Substrate specificity of penicillin amidase from E. coli.

1. The kinetic parameters of 12 substrates of penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11) from E. coli have been determined. Most of the penicillin amidase amide substrates containing a phenylacetyl group in the acyl moiety have been shown to have similar catalytic constants of 50 s-1. Substitution of the phenylacetyl group b 2-thienylacetyl group (cephalothin, cephaloridine) having a similar structure leads to a slight decrease in kcat. 2. Nonspecific penicillin amidase substrates, which contain a free amino group in their acyl moiety, are characterized by a strong dependence of kcat, on the structure of the leaving group with Km being constant. To investigate the free amino group influence on the reaction kinetics, pH-dependences of kcat/Km of enzymatic hydrolysis of phenylacetic and D-(-)-alpha-aminophenylacetic acid p-nitroanilides have been studied. It has been shown that enzyme binds the deprotonated form of the substrate only. 3. Under thermodynamically favourable conditions for the synthesis of beta-lactam antibiotics (at low pH), a concentration of the deprotonated substrate form is very low, and the reaction proceeds in the bimolecular regime. The value of the second-order rate constant for the substrate having a free amino group is small even at pH 7.5, and sharply decreases as does the pH. Hence, despite the favourable thermodynamic conditions for the production of all beta-lactam antibiotics, low reaction rate is the basic hindrance for enzymatic synthesis of penicillins and cephalosporins having a free amino group in the acyl moiety.

A randomized controlled trial of auricular acupuncture for cocaine dependence.

BACKGROUND: Partly because of a lack of a conventional, effective treatment for cocaine addiction, auricular acupuncture is used to treat this disorder in numerous drug treatment facilities across the country for both primary cocaine-dependent and opiate-dependent populations. OBJECTIVE: To evaluate the effectiveness of auricular acupuncture for the treatment of cocaine addiction. METHODS: Eighty-two cocaine-dependent, methadone-maintained patients were randomly assigned to 1 of 3 conditions: auricular acupuncture, a needle-insertion control condition, or a no-needle relaxation control. Treatment sessions were provided 5 times weekly for 8 weeks. The primary outcome was cocaine use assessed by 3-times-weekly urine toxicology screens. RESULTS: Longitudinal analysis of the urine data for the intent-to-treat sample showed that patients assigned to acupuncture were significantly more likely to provide cocaine-negative urine samples relative to both the relaxation control (odds ratio, 3.41; 95% confidence interval, 1.33-8.72; P =. 01) and the needle-insertion control (odds ratio, 2.40; 95% confidence interval, 1.00-5.75; P =.05). CONCLUSIONS: Findings from the current study suggest that acupuncture shows promise for the treatment of cocaine dependence. Further investigation of this treatment modality appears to be warranted.

Day treatment versus enhanced standard methadone services for opioid-dependent patients: a comparison of clinical efficacy and cost.

OBJECTIVE: This study examined the differential efficacy and relative costs of two intensities of adjunctive psychosocial services--a day treatment program and enhanced standard care--for the treatment of opioid-dependent patients maintained on methadone hydrochloride. METHOD: A 12-week randomized clinical trial with 6-month follow-up was conducted in a community-based methadone maintenance program. Of the 308 patients who met inclusion criteria, 291 began treatment (day treatment program: N=145; enhanced standard care: N=146), and 237 completed treatment (82% of those assigned to the day treatment program and 81% of those receiving enhanced standard care). Two hundred twenty of the patients participated in the 6-month follow-up (75% of those in the day treatment program and 73% of those in enhanced standard care provided a follow-up urine sample for screening). Both interventions were 12 weeks in duration, manual-guided, and provided by master's-level clinicians. The day treatment was an intensive, 25-hour-per-week program. The enhanced standard care was standard methadone maintenance plus a weekly skills training group and referral to on- and off-site services. Outcome measures included twice weekly urine toxicology screens, severity of addiction-related problems, prevalence of HIV risk behaviors, and program costs. RESULTS: Although the cost of the day treatment program was significantly higher, there was no significant difference in the two groups' use of either opiates or cocaine. Over the course of treatment, drug use, drug-related problems, and HIV risk behaviors decreased significantly for patients assigned to both treatment intensities. Improvements were maintained at follow-up. CONCLUSIONS: Providing an intensive day treatment program to unemployed, inner-city methadone patients was not cost-effective relative to a program of enhanced methadone maintenance services, which produced comparable outcomes at less than half the cost.

