Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis.

The PvuII restriction endonuclease is a homodimer that recognizes and cleaves the DNA sequence 5'-CAGCTG-3' in double-stranded DNA, and the structure of this enzyme has been reported. In the wild-type enzyme, Asp34 interacts with the internal guanine of the recognition sequence on the minor groove side. The Asp34 codon was altered to specify Gly (D34G), and in vitro studies have revealed that the D34G protein has lost binding specificity for the central G.C base-pairs, and that it cuts the canonical sequence with 10(-4)-fold reduced activity as compared to the wild-type enzyme. We have now determined the structure at 1.59 A resolution of the D34G PvuII endonuclease complexed with a 12 bp duplex deoxyoligonucleotide containing the cognate sequence. The D34G alteration results in several structural changes relative to wild-type protein/DNA complexes. First, the sugar moiety of the internal guanine changes from a C2'-endo to C3'-endo pucker while that of the 3' guanine changes from C3'-endo to C2'-endo pucker. Second, the axial rise between the internal G.C base-pairs is reduced while that between the G.C and flanking base-pairs is expanded. Third, two distinct monomeric active sites are observed that we refer to as being "primed" and "unprimed" for phosphodiester bond cleavage. The primed and unprimed sites differ in the conformation of the Asp58 side-chain, and in the absence from unprimed sites of four networked water molecules. These water molecules, present in the primed site, have been implicated in the catalytic mechanism of this and other endonucleases; some of them can be replaced by the Mg2+ necessary for cleavage. Taken together, these structural changes imply that the Asp34 side-chains from the two subunits maintain a distinct conformation of its DNA substrate, properly situating the target backbone phosphates and indirectly manipulating the active sites. This provides some insight into how recognition of the specific DNA sequence is linked to catalysis by the highly specific restriction endonucleases, and reveals one way in which the structural conformation of the DNA is modulated coordinately with that of the PvuII protein.

A mutation affecting the regulation of a secA-lacZ fusion defines a new sec gene.

It was shown previously that the secA gene of Escherichia coli is derepressed in cells that have a defect in protein export. Here it is demonstrated that the beta-galactosidase produced by a secA-lacZ gene fusion strain is regulated in the same way. Studies on the fusion strain reveal that the promoter or a site involved in regulation of the secA gene is located considerably upstream from the structural gene. The properties of the fusion strain provide a new selection for mutants that are defective in protein export. Selection for increased lac expression of a secA-lacZ fusion strain yields mutations in three of the known sec genes, secA, secD and prlA/secY. In addition, mutations in several genes not previously known to affect secA expression were obtained. A mutation in one of these genes causes a pleiotropic defect in protein export and a cold-sensitive growth defect; this gene, which maps at approximately 90 min on the bacterial chromosome, has been named secE.

Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein.

Vectors were constructed that allow foreign peptides to be expressed in Escherichia coli as fusion proteins. The peptides are fused to the C terminus of maltose-binding protein (MBP), which allows them to be purified by the MBP's affinity to cross-linked amylose (starch). The fusion protein can be directed to the periplasm by including the leader sequence from the phoA gene on the vector.

An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein.

A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.

LITMUS: multipurpose cloning vectors with a novel system for bidirectional in vitro transcription.

We describe the construction and uses of a set of four multipurpose cloning vectors: LITMUS 28, 29, 38 and 39. The vectors feature the high-copy pUC origin and an M13 origin for single-stranded DNA production as well as polylinker sites for most commercially available restriction enzymes that recognize nondegenerate hexanucleotide sites and yield 4-base sticky ends upon cleavage. Sites are arranged, without overlaps, to permit linker addition to blunt-ended fragments and unidirectional nested deletions and are within the lacZ alpha gene to facilitate blue-white screening. Finally, the polylinkers are flanked by a pair of opposing modified T7 promoters to allow in vitro transcription of either strand of a cloned insert with T7 RNA polymerase. Selective unidirectional transcription from one promoter is achieved by cleaving the other at an internal restriction site (AflII or SpeI). Both modified promoters are fully active under standard RNA probe synthesis conditions. In Southern blots of Dirofilaria immitis genomic DNA, an RNA probe prepared from LITMUS performed equivalently to the same RNA probe made from a wild-type promoter vector and a DNA probe prepared by random priming.

