Consumption, knowledge and attitudes of high school pupils towards alcohol and alcoholism: the Israeli experience.

Five hundred nine high school pupils from Holon (a city in the center of Israel) were surveyed about their consumption, knowledge and attitudes towards alcohol use and alcohol dependence. Two hundred fifty-nine pupils attended a vocational high school and 253 attended an academic high school. Forty percent of the pupils attending the academic school reported that they had drank beer between one to nine times during the last 2 months. In comparison with 72% of the vocational pupils, 42% of the academic pupils and 47% of the vocational pupils drank other alcoholic beverages (such as hard liquor, cognac, whisky or vodka) between one to nine times during the last 2 months. Boys drank alcohol more frequently than girls did. An earlier mean age of beer consumption was found among pupils in the vocational schools-12.8 years; as opposed to pupils in the academic school-13.4 years. Knowledge of most pupils concerning alcoholic beverages and its potential harmful effects was lacking and pupils in the academic school showed a higher level of knowledge in comparison with the vocational pupils. Pupils in the vocational school had more liberal attitudes concerning recurrent consumption of alcoholic beverages than pupils in the academic school. Among the three leading reasons for drinking in the two schools were helping foster a sense of belonging, wish to feel like an adult and desire to forget daily anxieties and conflicts. Pupils in vocational schools are a target population with a higher risk for consuming alcoholic beverages. Discussion groups should be held in school and include personal stories of recovering alcoholics.

Blood pressure, left ventricular mass, and lead exposure in battery manufacturing workers.

BACKGROUND: Although debate about the relationship between lead and blood pressure has focused on low environmental lead levels, industrial exposure remains a concern. METHODS: We measured blood pressure and left ventricular mass (LVM) in 108 battery manufacturing workers, and calculated cumulative and historic average measures of blood lead. RESULTS: Diastolic pressure increased with increasing lead levels, with a significant (P = 0.04) 5 mmHg difference in mean pressure between the highest and lowest cumulative exposure levels. Diastolic pressure increased with the log of cumulative lead (P = 0.06). Both hypertension (defined as currently medicated or systolic > 160 mmHg or diastolic > 95 mmHg) and LVM increased nonsignificantly with increasing lead exposure (P-values > or = 0.17 for hypertension and > or = 0.20 for LVM). CONCLUSIONS: We found a small effect of blood lead on diastolic blood pressure, particularly for a cumulative measure of exposure, but no convincing evidence of associations between lead and other blood-pressure-related outcomes. Published 2001 Wiley-Liss, Inc.

Effect of overexpression of a neutral sphingomyelinase on CD95-induced ceramide production and apoptosis.

We previously showed that ceramide (Cer) formed during the execution phase of apoptosis is derived from plasma membrane sphingomyelin (SM), most likely by a neutral sphingomyelinase activity (Tepper et al., J. Cell Biol. 150, 2000, 155-164). In this study, we investigated the involvement of a cloned putative human neutral sphingomyelinase (nSMase1) in this process. Site-directed mutagenesis of predicted catalytic residues (Glu(49), Asn(180), and His(272)) to Ala residues abolished the catalytic activity of nSMase1. Jurkat cells were retrovirally transduced with either wildtype or inactive (with all three point mutations) Myc-tagged nSMase1. Cells overexpressing wildtype nSMase1 showed dramatically elevated in vitro nSMase activity. However, nSMase1 gene transduction (wildtype or mutant) did not alter steady-state levels of SM, Cer, or glucosylceramide. Moreover, the Cer response and apoptosis sensitivity to ligation of the CD95/Fas receptor in cells overexpressing wildtype or mutant nSMase1 were identical to vector-transduced cells. We conclude that not nSMase1 but a different, yet to be identified, nSMase accounts for the generation of Cer during the execution phase of death receptor-induced apoptosis.

Glucosylceramide synthase does not attenuate the ceramide pool accumulating during apoptosis induced by CD95 or anti-cancer regimens.