Development of a nucleic acid capture probe with reverse transcriptase-polymerase chain reaction to detect poliovirus in groundwater.

There is a need to develop a practical method for the detection of viral contaminates in water supplies. In this study, poliovirus was used as a model to develop a nucleic acid capture technique. This technique was used to recover viral RNA from concentrated groundwater samples. Poliovirus RNA was isolated using magnetic bead technology. A biotinylated oligonucleotide probe was hybridized to poliovirus-RNA in solution. Streptavidin coated magnetic beads were then added to isolate the RNA-oligonucleotide hybrid. The procedure allows for the recovery of viral RNA suitable for amplification by reverse transcription-polymerase chain reaction (RT-PCR). This nucleic acid capture system was effective in both concentrating, and purifying poliovirus RNA while removing environmental RT-PCR inhibitors. A detection sensitivity of one plaque forming unit (PFU) in 250 microliters of a concentrated environmental sample was routinely attained. This was the same detection level found with seeded purified water. It was shown that the sensitivity of nucleic acid capture RT-PCR was significantly greater than direct RT-PCR, when applied to environmental samples. The amplified product was sequenced to ensure specificity. Furthermore, this technique is rapid, reliable and can be readily adapted to detect other viral pathogens.

A novel method for liberating viral nucleic acid for assay of water samples with cDNA probes.

Rapid and sensitive methods are needed for the detection of enteric viruses to ensure proper drinking water quality. Gene probes have been shown to be useful for this purpose. Previously, samples to be assayed were treated with a series of phenol-chloroform extractions to release the viral nucleic acid. We have developed a more rapid procedure for liberating or exposing the genome of poliovirus for probing. In this study, a poliovirus model was used to test the ability of heat (65 degrees C for 30 min) for release or exposure of viral nucleic acid. Several different RNase inhibitors were tested for their ability to prevent viral RNA degradation. A comparison of the two methods indicates phenol-chloroform extraction is not necessary before probing. In addition to saving 2-4 h of time, maximum sensitivity levels were consistently obtained using this novel procedure.

The efficacy of three common hospital liquid germicides to inactivate Cryptosporidium parvum oocysts.

We evaluated three commonly used hospital disinfectants against three concentrations of Cryptosporidium parvum oocysts (1.5 x 10(6), 1.5 x 10(5), 1.5 x 10(4)). A 10% phenol product, a 10% povidone-iodine product and a 2.5% glutaraldehyde product were tested against Cryptosporidium parvum oocysts without organic load. In-vitro excystation was used to determine viability and a cell culture assay was used to determine infectivity of germicide-treated oocysts. A 2.5% glutaraldehyde product was the most effective in halting excystation of sporozoites and infection in cell monolayers. However, this occurred only at the longest exposure time of 10 h and with the lowest concentration of oocysts (1.5 x 10(4)). The 10% phenol product and the 10% povidone-iodine product also decreased excystation, but were unable to halt infection. Although the ability of C. parvum to with-stand chemical treatment is well known, the ability of oocysts to remain viable and infectious after a 10 h treatment in glutaraldehyde is cause for concern. Endoscopic equipment that may come into contact with these organisms cannot be immersed into glutaraldehyde for this length of time due to its corrosive nature. Thus, the results of this research are cause for concern in hospital disinfection units.

When is less treatment better? The role of social anxiety in matching methadone patients to psychosocial treatments.

In response to a need to match drug users to the most appropriate and cost-effective level of care, it was hypothesized the socially anxious methadone-maintained patients would attain greater benefit from coping skills training provided in the context of a low-intensity enhanced standard methadone maintenance intervention (E-STD) than in the context of a high-intensity, socially demanding day treatment program (DTP). Social anxiety was assessed in 307 methadone-maintained patients using the Social Anxiety and Distress Scale prior to randomization to either E-STD or DTP. The hypothesis was supported: Socially anxious patients were drug free longer during treatment, were more likely to be abstinent at treatment completion, and had greater reductions in HIV risk behaviors if assigned to the lower intensity intervention, which was provided at 1/3 the cost of the DTP.

