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Tobacco use among Vietnamese adult males in the United States is higher than the general population. Less is known about the role of knowledge and attitudes of smoking in smoking status. This study describes the smoking prevalence, practices, support, knowledge, and attitudes among Vietnamese American men by smoking status. We administrated a cross-sectional in-person health questionnaire to randomly selected Vietnamese men (18-64 years of age) living in Seattle, Washington, using bilingual, bicultural Vietnamese male interviewers (N = 509). The response rate was 79%; the cooperation rate was 82%. Sixty-four percent of respondents had a history of smoking: 37% current, 27% former, and 36% never smokers. Smoking prevalence was lowest among men aged 18-29 years. Among smokers, 81% smoked 1 to 10 cigarettes per day, 69% wanted to quit, and 48% planned to do so in the next 6 months. Twelve percent of smokers reported smoking was allowed in the home. On average, respondents correctly answered six out of seven questions regarding health risks related to smoking. In logistic regression analyses, being a current smoker was negatively associated with a higher knowledge score (OR = 0.83, 95% CI 0.71-0.97). Adjusted odds of being a current smoker were 3.77 times higher among men who agreed with the attitude statement "It is appropriate for Vietnamese men to smoke when with friends." (OR = 2.15, 95% CI 1.28-3.61). The findings suggest a great need to develop appropriate tobacco-control interventions to lower smoking prevalence, improve tobacco-related health knowledge, and reduce the acceptance of smoking among Vietnamese American men.

Membrane-associated ATPase constitutes an essential element common to all secretion machineries in Gram-negative bacteria. How ATP hydrolysis by these ATPases is coupled to secretion process remains unclear. Here we identified R286 as a key residue in the type II secretion system (T2SS) ATPase XpsE of Xanthomonas campestris that plays a pivotal role in coupling ATP hydrolysis to protein translocation. Mutation of R286 to alanine made XpsE hydrolyse ATP at a rate five times that of the wild-type XpsE. Yet the mutant XpsE(R286A) is non-functional in protein secretion via T2SS. Detailed analyses indicated that the mutant XpsE(R286A) lost the ability co-ordinating the N- and C-domain of XpsE. Without significantly influencing XpsE binding affinity with ATP or its oligomerization, R286A mutation however, caused XpsE lose the ability to associate with the cytoplasmic membrane via XpsL(N). As a consequence, ATP hydrolysis by XpsE was uncoupled from protein secretion. Because R286 is highly conserved among members of the secretion NTPase superfamily, we speculate that its equivalent in other homologues may also play a critical energy coupling role for T2SS, type IV pilus assembly and type IV secretion system.

Background: Chinese North American women have high invasive cervical cancer rates and low screening rates. The cost-effectiveness of strategies to improve Pap testing rates for Chinese women living in Seattle, Washington and Vancouver, British Columbia was examined., Objectives: To calculate the costs and cost-effectiveness of implementing two strategies to motivate women to obtain a Pap smear., Research Design: A three-armed randomized, controlled trial was conducted. Women in each of two interventions (high-intensity outreach and low-intensity mailing intervention) were compared to a group of women who received usual care., Measures: Costs were captured via a group discussion of costs, accounting records, sampling of staff time logs, and estimation of costs and task times. Effectiveness was measured as the proportion of women in each intervention arm who reported receiving a Pap smear since the trial began. Cost-effectiveness was calculated as the incremental cost of screening each additional woman between an intervention arm and the control arm., Results: A greater percentage of women who received the outreach intervention had a Pap test than women who received mailed materials or women who were in the usual care arm. The intent-to-treat cost for each additional woman to be screened for a Pap test was $415 in the Outreach arm and $676 for the Direct Mailing arm. The outreach worker intervention, though more expensive overall, was more cost-effective than the mailing intervention., Conclusions: Outreach intervention is cost-effective for sponsors and should be considered as a strategy to motivate Chinese women living in North America to seek cervical cancer screening.