Ceramide (Cer) accumulating during the execution phase of apoptosis is generated from plasma membrane sphingomyelin (SM), which gains access to a sphingomyelinase due to phospholipid scrambling (Tepper, A. D., Ruurs, P., Wiedmer, T., Sims, P., Borst, J., and van Blitterswijk, W. J. (2000) J. Cell. Biol. 150, 155-164). To evaluate the functional significance of this Cer pool, we aimed to convert it to glucosylceramide (GlcCer), by constitutive overexpression of glucosylceramide synthase (GCS). Jurkat cells, retrovirally transduced with GCS cDNA, showed a 10-12-fold increase in GCS activity in vitro and a 7-fold elevated basal GlcCer level in vivo. However, Cer accumulating during apoptosis induced by ligation of the death receptor CD95, treatment with the anti-cancer drug etoposide, or exposure to gamma-radiation was not glycosylated by GCS. Likewise, Cer liberated at the plasma membrane by bacterial SMase was not converted by the enzyme. Thus, GCS, located at the Golgi, is topologically segregated from Cer produced in the plasma membrane. In contrast, de novo synthesized Cer as well as an exogenously supplied cell-permeable Cer analog were efficiently glycosylated, apparently due to different Cer topology and distinct physicochemical behavior of the synthetic Cer species, respectively. Exogenous cell-permeable Cer species, despite their conversion by GCS, effectively induced apoptosis. We also observed that GCS activity is down-regulated in cells undergoing apoptosis. In conclusion, GCS can convert de novo synthesized Cer but not SM-derived Cer, and, therefore, the ability of GCS overexpression to protect cells from possible detrimental effects of Cer accumulation is limited.

Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology.

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.

Ordering of ceramide formation, caspase activation, and mitochondrial changes during CD95- and DNA damage-induced apoptosis.

To evaluate the role of ceramide (Cer) in apoptosis signaling, we examined Cer formation induced by CD95, etoposide, or gamma-radiation (IR) in relation to caspase activation and mitochondrial changes in Jurkat T cells. The Cer response to all three stimuli was mapped in between caspases sensitive to benzoyloxycarbonyl-VAD-fluoromethylketone (zVAD-fmk) and acetyl-DEVD-aldehyde (DEVD-CHO). Cer production was independent of nuclear fragmentation but associated with the occurrence of other aspects of the apoptotic morphology. Caspase-8 inhibition abrogated Cer formation and apoptosis induced by CD95 but did not affect the response to etoposide or IR, placing CD95-induced Cer formation downstream from caspase-8 and excluding a role for caspase-8 in the DNA damage pathways. CD95 signaling to the mitochondria required caspase-8, whereas cytochrome c release in response to DNA damage was caspase-independent. These results indicate that the caspases required for the Cer response to etoposide and IR reside at or downstream from the mitochondria. Bcl-2 overexpression abrogated the Cer response to etoposide and IR and reduced CD95-induced Cer accumulation. We conclude that the Cer response to DNA damage fully depends on mitochondrion-dependent caspases, whereas the response to CD95 partially relies on these caspases. Our data imply that Cer is not instrumental in the activation of inducer caspases or signaling to the mitochondria. Rather, Cer formation is associated with the execution phase of apoptosis.

Thermodynamic analysis of halide binding to haloalkane dehalogenase suggests the occurrence of large conformational changes.

Haloalkane dehalogenase (DhlA) hydrolyzes short-chain haloalkanes to produce the corresponding alcohols and halide ions. Release of the halide ion from the active-site cavity can proceed via a two-step and a three-step route, which both contain slow enzyme isomerization steps. Thermodynamic analysis of bromide binding and release showed that the slow unimolecular isomerization steps in the three-step bromide export route have considerably larger transition state enthalpies and entropies than those in the other route. This suggests that the three-step route involves different and perhaps larger conformational changes than the two-step export route. We propose that the three-step halide export route starts with conformational changes that result in a more open configuration of the active site from which the halide ion can readily escape. In addition, we suggest that the two-step route for halide release involves the transfer of the halide ion from the halide-binding site in the cavity to a binding site somewhere at the protein surface, where a so-called collision complex is formed in which the halide ion is only weakly bound. No large structural rearrangements are necessary for this latter process.

1H NMR spectroscopy of the binuclear Cu(II) active site of Streptomyces antibioticus tyrosinase.