Chlorhexidine release from room temperature polymerising methacrylate systems.

A series of different methacrylate monomers (with either 1 or 2.5% dimethyl-p-toluidine, DMPT) was gelled with poly(ethyl methacrylate) powder (containing benzoyl peroxide) thus forming a room temperature curing system. When doped with 5.625% chlorhexidine diacetate the release from the tetrahydrofurfuryl methacrylate-based samples was considerably greater than that from other methacrylate monomers. This seems to be due to the formation of channels in the polymer. Nuclear magnetic resonance spectroscopy, of solutions that the samples were immersed in, showed chlorhexidine was indeed being released from the polymer. It also showed that doping the polymer with chlorhexidine hindered the polymerisation, resulting in a higher level of residual monomer and low molecular weight components being leached from the polymer. The DMPT also affected the polymerisation with greater leaching from the 2.5% DMPT sample.

Development of butyl elastomer/methacrylate monomer systems as denture soft lining materials.

Previous studies have shown elastomer/methacrylate monomer formulations to have good strength; however, water uptake was high and they also suffered from oxidation. This study has looked at the use of three different butyl elastomers, well known for their oxidation resistance, butyl (PB), chlorobutyl (PCB) and bromobutyl (PBB). The tensile and water uptake properties of the three elastomers gelled with ethyl hexyl methacrylate containing 1% ethylene glycol dimethacrylate and 1% lauryl peroxide (5+ formulation from previous studies) were studied. Water uptake of the pure elastomers was also measured. Tensile strengths were low (PCB5+ = 3.09+/-0.12 MPa and PBB5+ = 3.90+/-0.36 MPa); however, elongation to break values were high (PCB5+ = 797+/-17% and PBB5+ = 599+/-13%). Water uptake was high and protracted with none of the formulations reaching equilibrium. The PCB5+ had the highest uptake (approximately 6% at 203 days) with that for PBB5+ and PB5+ at a similar level (approxiamtely 4% at 203 days). None of the materials showed any sign of oxidation. The PBB proved to be the most suitable of the three elastomers for further development in soft lining formulations.

Influence of additives on the water uptake of hydrosilanized silicone rubbers.

Water uptake characteristics of a silicone polymer containing additives of varying solubility have been investigated. A hydrophobic silica, a sparingly soluble agent and a soluble catalyst were added to a stoichiometrically balanced hydrosilanized silicone polymer (the inherent absorption of which was 0.09 wt%). The diffusion coefficients for desorption of all materials were of the order 10(-6) cm2s-1, whereas the diffusion coefficients for absorption showed a decrease with increasing hydrophilicity and solubility of the additive. The greater the solubility of the additive, the more prolonged the uptake and the greater the deviation from classic diffusion theory.

Nuclear magnetic imaging of an osmotic water uptake and delivery process.

The water absorption of NH4F-doped silicone cylinders was studied by spin-echo Fourier nuclear magnetic resonance imaging, in conjunction with gravimetric (mass uptake) and ionic concentration (release of NH4F) measurements. The addition of NH4F greatly increases the water uptake as the inclusions are surrounded by droplets of water when wetted by water diffusing in the polymer. These droplets then expand due to the osmotic pressure difference between the internal droplet and external solution; this expansion is opposed by an elastic restraining force. The rate of ingress into the polymer will depend on the relative chemical potential of water associated with the polymer and the droplet, as well as the diffusion coefficient. Later stages of the absorption process are dominated by the droplets. The dilution of the droplets (from water diffusing into the droplets) slowly reduces the chemical potential driving the process. Hence, the later stages occur without any notable concentration gradient but a slight chemical potential gradient. If the droplet expansion exceeds that which can be restrained by the material, failure around the droplet occurs resulting in the release of the salt and the solution within the droplet.

Blends of isoprene-styrene/methacrylate monomer systems as denture soft lining material.

