myo-Inositol phosphorothioates, phosphatase-resistant analogues of myo-inositol phosphates. Synthesis of DL-myo-inositol 1,4-bisphosphate and DL-myo-inositol 1,4-bisphosphorothioate.

Syntheses of a metabolite of the second messenger myo-inositol 1,4,5-trisphosphate, myo-inositol 1,4-bisphosphate, and an analogue, the 1,4-bisphosphorothioate, are reported, by using phosphite chemistry on (+/-)-1,2:4,5-di-isopropylidene-myo-inositol. The synthesis of (+/-)-1,2:4,5-di-isopropylidene 3,6-bis[di-(2-cyanoethyl)]phosphite provides an intermediate that can be oxidized to either the corresponding bisphosphate or bisphosphorothioate. myo-Inositol phosphorothioates are proposed as novel analogues of myo-inositol phosphates; their resistance to phosphatase-catalysed breakdown is reported.     

Subcellular Location and Neuronal Release of Diazepam Binding Inhibitor

Diazepam binding inhibitor (DBI), a peptide located in CNS neurons, blocks the binding of benzodiazepines and beta-carbolines to the allosteric modulatory sites of gamma-aminobutyric acid (GABAA) receptors. Subcellular fractionation studies of rat brain indicate that DBI is compartmentalized. DBI-like immunoreactivity is highly enriched in synaptosomes obtained by differential centrifugation in isotonic sucrose followed by a Percoll gradient. In synaptosomal lysate, DBI-like immunoreactivity is primarily associated with synaptic vesicles partially purified by differential centrifugation and continuous sucrose gradient. Depolarization induced by high K+ levels (50 mM) or veratridine (50 microM) released DBI stored in neurons of superfused slices of hypothalamus, hippocampus, striatum, and cerebral cortex. The high K+ level-induced release is Ca2+ dependent, and the release induced by veratridine is blocked by 1.7 microM tetrodotoxin. Depolarization released GABA and Met5-enkephalin-Arg6-Phe7 together with DBI. DBI is also released by veratridine depolarization, in a tetrodotoxin-sensitive fashion, from primary cultures of cerebral cortical neurons, but not from cortical astrocytes. Depolarization fails to release DBI from slices of liver and other peripheral organs. These data support the view that DBI may be released as a putative neuromodulatory substance from rat brain neurons.     

Metabolic reduction of chromium,as related to its carcinogenic properties

At variance with Cr(III), Cr(VI) compounds easily cross cell membranes and exert genotoxic effects. No metabolic oxidation of Cr(III) could be detected, whereas Cr(VI) reduction was observed in the presence of body fluids and subcellular fractions of various tissues from several animal species. The differential efficiency of this process may account for the selection of target tissues in Cr(VI) carcinogenesis. For instance, reduction by saliva and gastric juice may explain a lack of carcinogenicity by the oral route; reduction inside erythrocytes may explain a lack of carcinogenicity at a distance from administration sites; reduction by the epithelial-lining fluid of terminal airways and by alveolar macrophages may be consistent with the occurrence of thresholds in lung carcinogenesis. Liver preparations displayed the top efficiency in reducing Cr(VI), whereas skeletal muscle, i.e., a typical target in experimental Cr(VI) carcinogenesis, had no detectable activity. Bronchial tree and peripheral lung parenchyma preparations from almost 100 individuals reduced Cr(VI) to a variable extent. The efficiency of lung parenchyma and of isolated alveolar macrophages was enhanced in cigarette smokers. In rats, Cr(VI) reduction by lung preparations was significantly stimulated by the repeated i.t. instillation of Cr(VI) itself. Among the electron donors (chiefly GSH) and enzymatic mechanisms responsible for the intracellular Cr(VI) reduction, such as cytochrome P-450 reductase, glutathione redactase, and aldehyde oxidase, an important role can be ascribed to cytosolic DT diaphorase activity, usually catalyzing a 2-electron reduction.     

A mite survey in the dumpsters for urban solid rubbish

Summary The presence of house dust mites and storage mites in dumpsters was investigated in 3 different areas of Venice hinterland. The aim of this study was to find a relationship between some reported respiratory symptoms among 6 workers who were responsible for moving the dumpsters to the truck and the bio-aerosol released from such operation. These symptoms were closely related to the work and after allergological evaluation we found sensitisation to house dust mites in all 6 employees. The mine exposure in the workplace was assessed by a standardized sampling and analysis of dust obtained after brushing the inside surfaces of dumpsters. In 50% of the specimens (10/20) we found mites belonging to the following families: Pyroglyphidae (Dermatophagoides spp.), Acaridae (Acarus siro) and a lower amount of Cheyletidae (Cheyletus spp.). An indirect test (guanine test) was also performed and gave positivity in 16 dust samples (80%). The insides of dumpsters showed to be an optimal environment for mites survival and reproduction because of cracks and grooves on the walls, the high humidity level and the presence of mould. These mites probably come from dwelling places and other environments (markets, slaughters-houses, farms, etc.). This suggests that mites allergens can be released during rubbish discharging from the dumpsters and represent a possible risk for the employees.     