The 600 MHz 1H NMR spectrum of tyrosinase (31 kDa) of Streptomyces antibioticus in the oxidized, chloride-bound form is reported. The downfield part of the spectrum (15-55 ppm) exhibits a large number of paramagnetically shifted signals. The paramagnetism is ascribed to a thermally populated triplet state. The signals derive from six histidines binding to the metals through their Nepsilon atoms. There is no evidence for endogenous bridges. The exchange coupling, -2J, amounts to 298 cm(-1). In the absence of chloride the peaks broaden. This is ascribed to a slowing down of the electronic relaxation. The exchange coupling decreases to -2J=103 cm(-1).

[Nutritional characteristics of cereal and peanut bars]. / Características nutricionales de barras de cereales y maní.

Snack with good nutritional value could play an important role in the physical and mental development of children and teenagers since they show a great preference for them. The tendency is increasing their nutritional value by supplying proteins, carbohydrates, fiber, vitamins and minerals in a balanced form. The purpose of this research was to evaluate the chemical, sensorial and nutritional quality of cereal and peanut bars. Three types of bars using different ratios of oat, wheat germ, peanut, toasted and expanded amaranthus and wheat extrudate were prepared. Bars proximate composition was determined according the AOAC methods, and their acceptability according Hedonic Scale. In the biological assays, rats fed with 10% protein diets, were used to obtain the Protein Efficiency Ratio (PER) Net Protein Ratio (NPR) and Apparent Digestibility (AD). Corrected PER, relative PER, relative AD, PER and NPR values did not showed difference between bars CM1 and CM2 (PER: 2.59-2.57; NPR: 3.99-3.95 respectively); CM3 bar showed a lower quality. There were not differences among bars in relation to AD. CM1 and CM2 bars had a better biological quality of the protein being CM3 bar of lower quality. From a chemical and sensorial point of view CM1 bar shows the highest protein content (14.23%) and acceptability (6.8) and CM2 bar shows a high raw fiber content (2.27%).

[Storage and use of antioxidants in cereal and peanut bars]. / Almacenamiento y uso de antioxidantes en barras de cereales y maní.

The use of fatty materials in cereal bars gives to them a good energetic value; however they are exposed to oxidative rancidity which can affect their acceptability and nutritional value. So, the purpose of this research was to determine the stability in storage and the effect of antioxidants on three tipes of cereal bars with peanuts. Cereal bars with 18% of peanuts were prepared, with and without antioxidants (BHA + BHT; 100 ppm). Bars were packed in polyprolpilene-aluminium-polythilene bags, and were stored at room temperature (18-20 degrees C) for 90 days. Each 30 days, analysis of water activity (Aw); moisture content, peroxides index, sensory quality (flavor, aroma and appearance) and acceptability, were carried out. Moisture content was similar in all bars (7.6-9.6%) and Aw was higher in the bar which contained expanded amaranthus and antioxidant. At the 60th day of storage, the peroxide values were lower in the bars with antioxidants; only the bar which included expanded amaranthus showed significant differences (16.4 meq/kg in the bar with antioxidant and 25.7 meq/kg for the control bar). The sensory parameters were kept within normal status without differences between the bars with antioxidants and the control ones, along all the storage period. Shelf life of bars CM1 and CM2 was at least of 60 days when they are kept at 18-20 degrees C.

Kinetic analysis and X-ray structure of haloalkane dehalogenase with a modified halide-binding site.

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. Trp175 forms a halogen/halide-binding site in the active-site cavity together with Trp125. To get more insight in the role of Trp175 in DhlA, we mutated residue 175 and explored the kinetics and X-ray structure of the Trp175Tyr enzyme. The mutagenesis study indicated that an aromatic residue at position 175 is important for the catalytic performance of DhlA. Pre-steady-state kinetic analysis of Trp175Tyr-DhlA showed that the observed 6-fold increase of the Km for 1,2-dibromoethane (DBE) results from reduced rates of both DBE binding and cleavage of the carbon-bromine bond. Furthermore, the enzyme isomerization preceding bromide release became 4-fold faster in the mutant enzyme. As a result, the rate of hydrolysis of the alkyl-enzyme intermediate became the main determinant of the kcat for DBE, which was 2-fold higher than the wild-type kcat. The X-ray structure of the mutant enzyme at pH 6 showed that the backbone structure of the enzyme remains intact and that the tyrosine side chain lies in the same plane as Trp175 in the wild-type enzyme. The Clalpha-stabilizing aromatic rings of Tyr175 and Trp125 are 0.7 A further apart and due to the smaller size of the mutated residue, the volume of the cavity has increased by one-fifth. X-ray structures of mutant and wild-type enzyme at pH 5 demonstrated that the Tyr175 side chain rotated away upon binding of an acetic acid molecule, leaving one of its oxygen atoms hydrogen bonded to the indole nitrogen of Trp125 only. These structural changes indicate a weakened interaction between residue 175 and the halogen atom or halide ion in the active site and help to explain the kinetic changes induced by the Trp175Tyr mutation.

