The influence of cytochrome P-450 induction on the disposition of carcinogenic benzo[a]pyrene 7, 8-dihydrodiol 9, 10-epoxide in isolated cells

The disposition of the carcinogenic (+)-7ß, 8-dihydroxy-9,10-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]has been studied in isolated hepatocytes obtained from 3-methylcholanthrene-pretreatedrats. In these cells different routes are acting in concertand contribute to diol-epoxide elimination. Conjugation of (+)-anti-BPDEwith glutathione (GSH) and cytochrome P-450c-mediated metabolismof the diol-epoxide to 1- and 3-hydroxy-anti-BPDE (triol-epoxides)appears to be equally important. The reactive triol-epoxidesundergo a number of secondary reactions, including covalentbinding to cellular constituents, e.g. protein and GSH, andhydrolysis to pentahydroxyderivatives. The effective intracellularlifetime of (+)-anti-BPDE is 1 min and comparable to that previouslyobserved in hepatocytes obtained from uninduced animals.     

Effects of glutathione transferase activity on benzo[a]pyrene 7,8-dihydrodiol metabolism and mutagenesis studied in a mammalian cell co-cultivation assay

This study deals with the role of glutathione transferase (GST)-mediatedconjugation of (+)-7ß,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) in two mammalian cell lines, human mammary carcinomacells (MCF-7) and rat hepatoma cells (H4IIE), in relation tothen-capacity to metabolize (–)-trans-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene[(–)-BP-7,8-diol] to products that induce mutations inco-cultivated V79 cells. Both MCF-7 and H4IIE cells metabolized(–)-BP-7,8-diol to BPDE, but mutations in co-cultivatedV79 cells were only detected with MCF-7 cells. However, depletionof glutathione (GSH) in H4HE cells increased the mutagenidtyof (– )-BP-7,8-diol to a similar level to that found withMCF-7 cells. Measurements of GST activity using GSH and post-microsomalsupernatants from H4IIE, V79 and MCF-7 cells indicated a substantialdifference in conjugation capacity. Although preparations fromall three cell-lines showed GST activity with l-chloro-2,4-dlnitro-benzeneas the substrate, GST activity towards BPDE could only be detectedin supernatants from H4HE cells. This is consistent with thepresence of GST 7-7 an isoenzyme highly efficient hi catalysingBPDE-GSH conjugation. The difference in GSH-conjugation activitytowards BPDE was confirmed using intact H4IIE and MCF-7 cellsin culture. These results indicate that GSH-conjugation playsa pivotal role in mutagenesis induced by polycyclk aromatichydrocarbons (PAH). Accordingly, a deficiency in GSH-conjugationcapacity may be regarded as one important factor in defininga target cell population with an increased risk for tumour initiationfollowing exposure to PAH.     

Treatment with an anti-CD18 monoclonal antibody in rabbits inhibits pneumococcal-induced leukocyte recruitment in the skin,but not in the meninges

Inflammatory recruitment of leukocytes into the cerebrospinal fluid (CSF) during bacterial meningitis has been shown to contribute to the neurological damage commonly associated with this disease. In this study we tested whether inhibition of firm leukocyte adhesion to vascular endothelium could reduce leukocyte recruitment into the subarachnoid space (SAS) and into the skin in rabbits challenged with pneumococcal cell wall (PCW) antigen. PCW was given either as an intracisternal or an intradermal (i.d.) injection. Intravenous (i.v.) treatment with a monoclonal antibody (mAb), IB4, against the leukocytic adhesion molecule CD18 has previously been documented to attenuate leukocyte CSF accumulation in experimental bacterial meningitis. In the present study, i.v. treatment with anti-CD18 mAbs (IB4) only tended to inhibit CSF leukocyte influx in animals with PCW-induced meningitis. However, if the antigen was injected i.d., treatment i.v. with the same mAb (IB4) dramatically reduced leukocyte accumulation in the skin. Our findings indicate that the mechanisms responsible for PCW-induced inflammatory accumulation of leukocytes in skin and meninges are different.     

Epitope of the vaccine-type Bordetella pertussis strain 186 lipooligosaccharide and antiendotoxin activity of antibodies directed against the terminal pentasaccharide-tetanus toxoid conjugate