Storage mites sensitivity and allergic respiratory disease

Summary In order to evaluate the frequency of skin sensitivity to Storage Mites and the role of such sensitization in respiratory allergic disease in workers with occupational exposure to stored items we studied 217 dock workers, 93 farmers and 104 white collars.From the results of skin prick tests the sensitization to sole Storage Mites appears significantly higher among people working in docks or farms, compared with a control group. This confirms the role of the working environment in inducing sensitization to Storage Mites. Rhinitis and asthma however affect nearly always (27/29 cases) people with an associated sensitization to House Dust Mites. Further studies are needed to define the allergenic importance of Storage Mites in working environments.     

Prevention by N-acetylcysteine of benzo[a]pyrene clastogenicity and DNA adducts in rats

The daily i.t. administration of benzo[a]pyrene (BP) to Sprague-Dawley rats, for 3 consecutive days, did not cause any toxicity or clastogenicity in bone marrow cells, as evaluated by monitoring the ratio of polychromatic to normochromatic erythrocytes and the frequency of micronucleated polychromatic erythrocytes. However, BP produced a considerable enhancement of binucleated and micronucleated pulmonary alveolar macrophages, as well as a significant increase in polymorphonucleates recovered by bronchoalveolar lavage. These effects were prevented by administering the thiol N-acetylcysteine (NAC) by gavage 5 h before each BP instillation. In addition, the i.t. treatment with BP resulted in the formation of BP diolepoxide (BPDE)-DNA adducts in lungs and liver, as assessed by synchronous fluorescence spectrophotometry, with fluorescence peaks of similar magnitude in the 2 tissues. Pretreatment with NAC by gavage completely prevented BPDE adducts to liver DNA and significantly decreased those to lung DNA.     

Increased membrane heterogeneity in stimulated human granulocytes

TMA-DPH fluorescence decay in human PMN before and after stimulation with FMLP was studied using frequency domain fluorometry. Membrane heterogeneity was assessed by the width of the continuous distributions of lifetime values of Lorentzian shape used to describe the fluorescence decay. In non-stimulated granulocytes TMA-DPH fluorescence decay is characterized by two distributions of lifetime values centered at 6.5 and 1.0 ns and full width at half maximum of 0.3 and 1.2 ns, respectively. Within 15 min after stimulation, the center values of the two distribution components were 5.1 and 0.8 ns and the distribution width was 0.8 and 0.6 ns, respectively. These results indicate changes of membrane domain organization which can be ascribed to compositional changes and redistribution of membrane components.     

Molecular cloning and characterization of the trpC gene from Penicillium chrysogenum

Summary We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. The gene ecodes a 2.7 kb poly(A)⁺ RNA. We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library. Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes. We found that a DNA fragment which codes only for the carboxy-terminal protion of the polypeptide is sufficient for complementation of the E. coli trpC9830 mutation.     

Impairment of the calcium pump of human erythrocytes by divicine

Divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, produces a remarkable and consistent inactivation of the Ca2+-ATPase activity of the erythrocyte calcium pump. The patterns of inactivation are similar in normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes. Inactivation of Ca2+-ATPase is apparently unrelated to the cellular GSH system, to the proteolytic machinery of mature erythrocytes, and to calmodulin, and also occurs in hemoglobin-free, unsealed erythrocytes membranes at 50-100 microM concentrations of divicine. Analysis of erythrocytes that have escaped destruction during the acute hemolytic crisis of a number of favic patients revealed a dramatic elevation of erythrocyte calcium and a significant decrease of Ca2+-ATPase activity. These results support the view that divicine plays a toxic role in the pathogenesis of favism and suggest that acute electrolyte imbalances, mostly affecting calcium homeostasis, are involved in the mechanisms of erythrocyte damage and destruction in this hemolytic disease.     

Modification of granulocyte chemotaxis during anesthesia for surgery

The PMNs of fourteen patients undergoing general anesthesia for surgical operations were examined to determine whether abnormal chemotaxis occurs during these procedures. Neutrophil chemotaxis was determined immediately before anesthesia and after the anesthetic somministration. Anesthetic agents included 0.2-1% enflurane, 0.3-1% halothane or N20-02 (60:40). Neutrophil chemotaxis was reduced an average of 10.9% by fluorinated anesthetics, no significant modification (+8.1 +/- 13.0%) was detected by N20-02.