CD95 (Fas/APO-1) induces ceramide formation and apoptosis in the absence of a functional acid sphingomyelinase.

CD95 is a potent inducer of apoptosis. It activates the caspase cascade, but also induces ceramide (Cer) production, reportedly involving acid sphingomyelinase (aSMase) activity. A role for Cer as a second messenger for apoptosis induction was proposed, based on the finding that synthetic Cer analogues can induce cell death. We have tested whether aSMase is required for 1) apoptosis induction and 2) Cer production by CD95. For this purpose, we have used cultured Niemann-Pick disease (NPD) lymphoid cells with a defined mutation (R600H) in the aSMase protein. Despite their inherited deficiency of aSMase, we found that these cells readily undergo apoptosis upon CD95 stimulation. After retrovirus-mediated gene transfer of the aSMase cDNA, the transduced (i.e. "corrected") NPD cells showed neither increased levels of apoptosis nor altered kinetics of caspase-8 and caspase-3 activation and apoptosis induction as compared with empty vector-transduced cells. The slow sustained elevation of Cer levels in response to CD95, which we have previously documented for Jurkat T cells (Tepper, A. D., Boesen-de Cock, J. G. R., de Vries, E., Borst, J., and van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 24308-24312), was similarly found in NPD cells. Moreover, the kinetics of Cer formation remained unaffected after aSMase transduction. These results indicate that this Cer does not result from aSMase activity. We conclude that aSMase is not required for and does not facilitate CD95-mediated apoptosis and that it is not responsible for the late Cer response.

CD95/Fas-induced ceramide formation proceeds with slow kinetics and is not blocked by caspase-3/CPP32 inhibition.

The current confusion regarding the relevance of endogenous ceramide in mediating CD95/Fas-induced apoptosis is based mainly on (i) discrepancies in kinetics of the ceramide response between different studies using the same apoptotic stimulus and (ii) the observation that late ceramide formation (hours) often parallels apoptosis onset. We investigated CD95-induced ceramide formation in Jurkat cells, using two methods (radiolabeling/thin layer chromatography and benzoylation/high performance liquid chromatography), which, unlike the commonly used diglyceride kinase assay, discriminate between ceramide species and de novo formed dihydroceramide. We demonstrate that ceramide accumulates after several hours, reaching a 7-fold increase after 8 h, kinetics closely paralleling apoptosis induction. No fast response was observed, not even in the presence of inhibitors of ceramide metabolism. The majority ( approximately 70%) of the ceramide response remained unaffected when apoptosis was completely inhibited at the level of caspase-3/CPP32 processing by the inhibitor peptide DEVD-CHO. Exogenous cell-permeable C2-ceramide induced the proteolytic processing of caspase-3, albeit with somewhat slower kinetics than with CD95. DEVD-CHO dose-dependently inhibited C2-ceramide- or exogenous sphingomyelinase-induced apoptosis. The results support the idea that ceramide acts in conjunction with the caspase cascade in CD95-induced apoptosis.

Coplanar PCBs and the relative contribution of coplanar PCBs, PCDDs, and PCDFs to the total 2,3,7,8-TCDD toxicity equivalents in human serum.

