Characterisation of non-toxigenic Clostridium spp. strains, to use as surrogates for non-proteolytic Clostridium botulinum in chilled food challenge testing.

Under many of the conditions studied, a two-strain cocktail of non-toxigenic Clostridium spp. was found to be suitable as a surrogate for non-proteolytic Clostridium botulinum, and has the potential for use in chilled food challenge tests measuring growth. Non-toxigenic surrogates could also be used in thermal process screening studies.

Sequence typing confirms that Campylobacter jejuni strains associated with Guillain-Barré and Miller-Fisher syndromes are of diverse genetic lineage, serotype, and flagella type.

Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by Campylobacter jejuni in up to 40% of cases. Nucleotide sequence-based typing of 25 C. jejuni isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800 C. jejuni isolates not associated with neuropathy. A total of 13 genetic lineages and 20 flaA short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the flaA short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS.

Molecular characterization of Campylobacter jejuni from patients with Guillain-Barré and Miller Fisher syndromes.

Campylobacter jejuni has been identified as the predominant cause of antecedent infection in Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS). The risk of developing GBS or MFS may be higher after infection with specific C. jejuni types. To investigate the putative clonality, 18 GBS- or MFS-related C. jejuni strains from The Netherlands and Belgium and 17 control strains were analyzed by serotyping (Penner and Lior), restriction fragment length polymorphism analysis of PCR products of the flaA gene, amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis, and randomly amplified polymorphic DNA analysis. Serotyping revealed 10 different O serotypes and 7 different Lior serotypes, thereby indicating a lack of serotype clustering. Two new O serotypes, O:35 and O:13/65, not previously associated with GBS or MFS were found. Serotype O:19 was encountered in 2 of 18 strains, and none was of serotype O:41. The results of all genotypic methods also demonstrated substantial heterogeneity. No clustering of GBS- or MFS-related strains occurred and no molecular marker capable of separating pathogenic GBS or MFS from non-GBS- or non-MFS-related enteritis strains could be identified in this study. Sialic-acid-containing lipopolysaccharides (LPS) are thought to be involved in the triggering of GBS or MFS through molecular mimicry with gangliosides in human peripheral nerves. Therefore, further characterization of GBS- or MFS-related C. jejuni should target the genes involved in the synthesis of LPS and the incorporation of sialic acid.

Kinetic studies on two isoforms of acetyl-CoA carboxylase from maize leaves.

The steady-state kinetics of two multifunctional isoforms of acetyl-CoA carboxylase (ACCase) from maize leaves (a major isoform, ACCase1 and a minor isoform, ACCase2) have been investigated with respect to reaction mechanism, inhibition by two graminicides of the aryloxyphenoxypropionate class (quizalofop and fluazifop) and some cellular metabolites. Substrate interaction and product inhibition patterns indicated that ADP and P(i) products from the first partial reaction were not released before acetyl-CoA bound to the enzymes. Product inhibition patterns did not match exactly those predicted for an ordered Ter Ter or a random Ter Ter mechanism, but were close to those postulated for an ordered mechanism. ACCase2 was about 1/2000 as sensitive as ACCase1 to quizalofop but only about 1/150 as sensitive to fluazifop. Fitting inhibition data to the Hill equation indicated that binding of quizalofop or fluazifop to ACCase1 was non-cooperative, as shown by the Hill constant (n(app)) values of 0.86 and 1.16 for quizalofop and fluazifop respectively. Apparent inhibition constant values (K' from the Hill equation) for ACCase1 were 0.054 microM for quizalofop and 21.8 microM for fluazifop. On the other hand, binding of quizalofop or fluazifop to ACCase2 exhibited positive co-operativity, as shown by the (napp) values of 1.85 and 1.59 for quizalofop and fluazifop respectively. K' values for ACCase2 were 1.7 mM for quizalofop and 140 mM for fluazifop. Kinetic parameters for the co-operative binding of quizalofop to maize ACCase2 were close to those of another multifunctional ACCase of limited sensitivity to graminicide, ACC220 from pea. Inhibition of ACCase1 by quizalofop was mixed-type with respect to acetyl-CoA or ATP, but the concentration of acetyl-CoA had the greater effect on the level of inhibition. Neither ACCase1 nor ACCase2 was appreciably sensitive to CoA esters of palmitic acid (16:0) or oleic acid (18:1). Approximate IC50 values were 10 microM (ACCase2) and 50 microM (ACCase1) for both CoA esters. Citrate concentrations up to 1 mM had no effect on ACCase1 activity. Above this concentration, citrate was inhibitory. ACCase2 activity was slightly stimulated by citrate over a broad concentration range (0.25-10 mM). The significance of possible effects of acyl-CoAs or citrate in vivo is discussed.