This work further develops the concept of using an elastomer gelled with methacrylate monomers to produce a methacrylate-based soft lining material without the use of a plasticizer. An isoprene-styrene (SIS) block copolymer was mixed with methyl methacrylate (MMA) and 1,6-hexandiol dimethacrylate (HDMA). The HDMA was used as a cross-linking agent. The elastomer/monomer ratios were maintained at 50/50 whereas the monomers ranged from 0 to 100%) HDMA. Mechanical properties and water absorption/desorption characteristics were used to assess the effect of varying the monomer compositions. The results indicated that phase separation took place, in particular at high HDMA content. This significantly increased the Young's modulus and decreased the elongation to break. Generally, the water uptake tended to decrease with increasing HDMA content, reflecting the effect of modulus. Second absorption cycles gave higher uptake values compared to the first. Formulations with a high amount of HDMA gave materials with modulus values too high for soft lining applications. This suggests that the optimum formulation requires a compromise between modulus and water uptake.

The water uptake of poly(tetrahydrofurfuryl methacrylate).

Poly(tetrahydrofurfuryl methacrylate) possesses some unique characteristics with respect to its biocompatibility and behaviour in water. The water uptake is high (>70%) and very slow (over 3 yr), but the material remains rigid throughout the process. The mechanism behind the uptake is in two stages; an initial Fickian stage, then as the matrix approaches saturation (about 3 wt%) a second clustering mechanism takes over. The rate of uptake of the second stage of the uptake is controlled by creep (or stress relaxation), and the chemical potential driving the uptake from clustering of the furfuryl rings of the polymer. If clustering or the creep is prevented (by appropriate co-polymerisation) the polymer behaves in an ideal, Fickian manner.

An open trial of pemoline in drug-dependent delinquents with attention-deficit hyperactivity disorder.

OBJECTIVE: Adolescents with conduct disorder and substance use disorders have high rates of comorbid attention-deficit hyperactivity disorder (ADHD); ADHD may contribute to the severity and persistence of substance use disorders and antisocial behaviors. Treatment of ADHD may help patients utilize substance and other behaviorally focused treatment. Yet little is known about the response of ADHD symptoms to psychopharmacological intervention in substance-dependent delinquents. METHOD: Pilot data are presented for 13 male adolescents with conduct disorder, substance use disorders, and ADHD, in a residential substance use treatment program. Patients were treated with pemoline. Scores from the Conners Hyperactivity Index and continuous performance tasks were obtained at baseline and after about 1 month of treatment with pemoline. Physical activity measurements were also assessed at baseline and 1 month. Postmedication assessments were obtained after at least 1 week at maximal dosage (1.2 to 3.3 mg/kg). RESULTS: Mean Conners Hyperactivity Index scores declined 13.9% (p < or = .002) and mean motility declined 7% (p < or = .04) with pemoline treatment. Continuous performance task scores did not change. CONCLUSIONS: Preliminary data indicate that pemoline may be a useful treatment for ADHD in substance-dependent delinquents; the authors propose a controlled trial of pemoline in such youths.

An open trial of bupropion for ADHD in adolescents with substance use disorders and conduct disorder.

OBJECTIVE: Adolescents with conduct disorder (CD) and substance use disorders (SUD) have higher rates of comorbid attention-deficit/hyperactivity disorder (ADHD) than those without CD and SUD. Comorbid ADHD may contribute to more severe SUD. Treatment of ADHD may enhance effective treatment of substance abuse and behavior problems, yet there are few data regarding pharmacological treatment of ADHD in such youths. METHOD: Pilot data are presented from a 5-week, open-label bupropian trial in 13 nondepressed adolescent boys in a residential treatment program focusing on substance and behavioral treatments. All had diagnoses of ADHD, CD, and SUD. Patients' doses were titrated to a maximum fixed daily dose of 300 mg of bupropion. The Conners Hyperactivity Index and Daydream Attention scores, along with Clinical Global Impressions Severity of Illness (CGI-S) ratings, were obtained at baseline and at the fifth week of treatment. RESULTS: Subjects' mean Conners Hyperactivity Index score declined from 75.5 to 65.4 (p < .01, Wilcoxon signed rank test) (13% decline). The mean Daydream Attention score declined from 59.5 to 53.6 (p < .02, Wilcoxon signed rank test) (10% decline). Mean CGI-S declined from 4.9 to 3.0 (p < .002, Wilcoxon signed rank test) (39% decline). CONCLUSIONS: These preliminary data suggest that bupropion may be a useful treatment for ADHD in adolescents with CD and SUD; they suggest the need for a controlled trial of bupropion in such youths.