Cocaine, HIV, and their cardiovascular effects: is there a role for ACE-inhibitor therapy?

Cocaine abuse and HIV disease each have potentially adverse effects upon the heart and cardiovascular system which may be exacerbated when these risk factors are combined. The development of a safe and effective agent to treat both cocaine addiction and its cardiovascular sequelae, that is well-tolerated by HIV patients, would thus be of considerable clinical utility. In this article we discuss the rationale for the investigation of angiotensin converting enzyme (ACE) inhibitors, commonly used to treat hypertension, for treatment in cocaine-abusing populations, based on their potential to reduce cocaine use by modulating levels of dopamine and corticotropin releasing factor in the brain, and on their ability to reverse cardiovascular and platelet abnormalities. We present preliminary findings from echocardiographic and platelet activation studies in 16 HIV-positive, cocaine abusing patients, as well as tolerability and efficacy studies of the ACE-inhibitor, fosinopril, for the treatment of cocaine abuse in both HIV-positive (n=6) and HIV-negative (n=5) methadone-maintained cocaine abusers. Findings suggest that HIV-positive cocaine-abusing patients possess abnormalities of diastolic heart function and platelet activation that are potentially reversible with ACE-inhibitor therapy. Findings also suggest that fosinopril is well-tolerated regardless of HIV serostatus, does not appear to cause hypotension, and may possess effectiveness for reducing cocaine use. We conclude that ACE-inhibitor therapy may offer a new pharmacologic approach to the treatment of cocaine abuse and its complications, and that controlled research of this class of agents may be promising.

A multicenter trial of bupropion for cocaine dependence in methadone-maintained patients.

We conducted a multi-site, placebo-controlled, randomized double-blind clinical trial comparing bupropion HCL (300 mg/day) to placebo for the treatment of cocaine dependence in methadone-maintained subjects. A total of 149 subjects at three sites participated in a 12-week study. Outcome measures included cocaine use, level of depression, and psychosocial functioning. Results showed no significant differences between placebo and bupropion. Exploratory analyses suggested a medication effect for the subset of subjects depressed at study entry. The need to target subgroups of cocaine abusers in future pharmacotherapy trials and the possible role of treatment readiness are discussed.

How injection drug users coped with testing HIV-seropositive: implications for subsequent health-related behaviors.

At entry into methadone maintenance treatment, 94 HIV-positive injection drug users (IDUs) completed the Coping Reponses Inventory, which asked them to "describe your feelings and experiences when you first learned you were HIV positive." Controlling for time since HIV testing, a reliance on avoidance coping following HIV testing was correlated with high levels of recent HIV risk behavior and poor health at entry into the study. The use of any coping strategy, particularly approach strategies, was related to medication adherence. Hierarchical regression analysis showed that avoidance coping accounted for a significant proportion of the variance in recent HIV risk behavior over and above that accounted for by the other variables. Other independent predictors of continued risk behavior were poor health, lack of social support, and low levels of HIV/AIDS knowledge. The need for interventions to help injection drug users (IDUs) cope subsequent to testing HIV seropositive is discussed.

Cue-elicited cocaine craving and autogenic relaxation. Association with treatment outcome.

Prior to entering a pharmacotherapy trial for the treatment of cocaine dependence, 19 patients participated in a pretreatment cue-reactivity protocol that concluded with a relaxation exercise. Measures included self-reported craving and skin conductance level (SCL). Post hoc exploratory analyses suggest that neither craving nor change in SCL in response to cocaine cues differentiated patients who subsequently achieved abstinence from those who did not. Craving following the relaxation procedure did differentiate the two groups: patients who subsequently initiated abstinence in treatment reported a reduction in cue-elicited craving to below baseline levels; craving reported by patients who did not successfully complete treatment remained elevated.

Changes concurrent with initiation of abstinence from cocaine abuse.

