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.

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.

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.

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.

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.

Quantitative detection of Streptococcus pneumoniae in nasopharyngeal secretions by real-time PCR.

Streptococcus pneumoniae is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pneumoniae may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of S. pneumoniae cells in which the quantities of DNA ranged from the amounts extracted from 1 to 10(6) cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient of S. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemophilus influenzae, Moraxella catarrhalis, or alpha-hemolytic streptococci other than S. pneumoniae were not amplified or were only weakly amplified when there were > or =10(6) cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of S. pneumoniae organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of S. pneumoniae present in NPSs. This assay may serve as a tool to study changes in the amounts of S. pneumoniae during lower respiratory tract infections.

2.8-A crystal structure of a nontoxic type-II ribosome-inactivating protein, ebulin l.

Ebulin l is a type-II ribosome-inactivating protein (RIP) isolated from the leaves of Sambucus ebulus L. As with other type-II RIP, ebulin is a disulfide-linked heterodimer composed of a toxic A chain and a galactoside-specific lectin B chain. A normal level of ribosome-inactivating N-glycosidase activity, characteristic of the A chain of type-II RIP, has been demonstrated for ebulin l. However, ebulin is considered a nontoxic type-II RIP due to a reduced cytotoxicity on whole cells and animals as compared with other toxic type-II RIP like ricin. The molecular cloning, amino acid sequence, and the crystal structure of ebulin l are presented and compared with ricin. Ebulin l is shown to bind an A-chain substrate analogue, pteroic acid, in the same manner as ricin. The galactoside-binding ability of ebulin l is demonstrated crystallographically with a complex of the B chain with galactose and with lactose. The negligible cytotoxicity of ebulin l is apparently due to a reduced affinity for galactosides. An altered mode of galactoside binding in the 2gamma subdomain of the lectin B chain primarily causes the reduced affinity.

An interaction between ricin and calreticulin that may have implications for toxin trafficking.

Here we demonstrate that ricin is able to interact with the molecular chaperone calreticulin both in vitro and in vivo. The interaction occurred with ricin holotoxin, but not with free ricin A chain; and it was prevented in the presence of lactose, suggesting that it was mediated by the lectin activity of the ricin B chain. This lectin is galactose-specific, and metabolic labeling with [(3)H]galactose or treating galactose oxidase-modified calreticulin with sodium [(3)H]borohydride indicated that Vero cell calreticulin possesses a terminally galactosylated oligosaccharide. Brefeldin A treatment indicated that the intracellular interaction occurred initially in a post-Golgi stack compartment, possibly the trans-Golgi network, whereas the reductive separation of ricin subunits occurred in an earlier part of the secretory pathway, most probably the endoplasmic reticulum (ER). Intoxicating Vero cells with ricin whose A chain had been modified to include either a tyrosine sulfation site or the sulfation site plus available N-glycosylation sites, in the presence of Na(2)35SO(4), confirmed that calreticulin interacted with endocytosed ricin that had already undergone retrograde transport to both the Golgi and the ER. Although we cannot exclude the possibility that the interaction between ricin and calreticulin is an indirect one, the data presented are consistent with the idea that calreticulin may function as a recycling carrier for retrograde transport of ricin from the Golgi to the ER.

Ribosome-mediated folding of partially unfolded ricin A-chain.

After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.

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.

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.

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.

The deoxyribonuclease activity attributed to ribosome-inactivating proteins is due to contamination.

The mode of action of ribosome-inactivating proteins (RIPs) has, for many years, been considered to be depurination of a specific adenyl residue of ribosomal RNA, resulting in inhibition of protein synthesis. Recently, this view has been challenged by the observation that many RIP preparations have significant DNase activity in addition to their N-glycosidase activity. In this study, we have investigated the putative DNase activity of two RIPs, ricin and pokeweed antiviral protein (PAP), and show that, in both cases, the DNase activity is due to the presence of contaminating nucleases. The N-glycosidase and DNase activities of PAP were separately and specifically inactivated by chemical modification and heat. Gel filtration of ricin allowed physical separation of the two activities. Furthermore, neither recombinant PAP nor recombinant ricin A-chain purified from Escherichia coli displayed DNase activity.

Kinetic analysis of barley chitinase.

The endochitinase from barley is the archetypal enzyme for a large class of plant-derived antifungal chitinases. The X-ray structure was solved previously in our laboratory and a mechanism of action proposed based on structural considerations. In this manuscript we report the use of a defined soluble substrate, 4-methylumbelliferyl beta-N,N',N"-triacetylchitotrioside, to characterize kinetic parameters of the enzyme. The pH profile shows that activity is controlled by a base with a pKa of 3.9 (Glu 89) and an acid with a pKa of 6.9 (Glu 67). The Km using the synthetic substrate is 33 microM, and the k(cat) is 0.33 min(-1), while the Km for (GlcNAc)4 is 3 microM and k(cat) is 35 min(-1). Binding constants were measured for beta-linked oligomers of N-acetylglucosamine. The monomer does not bind and dissociation constants for the dimer, trimer, and tetramer are 43, 19, and 6 microM, respectively. Analysis of kinetic and dissociation constants proves the mechanism of barley chitinase is consistent with a Bi-Bi kinetic model for hydrolysis, with (GlcNAc)4 and water as substrates and (GlcNAc)2 as products. Substrate cleavage patterns show that (GlcNAc)6 is cleaved in half to (GlcNAc)3 as well as into (GlcNAc)4 and (GlcNAc)2 with almost equal efficiency. NMR analysis of cleavage products confirms that the enzyme proceeds with anomeric inversion of products.

