regular blood counts, serial measurements of serum procollagen I and III and periodic bone marrow examinations. This report brings to 9 the number of well-documented cases of systemic lupus erythematosis and myelofibrosis in the literature; we discuss the pathogenesis and management of patients with this apparently rare disorder, with particular reference to the role of serum procollagen measurements as markers of myelofibrosis.

Effects of soybean polysaccharide on plasma lipids.

Free-living volunteers with mild to moderate hypercholesteremia added 25 gm soybean polysaccharide or starch placebo in crouton or cookie form to their normal, daily diets. A total of 31 persons completed the blind, crossover design, 8-week, experimental protocol. Subjects ingesting soybean polysaccharide prior to placebo showed an 11% decrease (from 252 to 224 mg/dl) in total plasma cholesterol; those who followed placebo with fiber showed a 5% decrease (from 241 to 230 mg/dl). Starch placebo was associated with a 2% decrease in total cholesterol when consumed first and a 4% increase when consumed following the fiber consumption period. High-density-lipoprotein (HDL) cholesterol decreased 8% and 6% from initial values during the first period for the fiber and starch groups, respectively. HDL cholesterol increased 2% but decreased 1% during the second period for starch and fiber, respectively. No significant changes in triglyceride levels occurred. The data indicate that soybean polysaccharide fiber promotes a significant decrease in plasma cholesterol in persons with mild to moderate hypercholesteremia. The addition of fiber may represent an important adjunct to traditional fat- and cholesterol-controlled diets for such persons.

Catherine Burns 'expendable'?

The resignation of the Health Visitors' Association General Secretary Catherine Burns must be of grave concern to all those members who understood what she was fighting for, which was typified in the proposal that there should be only one seat for health visitors on the UKCC (News, March 11).

Protein profiling of keloidal scar tissue.

Clinically, keloids are defined as scars that invade adjacent healthy tissue and are caused by tissue injury; however, the clear distinction relating keloids to wound healing or to cancer remains elusive. This study profiled protein extracts from the regions of keloid tissues to determine the activities within these different zones, and to help underpin the activities apparent within different regions of the disease. Two-dimensional gel electrophoresis, liquid chromatography mass spectrometer (LCMS) and a Mascot online database search were employed for comparative proteomic analysis between four sites in keloidal scars (KS). Out of 400 spots identified in all gels, 21 were unique to given KS sites. These were identified using LCMS and the results showed the presence of mitochondrial and structural proteins in the margin, while the top contained keratin II. Heat shock protein was the only protein present in the internal normal control and margin, while the external normal contain keratin II and the voltage-dependent anion-selective channel protein, annexin A6, plus glial fibrillary acidic protein. This work is novel since it has identified differentially expressed proteins across the tested keloidal scar sites. The presence of mitochondrial-associated proteins at the margins suggests that this is the most active part of the scar.

Levels of 5' RNA tags in plasma and buffy coat from EDTA blood increase with time.

BACKGROUND: For biological sample banking it is important to precisely document sample treatment prior to extraction and storage. A major variable is the interval between blood sampling and subsequent processing and storage. We have determined the relationship between this time interval and frequency of 5' transcript tags. This study was designed to establish guidelines for collecting RNA from blood in prospective studies and ensure maximum availability of RNA analytes. METHODS: Venous blood was collected from 40 healthy volunteers. Samples were processed immediately, 12, 24 and 36 h post collection and buffy coat and/or plasma removed. Total RNA was extracted and reverse transcribed, assays were optimized and levels of 5' RNA tags quantified by qPCR. RESULTS: Stably expressed reference genes were selected to examine 5' tags in plasma and buffy coat blood fractions. Whole blood was processed at various time points post collection to determine the affect on the presence and stability of 5' RNA tags. A significant increase (P < 0.05 to P < 0.001) in 5' RNA tags was observed at 12 h and up to 36 h in plasma and buffy coat samples isolated from EDTA blood which was maintained at 4 degrees C prior to processing when compared with plasma and buffy coat isolated from EDTA blood processed immediately. CONCLUSIONS: Over time 5' RNA tags increase in both plasma and buffy coat samples. It has been previously shown that removing cells from their normal environment produces cellular activation and up-regulation of pathways resulting in increased transcript expression. Positive correlation was observed between the time interval from sample collection to storage and amount of 5' transcript tags present. This increase could be due to white blood cells undergoing necrosis and lysis, or from RNA protected within apoptotic bodies. As 5' RNA tags were targeted using random primers for reverse transcription, even RNA partly degraded by RNases would have been detected.

Gene expression profiling of normal and ruptured canine anterior cruciate ligaments.

OBJECTIVE: To identify genes which may be involved in the development of anterior cruciate ligament (ACL) laxity and rupture in a naturally occurring canine osteoarthritis (OA) model. DESIGN: Three groups of dog were studied: (1) dogs with ACL rupture; (2) dogs with intact ACLs from a breed predisposed to ACL rupture; (3) dogs with intact ACLs from a breed at very low risk of rupture. The transcriptomes of the ACLs from each group were compared using a whole genome microarray and quantitative reverse transcriptase polymerase chain reaction. Differential gene expression in ruptured canine ACLs was compared with that published in the literature for ruptured human ACLs. RESULTS: No significant differences were identified between the gene expression profiles of normal ACLs of a breed predisposed to ACL rupture when compared to a breed relatively resistant to ACL rupture. A general pattern of increased protease and extracellular structural matrix gene expression was identified in the ruptured ACLs when compared to intact ACLs. The gene expression profiles of ruptured canine ACLs demonstrate similar patterns to those previously reported for ruptured human ACLs. CONCLUSIONS: A transcriptomic basis to breed specific risk for the development of canine ACL rupture was not identified. Although changes in matrix associated gene expression in the ruptured ACL are similar between humans and dogs, the molecular events which may predispose to ACL laxity and rupture were not defined.

Acetyl coenzyme A binding by chloramphenicol acetyltransferase. Hydrophobic determinants of recognition and catalysis.

The preponderance of nonpolar contacts between CoA and chloramphenicol acetyltransferase in the high resolution structure of the binary complex prompted a study of selected hydrophobic residues by site-directed mutagenesis and steady-state kinetic analysis. Substitutions of three aromatic residues were used to evaluate binding contacts with the adenine moiety of CoA (Tyr-178), the pantetheine arm of the coenzyme (Tyr-56), and the S-acyl substituent (Phe-33). For those substitutions at residues 56 and 178 that cannot promote alternative polar interactions there is a correlation between residue hydrophobicity and the free energy of formation of the binary and ternary complexes of acetyl-CoA and chloramphenicol acetyltransferase and of the transition-state complex. Substitutions at Tyr-178 destabilize all such complexes to approximately the same extent (uniform binding changes), whereas those at Tyr-56 and Phe-33 cause differential binding changes, having a greater effect on the transition state than on either of the other complexes with acetyl-CoA.