Depression in substance-dependent delinquents.

OBJECTIVE: Depression often is comorbid with conduct disorder. The purpose of this study is to assess whether, among youths with conduct disorder, those with depression differ in other ways from those without depression. METHOD: Ninety-nine delinquent boys (aged 13 through 19 years) were evaluated with the Diagnostic Interview Schedule for Children and other instruments. All boys had conduct disorder and substance use disorders. RESULTS: Staff-rated and self-rated depression scores correlated significantly. Twenty-one boys had major depression and/or dysthymia. Depressed boys had more substance dependence diagnoses and were more likely to have attention-deficit hyperactivity disorder, posttraumatic stress disorder, and anxiety disorders, compared with the nondepressed boys. Depressed boys tended to develop conduct symptoms earlier than did the nondepressed boys. Depression scores did not change after at least 4 weeks of abstinence, for either depressed or nondepressed boys. CONCLUSIONS: Depressed delinquents have more substance dependence diagnoses, tend to initiate behavioral problems at an earlier age, have increased anxiety and attentional problems, and more trauma effects, than nondepressed delinquents. Depression does not appear to be related to substance intoxication, since it is not alleviated after 4 weeks of abstinence. Such boys may require combined psychiatric and substance treatment.

3He ion-beam analysis of water uptake and drug delivery.

3He ion-beam analysis utilises a micronuclear reaction analysis (of deuterium and carbon) and microparticle induced X-ray emission (in this case silicon and chlorine), to determine an elemental map of the surface of a sample. This study used D(2)O to model the behaviour of water in poly(tetrahydrofurfuryl methacrylate-polyethyl methacrylate) and chlorhexidine diacetate doped silicone elastomers. The poly(tetrahydrofurfuryl methacrylate-polyethyl methacrylate) systems demonstrated an initial Fickian absorption process (diffusion coefficient 1.1 x 10(-11) m(2)s(-1)) which indicated the 2-stage nature of the polymer's absorption kinetics. The doped silicone samples demonstrated an osmotic mechanism for the controlled release of drugs, with correlation between the D(2)O and the chlorhexidine diacetate inclusions increasing during the experiment. The technique proved valuable in the analysis of delivery polymers and will undoubtedly have further applications in the development of drug delivery systems.

Relationship of ADHD, depression, and non-tobacco substance use disorders to nicotine dependence in substance-dependent delinquents.

This study used standardized interviews to examine the relationship of attention deficit hyperactivity disorder (ADHD), major depression (MDD), and other illicit substance use disorders (SUD) to onset and severity of nicotine dependence in 82 female and 285 male adolescents with conduct disorder (CD) and SUD. Results indicate that both ADHD and MDD significantly contribute to severity of nicotine dependence in delinquents with SUD. ADHD is further associated with earlier onset of regular smoking in males. Severity of non-tobacco SUD also was related directly to nicotine dependence severity in both males and females, and to earlier onset of smoking in males. Our findings illuminate the contribution of comorbidity to nicotine dependence and its relationship to other SUD severity among adolescents with CD and SUD and highlight the need for coordinated assessment and treatment of smoking cessation along with concurrent treatment of other drug use and psychiatric comorbidity such as ADHD and MDD in such youths.

Influences on adolescent substance dependence: conduct disorder, depression, attention deficit hyperactivity disorder, and gender.