The purpose of this study was to report the prevalence of Vietnamese households with smokers and examine Papanicolau (Pap) testing among Vietnamese American women living in households with and without smokers. In 2002, we surveyed Vietnamese between 18 and 64 years of age from a population-based sample of randomly selected households in Seattle, Washington zip codes known to have a high density of Vietnamese residents. The response rate among eligible households was 82%, and our sample included 418 households. We used two measures of Pap testing: ever had a Pap test and had one in the last two years. Household smoking status was categorized as current smoker in the house vs. no current smoker in the house. Overall, 47% of Vietnamese American women lived with a current smoker in the household, 73% had ever received a Pap test, and 63% received one in the last two years. Pap testing behavior varied only slightly by household smoking status, and the findings were not statistically significant. With nearly half of Vietnamese women in our study currently living with smokers, future studies should examine the relationship between secondhand smoke at home and other health behaviors in Vietnamese American households.

Prostacyclin synthase (PGIS) catalyzes an isomerization of prostaglandin H(2) to prostacyclin, a potent mediator of vasodilation and anti-platelet aggregation. Here, we report the crystal structure of human PGIS at 2.15 A resolution, which represents the first three-dimensional structure of a class III cytochrome P450. While notable sequence divergence has been recognized between PGIS and other P450s, PGIS exhibits the typical triangular prism-shaped P450 fold with only moderate structural differences. The conserved acid-alcohol pair in the I helix of P450s is replaced by residues G286 and N287 in PGIS, but the distinctive disruption of the I helix and the presence of a nearby water channel remain conserved. The side-chain of N287 appears to be positioned to facilitate the endoperoxide bond cleavage, suggesting a functional conservation of this residue in O-O bond cleavage. A combination of bent I helix and tilted B' helix creates a channel extending from the heme distal pocket, which seemingly allows binding of various ligands; however, residue W282, placed in this channel at a distance of 8.4 A from the iron with its indole side-chain lying parallel with the porphyrin plane, may serve as a threshold to exclude most ligands from binding. Additionally, a long "meander" region protruding from the protein surface may impede electron transfer. Although the primary sequence of the PGIS cysteine ligand loop diverges significantly from the consensus, conserved tertiary structure and hydrogen bonding pattern are observed for this region. The substrate-binding model was constructed and the structural basis for prostacyclin biosynthesis is discussed.

Perillyl alcohol (POH) is a dietary monoterpene with potential applications in chemoprevention and chemotherapy. Although clinical trials are under way, POH's physiological and pharmacological properties are still unclear. In the present study, the effect of POH on polycyclic aromatic hydrocarbon (PAH)-induced genotoxicity, and the related expression were examined in MCF-7 cells. Exposure to environmental toxicant increases the risk of cancer. Many of these compounds are pro-carcinogens and are biotransformed into their ultimate genotoxic structures by xenobiotic metabolizing enzymes. CYP1A1 and 1B1 are enzymes that catalyze the biotransformation of dimethylbenz[a]anthracene (DMBA). Our data revealed that 0.5 microM of POH was effective in blocking DMBA-DNA binding. Ethoxyresorufin-O-deethylase (EROD) assay indicated that the administration of POH inhibited the DMBA-induced enzyme activity in MCF-7 cells. Enzyme kinetic analysis revealed that POH inhibited CYP1B1 but not CYP1A1 activity. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay also demonstrated that the monoterpene reduced CYP1B1 mRNA abundance induced by DMBA. The present study illustrated that POH might inhibit and downregulate CYP1B1, which could protect against PAH-induced carcinogenesis.

GspE belongs to a secretion NTPase superfamily, members of which are involved in type II/IV secretion, type IV pilus biogenesis and DNA transport in conjugation or natural transformation. Predicted to be a cytoplasmic protein, GspE has nonetheless been shown to be membrane-associated by interacting with the N-terminal cytoplasmic domain of GspL. By taking biochemical and genetic approaches, we observed that ATP binding triggers oligomerization of Xanthomonas campestris XpsE (a GspE homolog) as well as its association with the N-terminal domain of XpsL (a GspL homolog). While isolated XpsE exhibits very low intrinsic ATPase activity, association with XpsL appears to stimulate ATP hydrolysis. Mutation at a conserved lysine residue in the XpsE Walker A motif causes reduction in its ATPase activity without significantly influencing its interaction with XpsL, congruent with the notion that XpsE-XpsL association precedes ATP hydrolysis. For the first time, functional significance of ATP binding to GspE in type II secretion system is clearly demonstrated. The implications may also be applicable to type IV pilus biogenesis.