In an exploration of changes that occur with initiation of cocaine abstinence, this study examined treatment outcomes in four domains--behavior (cocaine use), affect (depression), cognition (schematic self-representations), and psychophysiology (reactivity to drug cues). Nineteen patients initially entered this 8-week outpatient psychopharmacotherapy trial for the treatment of cocaine dependence. Thirteen completed treatment, and eleven maintained abstinence during the last four weeks of the study. In addition to a demonstrated change in their cocaine-using behavior, these "abstainers" reported reduced depression, increased congruency with "ideal" self-schemas, increased discrepancy with "addict" self-schemas, and generally more positive self-representations. In addition, "abstainers" reported reduced posttreatment craving in the presence of cocaine cues, but evidenced no change in physiological reactivity. Implications of these findings for research and treatment are discussed.

Continuation of high-risk behavior by HIV-positive drug users. Treatment implications.

Drug users who are positive for the human immunodeficiency virus (HIV) represent a major vector of HIV transmission, yet relatively little is known about their continued drug- and sex-related HIV-risk behavior, which may impede the development of effective risk-reduction interventions. In this study, 50 HIV-seropositive injection drug users entering methadone maintenance treatment completed a comprehensive risk assessment battery, including self-report of HIV-risk behavior since learning HIV serostatus, and measures of risk-reduction information, motivation, and behavioral skills. We found that a disconcertingly high proportion of patients (66%) reported having engaged in HIV-risk behavior since learning their HIV-seropositive status. Level of HIV-related knowledge did not predict high-risk behavior. Drug-related risk behavior was predicted by psychiatric severity and poor behavioral skills. Sex-related risk was predicted by low levels of motivation and poor behavioral skills. Implications of these findings for treatment are discussed.

A comprehensive pharmacologic-psychosocial treatment program for HIV-seropositive cocaine- and opioid-dependent patients. Preliminary findings.

HIV-seropositive opioid-dependent patients maintained on an opiate-agonist who continue to use cocaine and to engage in other high-risk behaviors may benefit from enhanced treatment services; however, there is currently little data to guide the formulation of such services. We report on a preliminary study in which six HIV-seropositive opioid-and cocaine-dependent patients were provided a 12-week comprehensive pharmacologic/psychosocial treatment program developed specifically to meet the treatment needs of HIV-seropositive drug users. This program was comprised of buprenorphine (12 mg/day), bupropion (150 mg/day), and twice weekly manual-guided group therapy. Results showed significant decreases in intravenous cocaine use, cocaine craving, and symptoms of depression. A post-hoc comparison to outcomes of eight HIV-seropositive patients receiving standard methadone-maintenance found no improvements for patients receiving standard care. Controlled investigation of enhanced drug treatment programs for HIV-seropositive patients may be warranted.

Methodological investigations for a multisite trial of auricular acupuncture for cocaine addiction: a study of active and control auricular zones.

We evaluated objective criteria for defining points for needle insertion prior to conducting a multisite clinical trial of auricular acupuncture for cocaine addiction. Thirty-four cocaine-abusing subjects participated in a study in which the trial's active zones (Shenmen, Liver, Lung, and Sympathetic) and control zones (located on the ear helix) were divided into quadrants and assessed along four dimensions: electrical resistance, skin discoloration, skin topography, and tenderness. Acute effects of needles inserted into points of low electrical resistance in one ear and high electrical resistance in the other were also assessed. Results showed that the active zones had lower overall electrical resistance and more subcutaneous ridges than control zones. Zones did not possess significant variability along any single dimension. Acute effects of needling high and low resistance points were similar, differing only for "fullness." Based on these findings, and in view of the difficulty of accurately measuring electrical resistance at ear points, we do not recommend the use of electrical devices for point determination in the multisite study. At present, there seems to be little scientific basis for the preselection of specific points for needle insertion within auricular zones. Needle placement should be based upon clinical judgement.

Acupuncture for the treatment of cocaine addiction. Investigation of a needle puncture control.

We conducted a 6-week, single-blind study of acupuncture for cocaine dependence in methadone-maintained patients (N = 40) for the purpose of identifying an appropriate needle puncture control for use in future large-scale clinical trials. Patients were randomly assigned to receive daily acupuncture in three auricular sites plus one body site (LI-4), or in control sites within 2-3 mm of the four active sites. Overall, there was a positive response to treatment on a variety of drug-related and psychosocial measures. Cocaine use decreased significantly for patients in both needle puncture groups. The only statistically significant difference between the two types of needle puncture was on ratings of craving. Subjects rated each type of needle puncture as equally credible and perceived no significant differences on the acute effects of the two types of needle insertions. Power calculations based on these findings suggest that very large sample sizes would be required to detect treatment differences between this control condition and active acupuncture. Alternative controls are suggested, and the challenges inherent in implementing controlled clinical trials of acupuncture are discussed.