Coplanar PCBs in human serum were measured by high-resolution gas chromatography/isotope-dilution high-resolution mess spectrometry in 46 pulp and paper mill workers and 16 community residents with no specific known source of PCB exposure. The relative contribution of coplanar PCBs, PCDDs, and PCDFs to the total 2,3,7,8-TCDD toxicity equivalents (TEQs) were compared using the toxic equivalency factors proposed by Safe [1] and the factors recently proposed by WHO [2]. The mean concentrations of PCB-126 and PCB-169 were higher in paper mill workers than in community residents. However, these differences were not statistically significant. Serum PCB-126, but not PCB-169, was correlated with body mass index (Spearman's r = 0.40, p = 0.002). Serum PCB-169, but not PCB-126, was correlated with age (Spearman's r = 0.54, p = 0.0001). Multiple linear regression analysis for log-transformed combined PCBs showed that age (p = 0.008), body mass index (p = 0.031), and eating locally caught fish (p = 0.019) were statistically significant predictors. The majority of the total TEQ in serum is due to PCDDs (63%), whereas PCDFs account for 21% and coplanar PCBs account for 15% when calculated using the TEFs proposed by Safe. The percent contributions from PCDDs, PCDFs, and coplanar PCBs were 66%, 24%, and 10% respectively when calculated based on the TEFs proposed by WHO. Age, body mass index, and consumption of locally caught fish are significant predictors for coplanar PCB levels in human serum. Serum PCDDs were the major contributors to the total 2,3,7,8-TCDD equivalent toxicity in this study.

Serum levels of polychlorinated dibenzo-p-dioxins and dibenzofurans in pulp and paper mill workers.

Serum levels of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) among 46 long-term workers at a pulp and paper mill were compared to the levels in 16 community residents who never worked at the mill. Overall, there were no appreciable differences among the three exposure groups (community resident, low-exposure-potential worker, high-exposure-potential worker) for specific PCDDs or PCDFs. Neither exposure group nor duration in high-exposure-potential-jobs was related to total toxic equivalents (I-TEQ). Serum levels of PCDDs and PCDFs in this study generally were within the range previously reported for persons with no known occupational exposure.

Monitoring of exposure to benomyl in nursery workers.

We compared urinary levels of the metabolite methyl-5-hydroxy-2-benzimidazole carbamate (5-HBC) among nursery workers exposed to the fungicide benomyl (specifically Benlate 50 DF [DuPont, Wilmington, DE]) and workers not exposed to benomyl. Environmental exposures were quantitated from gloves, body patches, and air samples collected with area and personal monitors. The median concentration of 5-HBC in the urine of benomyl-exposed workers was 23.8 mumol of 5-HBC per mole of creatinine. No 5-HBC was detected in the reference group. Industrial hygiene results and biological monitoring findings indicate that use of Benlate 50 DF in the ornamental industry can lead to absorption of the active ingredient, benomyl. Weighing, mixing, and application activities involved the highest exposures. Dermal contact appeared to be the primary route of exposure.

Work activities and musculoskeletal complaints among preschool workers.

The potential for musculoskeletal trauma among preschool workers has been largely unexplored in the United States. This case report describes an investigation conducted to identify and evaluate possible causes of back and lower extremity pain among 22 workers at a Montessori day care facility. Investigators met with and distributed a questionnaire to school employees, and made measurements of workstation and furniture dimensions. Investigators also recorded the normal work activities of school employees on videotape, and performed a work sampling study to estimate the percentage of time employees spend performing various tasks and in certain postures. Questionnaire results from 18 employees indicated that back pain/discomfort was a common musculoskeletal complaint, reported by 61% of respondents. Neck/shoulder pain, lower extremity pain and hand/wrist pain were reported by 33, 33 and 11% of respondents, respectively. Observation and analysis of work activities indicated that employees spend significant periods of time kneeling, sitting on the floor, squatting, or bending at the waist. Furthermore, staff members who work with smaller children (i.e. six weeks to 18 months of age) performed more lifts and assumed more awkward lower extremity postures than employees who work with older children (3-4 years of age). Analysis of two lifting tasks using the revised NIOSH lifting equation indicated that employees who handle small children may be at increased risk of lifting-related low back pain. Investigators concluded that day care employees at this facility are at increased risk of low back pain and lower extremity (i.e. knee) injury due to work activities that require awkward or heavy lifts, and static working postures. Recommendations for reducing or eliminating these risks by modifying the workplace and changing the organization and methods of work are presented.