Secretion of fully folded extracellular proteins across the outer membrane of Gram-negative bacteria is mainly assisted by the ATP-dependent type II secretion system (T2SS). Depending on species, 12-15 proteins are usually required for the function of T2SS by forming a trans-envelope multiprotein secretion complex. Here we report crystal structures of an essential component of the Xanthomonas campestris T2SS, the 21-kDa N-terminal domain of cytosolic secretion ATPase XpsE (XpsEN), in two conformational states. By mediating interaction between XpsE and the cytoplasmic membrane protein XpsL, XpsEN anchors XpsE to the membrane-associated secretion complex to allow the coupling between ATP utilization and exoprotein secretion. The structure of XpsEN observed in crystal form P4(3)2(1)2 is composed of a 90-residue alpha/beta sandwich core domain capped by a 62-residue N-terminal helical region. The core domain exhibits structural similarity with the NifU-like domain, suggesting that XpsE(N) may be involved in the regulation of XpsE ATPase activity. Surprisingly, although a similar core domain structure was observed in crystal form I4(1)22, the N-terminal 36 residues of the helical region undergo a large structural rearrangement. Deletion analysis indicates that these residues are required for exoprotein secretion by mediating the XpsE/XpsL interaction. Site-directed mutagenesis study further suggests the more compact conformation observed in the P4(3)2(1)2 crystal likely represents the XpsL binding-competent state. Based on these findings, we speculate that XpsE might function in T2SS by cycling between two conformational states. As a closely related protein to XpsE, secretion ATPase PilB may function similarly in the type IV pilus assembly.

Site-directed mutagenesis was performed to change the substrate specificity of Escherichia coli aspartate aminotransferase (AAT). A double mutant, R292E/L18H, with a 12.9-fold increase in the specific activity toward L-lysine and 2-oxo-4-phenylbutanoic acid (OPBA) was identified. E. coli cells expressing this mutant enzyme could convert OPBA to L-homophenylalanine (L-HPA) with 97% yield and more than 99.9% ee using L-lysine as amino donor. The transamination product of L-lysine, 2-keto-6-aminocaproate, was cyclized nonenzymatically to form Delta(1)-piperideine 2-carboxylic acid in the reaction mixture. The low solubility of L-HPA and spontaneous cyclization of 2-keto-6-aminocaproate drove the reaction completely toward L-HPA production. This is the first aminotransferase process using L-lysine as inexpensive amino donor for the L-HPA production to be reported.

Bacteria possess two closely related yet functionally distinct essential type IIA topoisomerases (Topos). DNA gyrase supports replication and transcription with its unique supercoiling activity, whereas Topo IV preferentially relaxes (+) supercoils and is a decatenating enzyme required for chromosome segregation. Here we report the crystal structure of the C-terminal domain of Topo IV ParC subunit (ParC-CTD) from Bacillus stearothermophilus and provide a structure-based explanation for how Topo IV and DNA gyrase execute distinct activities. Although the topological connectivity of ParC-CTD is similar to the recently determined CTD structure of DNA gyrase GyrA subunit (GyrA-CTD), ParC-CTD surprisingly folds as a previously unseen broken form of a six-bladed beta-propeller. Propeller breakage is due to the absence of a DNA gyrase-specific GyrA box motif, resulting in the reduction of curvature of the proposed DNA binding region, which explains why ParC-CTD is less efficient than GyrA-CTD in mediating DNA bending, a difference that leads to divergent activities of the two homologous enzymes. Moreover, we found that the topology of the propeller blades observed in ParC-CTD and GyrA-CTD can be achieved from a concerted beta-hairpin invasion-induced fold change event of a canonical six-bladed beta-propeller; hence, we proposed to name this new fold as "hairpin-invaded beta-propeller" to highlight the high degree of similarity and a potential evolutionary linkage between them. The possible role of ParC-CTD as a geometry facilitator during various catalytic events and the evolutionary relationships between prokaryotic type IIA Topos have also been discussed according to these new structural insights.