Enzymes in the synthesis of chiral drugs.

The majority of synthetic chiral drugs are now marketed as racemates. This situation is rapidly changing due to the recent advances in asymmetric chemical synthesis and biocatalytic methods. This article reviews the use of enzymes in the synthesis and modification of optically pure drugs. Special attention is focused on the synthesis of new pharmaceuticals which may require efficient procedures for large-scale synthesis in the future.

In vitro and in vivo characterization of avian reoviruses. II. Clinical evaluation of chickens infected with two avian reovirus pathotypes.

The effect of two avian reovirus isolates (2408 and 1733) on digestion and nutrient metabolism in infected chickens was assessed by an in vitro absorption assay and clinical blood chemistry analysis. Birds of various ages were inoculated orally and intratracheally with reovirus and sampled periodically for the respective assays. Transitory malabsorption was observed in the duodenum of birds infected with reovirus 2408. Conversely, increased absorption was detected in the ileum of these same birds. Clinical blood chemistry analyses of birds infected with both isolates revealed that severely affected birds had abnormally elevated plasma total protein, plasma albumin, and calcium levels. Decreases were found in percent bone ash and, due to abnormally high globulin levels, in albumin:globulin (A:G) ratios. A significant (P less than 0.05) correlation between body weights and total protein, albumin, A:G ratio, and bone ash was found in infected birds. The most pronounced metabolic and physiologic changes occurred in the severely affected birds, and, in general, pathogenicity of the isolates was reflected by the degree of metabolic change.

In vitro and in vivo characterization of avian reoviruses. I. Pathogenicity and antigenic relatedness of several avian reovirus isolates.

Pathogenicity, pathogenesis, and antigenic relatedness of four avian reovirus isolates obtained from commercially reared broilers were investigated. Chickens of various ages were inoculated both orally and intratracheally with reovirus. Based on disease signs, mortality, weight depression, tissue lesions, invasiveness, and viral persistence in chickens inoculated at 1 day of age, the isolates were classified as being of low, intermediate, or high pathogenicity. The low-pathogenicity isolate (2177) did not cause mortality, weight depression, or clinical disease. The isolate of intermediate pathogenicity (2035) produced low mortality rates (8%), some weight reduction by 7 weeks postinoculation, and microscopic lesions in the intestine and gastrocnemius tendons. The pathogenic isolates, 2408 and 1733, caused severe clinical disease characterized by stunting, feathering abnormalities, mortality as high as 84%, and microscopic lesions in the liver, intestine, pancreas, and/or gastrocnemius tendon. Highly pathogenic isolates also persisted longer in tissues of infected birds and elicited a more prompt and prolonged antibody response. Birds inoculated at 1 day or 1 week of age were more susceptible to reovirus-induced disease than birds inoculated at 2 weeks, suggesting an age-associated resistance. All isolates produced mortality with equal frequency in embryos. The isolates characterized were found to be antigenically similar based on cross-neutralization and cross-protection studies.

Association between self-report of cognitive impairment, HIV status, and cocaine use in a sample of cocaine-dependent methadone-maintained patients.

HIV-disease as well as chronic cocaine abuse may both produce neuropsychological deficits that could potentially interfere with psychoeducational treatments for drug abuse. In this study, the Neuropsychological Impairment Scale (NIS), a 95-item self-report assessment instrument, was administered to 120 cocaine-dependent methadone-maintained patients (59 HIV-seropositive; 61 seronegative) to assess self-awareness of cognitive deficits in this patient population. HIV-seropositive cocaine users reported significantly more impairment than did HIV-seronegative cocaine users on all summary scores and six of seven clinical subscales. Controlling for the influence of sociodemographic variables (age, sex, ethnicity, and education), acute and chronic cocaine use, and effective distress, there was still a significant relationship between HIV status and self-report of neuropsychological impairment. Relative to patients with known neuropsychological deficits, 41% of HIV-seropositive cocaine users and 31% of HIV-seronegative cocaine users scored in the impaired range on the Global Impairment Index. Implications for treatment are discussed.