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.

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/.

Structure and activity of an active site substitution of ricin A chain.

The A chain of ricin (RTA) is an N-glycosidase which inactivates ribosomes by removing a single adenine base from a conserved region of rRNA. X-ray structures and site-directed mutagenesis revealed that Arg 180 interacts with the target adenine hydrogen bonding with N3. It may fully or partially protonate that atom as part of the hydrolysis mechanism. Arg 180 was previously converted to His (R180H) and shown to greatly reduce activity. Here R180H is shown to reduce overall activity 500-fold against Artemia salina ribosomes. A 2.2 A crystal structure reveals the mutation causes a rearrangement of the active site cleft, with Tyr 80 moving to block access to the adenine recognition site. His 180 forms a strong aromatic interaction with Trp 211, Tyr 80, and Tyr 123. A complex is formed with 250 mM AMP. The nucleotide binds in the active site region, but in an apparently nonproductive orientation. His 180 cannot bond to N3 and is screened from the substrate analog by the intervening Tyr 80. It may be that natural polynucleotide substrates, using additional interactions, can displace Tyr 80 and effect a productive binding.

The consistent association between Epstein-Barr virus and Hodgkin's disease in children in Kenya.

Recent studies have suggested that Epstein-Barr virus (EBV) may play a role in the etiology of Hodgkin's disease (HD). In a previous study, we used a latent membrane protein 1 (LMP1)-specific antibodies to examine archival material from 74 British children with HD and found 50% of cases to be positive. It is known that there are geographic and ethnic variations in the incidence of HD. We have investigated LMP1 status in formalin-fixed, paraffin wax-embedded lymph nodes with HD involvement from 53 children and 48 adults from Kenya using immunohistochemical staining. We also developed sensitive and specific in vitro gene amplification protocols for examining the EBV strain type in such material using several combinations of primers derived from the EBNA 2 and EBNA 3 coding regions. LMP1 positivity was present in 100% of the pediatric cases (two lymphocyte-predominant, 25 nodular sclerosis, 16 mixed cellularity, 5 lymphocyte depletion, and 5 unclassified) and in 66% of the adult cases (two of three lymphocyte-predominant, 26 of 39 nodular, sclerosis, two of two mixed cellularity, and two of four lymphocyte depletion). Tests to type the EBV strain were undertaken in 25 EBV-positive pediatric cases. A combination of type-specific polymerase chain reactions for EBNA 2 and EBNA 3C genes indicated that seven patients had type 1, eight had type 2, and 10 had dual infections with both types. Five cases with dual infections were further investigated using a sensitive in situ hybridization for the EBV-encoded, small nuclear nonpolyadenylated RNAs (EBERs). EBER transcripts were detected in Reed-Sternberg and Hodgkin cells and in occasional infiltrating lymphocytes. These observations indicate that in Kenya EBV is consistently associated with pediatric cases of HD, and that biopsies from a number of such cases appear to carry both type 1 and type 2 viral sequences.

The role of Epstein-Barr virus in Hodgkin's disease from different geographical areas.

Recent studies have suggested that Epstein-Barr virus (EBV) may play a role in the aetiology of Hodgkin's disease. To determine the role of EBV in childhood Hodgkin's disease in different geographical areas, immunohistochemical staining and in situ hybridisation were used to analyse latent membrane protein 1 (LMP 1) and small nuclear non-transcribed RNAs (EBER-1) respectively. Testing for EBV within the Reed-Sternberg and Hodgkin's cells was carried out in childhood Hodgkin's disease from 10 different countries. The proportion of LMP 1 positive cases varied significantly, being 50% of cases from the United Kingdom (38/75), South Africa (9/18), Egypt (7/14), and Jordan (8/16), 60% from the United Arab Emirates (6/10), 70% from Australia (11/16), 81% from Costa Rica (34/42), 88% from Iran (7/8), 90% from Greece (20/22), and 100% of the 56 cases from Kenya. A sensitive polymerase chain reaction based EBV strain typing technique was established using archival tissues. EBV strain type 1 was shown to be predominant in childhood Hodgkin's disease from the United Kingdom, South Africa, Australia, and Greece. Type 2 was predominant in Egypt. EBV strain types 1 and 2 were both detected in some cases of childhood Hodgkin's disease in the United Kingdom, Costa Rica, and Kenya. The high incidence of EBV and the presence especially in developing countries of dual infection with both strain types 1 and 2 may reflect socioeconomic conditions leading to malnutrition induced immunological impairment. The possibility of HIV infection also needs to be explored.