OBJECTIVE: In adolescents, conduct disorder (CD), attention deficit hyperactivity disorder (ADHD), and depression are frequently comorbid with substance dependence (SD). We hypothesized that the prevalence and severity of CD, major depressive disorder (MDD), and ADHD would differ by gender, and that these conditions would associate differentially with severity of SD in males and females. METHODS: We examined these issues, using standardized diagnostic interviews, in 285 male and 82 female adolescents referred for comorbid CD and SD. RESULTS: Males and females did not differ significantly in severity of substance involvement, MDD, or ADHD, but males had more severe CD. MDD severity was the only variable significantly associated with SD severity for females, while for males, severity of CD combined with MDD and ADHD was significantly associated with SD severity. CONCLUSIONS: Among referred adolescents, CD, MDD, and ADHD may all be important concomitants of SD in males, while in females, depression may be the primary variable related to SD.

Fluoxetine in drug-dependent delinquents with major depression: an open trial.

Although fluoxetine might be more effective than placebo for treating adolescent depression without major comorbidity, little is known about the response of depressive symptoms to antidepressants in adolescents with comorbid conduct disorder (CD) and substance use disorders (SUD). Male adolescents, who remained or became depressed after > or = 1 month of abstinence from abused substances during residential treatment for SUD, were treated in an open trial for > or = 7 weeks with a fixed dose of 20 mg of fluoxetine. The eight adolescents (ages 14-18 years) with CD, SUD, and major depression were not in drug withdrawal or receiving other pharmacotherapy. A > or = 50% improvement was observed in mean scores on Ten Point Depression Scale rated by clinician (p < 0.01) and patients (p < 0.01), Carroll Self-Ratings for depression (p < 0.02), and Severity of Illness scores on the Clinical Global Impression (p < 0.01). Of the eight adolescents, seven showed marked improvement and wished to continue fluoxetine after the trial. Side effects were mild and transient. No subject required dosage reduction or discontinuation of medication because of side effects. Fluoxetine appeared useful in treating substance-dependent delinquents whose major depressions persisted or emerged after 4 weeks of abstinence. These preliminary findings justify a controlled trial in such youths.

Contribution of ADHD symptoms to substance problems and delinquency in conduct-disordered adolescents.

We examined adolescents with conduct disorder (CD) and substance problems to determine if those with attention deficit hyperactivity disorder (ADHD) symptomatology had more severe delinquency and substance involvement. ADHD symptomatology was assessed in two ways: (1) by self-reports using the Diagnostic Interview Schedule for Children (DISC) and (2) by use of DISC plus reports of others (parents, program staff, and program teacher). We divided boys into three ADHD groups based on DISC: those who met criteria, those who reported at least eight current symptoms, and those who reported fewer than eight symptoms. We also divided the same boys into two groups: those with reports of ADHD by two or more sources and those without this multisource ADHD. Examining these definitions of ADHD revealed that boys with either self- or multisource ADHD had more CD symptoms, earlier age of CD onset, more substance dependence diagnoses, and more comorbid depression and anxiety.

Water uptake of soft lining materials from osmotic solutions.

OBJECTIVES: The water uptake characteristics of soft lining materials are of obvious importance in that they are expected to function in the oral environment. Results for Novus (Hygenic Corp., Akron, OH, USA) show a very high uptake from distilled water. Despite this high uptake, Novus appears to function satisfactorily in the mouth. High water uptake of soft lining materials has been attributed to the presence of water soluble impurities that, on immersion, form solution droplets; the driving force for the uptake being the osmotic gradient between the droplets and the external solution. Uptake should therefore be less from ionic solutions. The object of this study was to test the applicability of this theory to Novus and two experimental soft lining materials. METHODS: Water uptake of two experimental materials and Novus has been determined from distilled water and two saline solutions (0.45 and 0.9 M). After 196 days specimens were desorbed to constant weight and then subjected to a second sorption cycle. RESULTS: Novus had the highest uptake from distilled water at approximately 18%, the experimental materials having an uptake approximately 7%. Desorptions were all rapid, minimum weight being reached within 1-2 days. Uptakes of the second sorptions from water were all higher. Uptake from saline solutions was approximately 12% for all materials, uptake from 0.9 M saline being the lowest. Second sorption results from solution were similar to the first. CONCLUSION: The results obtained support the theory that the high water uptake of elastomeric materials is osmotically driven.