Lipooligosaccharides (LOS) isolated from Bordetella pertussis strains 186 and 606 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-resolution magic angle spinning nuclear magnetic resonance (NMR). These analyses distinguished between the LOS of strains 186 and 606, suggesting that the structure of LOS in B. pertussis is heterogeneous. The pentasaccharide was selectively cleaved from LOS of B. pertussis strain 186, purified, and covalently linked to a monomer fraction of tetanus toxoid. Injection of rabbits with the neoglycoconjugate emulsified in complete Freund's adjuvant yielded immunoglobulin G antibodies that were reactive with the LOS. These antibodies reacted strongly with B. pertussis LOS possessing the complete dodecasaccharide, as determined by an enzyme-linked immunosorbent assay, immunoblotting, and flow cytometry with intact, live bacterial cells. The binding epitope within the pentasaccharide was investigated by saturation transfer difference (STD) NMR spectroscopy. Protons H-1 and H-4 of the terminal alpha-D-GlcpNAc and proton H-6 and protons of an N-methyl group at H-4 of 3-substituted beta-L-FucpNAc4NMe exhibited the largest saturation transfers. STD NMR experiments confirmed that the immunodominant epitope recognized by the antineoglycoconjugate antibodies is located predominantly in the distal trisaccharide of B. pertussis 186 LOS. The antipentasaccharide antibodies induced by the conjugate inhibited the secretion of tumor necrosis factor alpha, interleukin-6, and NO by LOS-stimulated J774A.1 cells.     

The Excitatory Action of Acetylcholine on Intradental Sensory Units

In order to test the hypothesis that ACh mediates the transmission of pain stimuli from dentin to sensory intradental nerve endings the following experiments were performed. Intradental nerve impulses were recorded by means of low impedance electrodes inserted in dentinal cavities in the tooth of the cat. An air blast proved to be an efficient physical stimulus to excite the intradental nerves. Local application of acetylcholine caused a similar response. This response to acetylcholine was followed by a transient blockage to repeated application. The response to acetylcholine could be blocked by d-tubocurarine, atropine, succinylcholine and hexamethonium administered locally. In contrast, the response to physical stimuli (air blasts) could not be blocked by these drugs. Moreover, during the period of depression following acetylcholine the preparation responded to physical stimuli. These findings suggest that acetylcholine is not a mediator in the intradental pain transmission provoked by physical stimuli.     

Multilocus sequence typing of methicillin-resistant Staphylococcus aureus from an area of low endemicity by real-time PCR

A protocol for multilocus sequence typing (MLST) of methicillin-resistant Staphylococcus aureus (MRSA) was adapted to real-time LightCycler System PCR for efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products. The method was evaluated on a representative and well-characterized collection of clinical MRSA isolates (n = 57) obtained from an area of low endemicity. Twenty sequence types (STs) and nine clonal complexes were identified. Combining STs and the staphylococcal cassette chromosome mec (SCCmec) type identified 27 different genotypes, and type IV SCCmec was present in 11 different STs. The presence of the Panton Valentine leukocidin (PVL) genes was found in isolates of four different STs. Eleven different STs were found among the community-acquired as well as among the hospital-acquired MRSA. The genetic heterogeneity was also denoted by pulsed-field gel electrophoresis analysis that showed 24 different pulsotypes among the 57 MRSA isolates. The presence of more than one different type of SCCmec in the same ST indicates that the MRSA clones have arisen at several occasions in the same genetic background by independent acquisition of SCCmec into methicillin-sensitive strains. This circumstance shows the importance of combining MLST data with SCCmec-typing results when investigating the origins of MRSA.     

Monosomy and trisomy of 15q24→qter in a family with a translocation t(6;15)(P25;q24)

A child with multiple anomalies, including growth retardation, a left-sided diaphragmatic hernia with lung hypoplasia, and cerebral malformations is described. Cytogenetic investigation demonstrated a deletion of the distal part of one chromosome 15, del(15)(q24qter), an aberration not previously described. Family studies revealed that the mother had a balanced translocation, t(6;15)(p25;q24). Two of her subsequent pregnancies resulted in abortions after prenatal diagnosis: one fetus was trisomic for 15q24→qter, while the other had monosomy 15q24→qter and a left-sided diaphragmatic hernia similar to the first child.     

Rate Limiting Factors in Sympathetic Neurotransmitter Secretion

Isolated superfused field stimulated preparations of guinea-pig vas deferens, in which the neural stores of noradrenaline (NA) had been labelled by preincubation with tritium-marked (-)-NA, were used to study the factors regulating the amount of NA secreted from the nerves, per applied shock. The results indicate that ‘stimulation secretion coupling’ in this tissue is subject to 2 different kinds of facilitation, one independent and one dependent on nerve stimulation frequency. Kinetic analysis of the calcium dependence of transmitter secretion, after removal of α-adrenoceptor mediated negative feedback control, suggests that there is a definite upper limit to the amount of transmitter which can be secreted, per pulse, from each ‘secretory area’ of the nerves. The low Q₁₀ value of this apparent V_max suggests that the rate limiting factor is non-enzymatic in nature. It is proposed that the ultimately rate limiting factor in NA secretion may rather be the number of vesicles in each potential ‘secretory area’ which are in a suitable position within the nerve for active participation in the secretion of transmitter.