New energy transfer dyes for DNA sequencing

We have synthesized a set of four energy transfer dyes and demonstrated their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a novel set of four 4,7-dichloro-substituted rhodamine dyes which have narrower emission spectra than the standard, unsubstituted rhodamines. A rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequencing instrument equipped with a dual line argon ion laser (488 and 514 nm excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvement in signal-to-noise was 4- to 5-fold. The utility of the new dye set was demonstrated by sequencing of a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients and the relative quantum yields of the dichlororhodamine components of the energy transfer dyes showed their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.     

DSM-IV field trials for oppositional defiant disorder and conduct disorder in children and adolescents

OBJECTIVE: The purpose of the field trials for oppositional defiant disorder and conduct disorder was to select valid diagnostic thresholds for these disorders and to compare the psychometric properties of DSM-IV criteria for oppositional defiant disorder and conduct disorder with previous DSM diagnostic formulations. METHOD: Structured diagnostic interviews, standardized clinician's validation diagnoses, and multiple measures of impairment were obtained for 440 clinic-referred children and adolescents aged 4-17 years. RESULTS: A diagnostic threshold of four symptoms of oppositional defiant disorder optimized identification of impaired children, improved agreement somewhat with the clinician's validation diagnosis, and had somewhat better test-retest agreement than DSM-III-R. In the case of conduct disorder, the optimal time window for ascertainment of symptoms was clarified. A diagnostic threshold of three symptoms of conduct disorder maximized accurate identification of impaired children and agreement with the clinician's validation diagnosis and resulted in slightly better test-retest agreement than DSM-III-R. Compared with the DSM-III-R definition, the DSM-IV definition of oppositional defiant disorder was somewhat more prevalent, but the prevalence of conduct disorder was essentially unchanged. CONCLUSIONS: DSM-IV definitions of oppositional defiant disorder and conduct disorder are somewhat better than DSM-III-R definitions in terms of internal consistency and test-retest agreement, and the validity of the DSM-IV definition of oppositional defiant disorder is slightly better than that of DSM-III-R.     

Protein translocation: delivering virulence into the host cell

A wide variety of plant and human bacterial pathogens use a specialized 'type III' protein secretion system to deliver virulence factors into host cells. Appendage-like surface structures have recently been identified on several bacterial pathogens and there are indications that these may be conduits for virulence factor delivery.     

Fast and one-step folding of closely and distantly related homologous proteins of a four-helix bundle family

Bovine acyl-coenzyme A binding protein is a four-helix bundle protein belonging to a group of homologous eukaryote proteins that binds medium and long-chain acyl-coenzyme A esters with a very high affinity. The three-dimensional structure of both the free and the ligated protein together with the folding kinetics have been described in detail for the bovine protein and with four new sequences reported here, a total of 16 closely related sequences ranging from yeasts and plants to human are known. The kinetics of folding and unfolding in different concentrations of guanidine hydrochloride together with equilibrium unfolding have been measured for bovine, rat and yeast acyl-coenzyme A binding protein. The bovine and rat sequences are closely related whereas the yeast is more distantly related to these. In addition to the three natural variants, kinetics of a bovine mutant protein, Tyr31 --> Asn, have been studied. Both the folding and unfolding rates in water of the yeast protein are 15 times faster than those of bovine. The folding rates in water of the two mammalian forms, rat and bovine, are similar, though still significantly different. A faster unfolding rate both for rat and the bovine mutant protein results from a lower stability of the native states of these. These hydrophobic regions, mini cores, have been identified in the three-dimensional structure of the bovine protein and found to be formed primarily by residues that have been conserved throughout the entire eukaryote evolution from yeasts to both plants and mammals as seen in the sample of 16 sequences. The conserved residues are found to stabilize helix-helix interactions and serve specific functional purposes for ligand binding. The fast one-step folding mechanism of ACBP has been shown to be a feature that seems to be maintained throughout evolution despite numerous differences in sequence and even dramatic differences in folding kinetics and protein stability. The protein study raises the question to what extent does the conserved hydrophobic residues provide a scaffold for an efficient one-step folding mechanism.     

An automated, parallel processing approach to biomolecular sample preparation

Sample preparation for DNA and RNA assays is a prime candidate for laboratory automation. A novel, parallel processing device that performs the three separate liquid-handling functions necessary for such sample preparation-dispensing, pipetting, and pressurizing-is presented. The device comprises an array of fine nozzles connected by fluidic channels to automatically and precisely distribute flow between one source and an array of points. The design principles, as well as the experimental and computational methods used to develop the device, are described. Test results, including accuracy, uniformity, volume range, and timing, are presented. The functionality of the device is demonstrated by performing a solid-phase extraction of DNA with two types of microcolumns.     

The inhibitive effect of extraneous ion addition on stress corrosion cracking of alpha-brass

The addition of ammonium chloride, cobalt sulphate, cesium sulphate and ethylene diamine to a Mattsson solution and the addition of ammonium sulphate to the Pugh solution have been found to produce an inhibitive effect on the stress corrosion cracking of alpha-brass in these solutions. Ammonium chloride and ammonium sulphate additions affect the tarnish formation on the specimen as well as the cracking pattern. Electrochemical polarization studies have also been carried out with these two additives. The observations have been explained in the light of adsorption of these ionic species at selected sites on the metal surface and their possible interference with the cathodic reactions.     

Mass transfer from a carbon dioxide jet to tap water

When a jet of soluble gas is directed on to liquid surface, the kinetic energy of the jet is transferred to the liquid giving rise to circulation, which in turn enhances mass transfer. Consequently, the mass transfer coefficient is related to the characteristics of the jet.     

The beneficial effect of small amount of water in the ambient temperature atom transfer radical homo and block co-polymerization of methacrylates

ATRP of several methacrylates viz. methyl methacrylate (MMA), ethyl methacrylate (EMA), n-butyl methacrylate (nBMA), t-butyl methacrylate (tBMA), benzyl methacrylate (BzMA) and (N,N-dimethylamino)ethyl methacrylate (DMAEMA) has been studied in neat as well as aqueous (up to 12 vol% water) acetone at 35 °C using CuCl/bipyridine (bpy) catalyst and ethyl 2-bromoisobutyrate as the initiator. Addition of water significantly enhances the rate of polymerization without losing control. Unlike CuCl/bpy the CuBr/bpy catalyst gives poor control which is attributed to the lower solubility and consequent heterogeneity in the latter case. Of the other ligands used with the CuCl catalyst viz. o-phenanthroline (o-phen), 1,1,4,7,7-pentamethyldiethylenetriamine (PMDETA), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), Me₆TREN only o-phen offers reasonably good control. The CuCl/bpy catalyst system has been used also in preparing some di- and tri-block copolymers with reasonably low polydispersity index (PDI) at ambient temperature (35 °C) using aqueous acetone as the solvent. The following block copolymers have been prepared PMMA-tBMA, PMMA-b-tBMA-b-MMA, PMMA-DMAEMA, by this method.     

Estimation of impact forces during multi-point collisions involving small deformations

Multibody System Dynamics - Collision between hard objects causes abrupt changes in the velocities of the system, which are characterized by very large contact forces over very small time...