Diagnosis of pediatric human immunodeficiency virus infection by means of a commercially available polymerase chain reaction gene amplification.

OBJECTIVE: To assess the sensitivity and specificity of polymerase chain reaction (PCR) in infants and children at risk for human immunodeficiency virus (HIV) infection. DESIGN: A prospective, blinded study of 286 HIV-seropositive infants and children. Infection was diagnosed by antibody detection after 18 months of age, two positive direct tests (p24 antigen and HIV culture), or the presence of an illness that defines the acquired immunodeficiency syndrome. SETTING: University of South Florida and All Children's Hospital, St Petersburg, inpatient and outpatient centers. PARTICIPANTS: Two hundred eighty-six infants and children seropositive for HIV who were examined between July 1988 and September 1992. MAIN OUTCOME MEASURES: Sensitivity, specificity, and predictive values of a commercially available PCR test. RESULTS: Five hundred sixty-seven PCR tests were performed on samples from 286 seropositive subjects followed up for a minimum of 16 months. Of the subjects, 105 were confirmed to be infected and 181 uninfected. Overall, 96 of 105 initial PCRs in infected subjects were positive (sensitivity, 91.4%; positive predictive value, 99%). If samples obtained during the first week of life are excluded, 95 to 100 samples were positive (sensitivity, 95%). Of 181 initial PCR tests from seropositive subjects who seroreverted, 180 were negative (specificity, 99.4%,; negative predictive value, 95.2%). The predictive value of a positive test was 90.9% and that of a negative test was 93.1% in the first month of life. All 145 initial samples obtained between 5 weeks and 12 months of age correctly predicted infection status (positive predictive value, 100%). CONCLUSIONS: Gene amplification by means of a commercially available PCR is useful in the diagnosis of HIV infection for infants born to seropositive mothers. Between day 7 through 1 year of age, HIV infection is accurately diagnosed by the PCR assay.

In the shadow of biomedicine: self medication in two Ecuadorian pharmacies.

In Ecuador, as in most Third World nations, thousands of different prescription-only pharmaceuticals can be bought without a doctor's prescription. But how often does self medication actually occur? This study documents 619 prescription drug sales in two Ecuadorian pharmacies. In 51% of these sales, customers in fact present no prescription. Many of the drugs sold this way have serious side effects and must be used with care. Ecuadorian law greatly restricts information on drug packaging about side effects, indications, contraindications, schedule and dosage. Although the pharmacies differ with respect to self medication rates, drug choices, and clerk-customer interactions, both show the existence of a 'shadow system of biomedicine' in which prescription drugs are used without physician consultation. In view of the dominant role that transnational corporations play in Third World pharmaceuticals usage, this analysis incorporates a political economic perspective.

Flurazepam effects on sleep EEG. Visual, computer, and cycle analysis.

Analysis of sleep effects of flurazepam hydrochloride on four normal subjects confirmed that this drug substantially suppresses both REM and stage 4 sleep. Computer analysis disclosed that delta wave amplitude was greatly reduced by flurazepam. However, low density delta wave activity (ie, stage 2 sleep, which was increased in duration beyond the reduction in stage 4), permitted the number of delta waves and the time they occupied per night to remain at baseline levels. This finding suggests that sedative-hypnotics increase total sleep time by slowing the metabolic processes of sleep so that a longer sleep duration is required for the same biological effects. New observations on the induction times of REM and stage 4 effects are also presented. In general, the distortions in sleep EEG produced by flurazepam qualitatively resemble, but are quantitatively greater than, those produced by barbiturates in equivalent hypnotic doses.

Flurazepam effects on slow-wave sleep: stage 4 suppressed but number of delta waves constant.

Repeated administration of flurazepam reduced stage 4 sleep (high delta-wave concentration) but produced a greater increase in stage 2 duration so that total sleep time was increased. Computer analysis revealed that the increased amount of stage 2 (low delta-wave concentration) sleep provided a number and duration of delta waves sufficient to offset the loss of delta activity in stage 4. However, the amplitude of the average delta wave was reduced. These results demonstrate the value of direct quantification of delta-wave activity, the variable that underlies visual classification of slow-wave sleep into stages 2 to 4. They also give rise to new hypotheses regarding the relative absence of side effects in spite of profound stage 4 suppression by flurazepam and the mechanisms by which total sleep time is increased by this drug.