Type IIA topoisomerases are multidomain enzymes composed of four major domains: the ATPase domain, the TOPRIM domain, the DNA-cleavage/religation domain and the C-terminal domain (CTD). Although crystal structures of the first three domains are available, the three-dimensional structure of the less-conserved CTD has yet to be determined. In order to provide a three-dimensional structure of this structurally uncharacterized region, the 36 kDa CTD of ParC protein, the DNA-cleavage/religation subunit of topoisomerase IV, from Bacillus stearothermophilus has been cloned, purified and crystallized. The crystals belonged to the trigonal space group P3(1) (or P3(2)), with unit-cell parameters a = b = 83.5, c = 45.1 A. The asymmetric unit contains one molecule and the solvent content is 51.2%. A 98.9% complete native data set has been collected from a frozen crystal to 2.0 A resolution with an overall R(merge) of 6.5%.

Hereditary haemorrhagic telangiectasia, HHT, is an autosomal dominant disorder that affects approximately 1 in 8000 people. HHT1 is associated with mutations in the ENG (Endoglin) gene and with haploinsufficiency. The disorder is characterized by focally dilated vessels, which can lead to arteriovenous malformations and serious complications even in young children. In the current study, umbilical cord and placenta samples from newborns with ENG mutations were analyzed to estimate the level of corresponding protein and look for potential vascular dysplasia. We confirmed, using metabolic labelling and flow cytometry, that endoglin levels were significantly reduced to median values of 47 per cent (range 32-56 per cent) and 58 per cent (46-90 per cent), respectively, in human umbilical vein endothelial cells derived from newborns with ENG mutations (HHT1 group; n=18) relative to samples from newborns shown not to have the familial mutation (non-HHT group). We also quantified the relative expression of endoglin by estimating the endoglin/PECAM-1 staining ratio in tissue sections. We observed significantly lower values in the HHT1 group, compared to the non-HHT group for the umbilical vein (n=9; median 0.6 vs 0.9; ranges 0.2-1.0 and 0.5-1.5) and for placental stem villus vessels (n=9 and 10; median 0.42 vs 0.93; ranges 0.24-0.58 and 0.56-1.18). No differences in the estimated umbilical vein cross-sectional area and in the proportion of vessels present in placental villi were observed in sections from the HHT1 group relative to the non-HHT group. Thus, blood vessels from HHT1 individuals are maintained intact in the umbilical vein and placenta during pregnancy and delivery, despite a significant reduction in endoglin expression.

In addition to interacting with hemoglobin as a heme ligand to form nitrosylhemoglobin, NO can react with cysteine sulfhydryl groups to form S-nitrosocysteine or cysteine oxides such as cysteinesulfenic acid. Both modes of interaction are very sensitive to the quaternary structure of hemoglobin. To directly view the interaction of NO with quaternary-T deoxyhemoglobin, crystallographic studies were carried out on crystals of deoxyhemoglobin that were exposed to gaseous NO under a variety of conditions. Consistent with previous spectroscopic studies in solution, these crystallographic studies show that the binding of NO to the heme groups of crystalline wild-type deoxyhemoglobin ruptures the Fe-proximal histidine bonds of the alpha-subunits but not the beta-subunits. This finding supports Perutz's theory that ligand binding induces tension in the alpha Fe-proximal histidine bond. To test Perutz's theory, deoxy crystals of the mutant hemoglobin betaW37E were exposed to NO. This experiment was carried out because previous studies have shown that this mutation greatly reduces the quaternary constraints that oppose the ligand-induced movement of the alpha-heme Fe atom into the plane of the porphyrin ring. As hypothesized, the Fe-proximal histidine bonds in both the beta- and the alpha-subunits remain intact in crystalline betaW37E after exposure to NO. With regard to S-nitrosocysteine or cysteine oxide formation, no evidence for the reaction of NO with any cysteine residues was detected under anaerobic conditions. However, when deoxyhemoglobin crystals are first exposed to air and then to NO, the appearance of additional electron density indicates that Cys93(F9)beta has been modified, most likely to cysteinesulfenic acid. This modification of Cys93(F9)beta disrupts the intrasubunit salt bridge between His146(HC3)beta and Asp94(FG1)beta, a key feature of the quaternary-T hemoglobin structure. Also presented is a reanalysis of our previous crystallographic studies [Chan, N.-L., et al. (1998) Biochemistry 37, 16459-16464] of the interaction of NO with liganded hemoglobin in the quaternary-R2 structure. These studies showed additional electron density at Cys93(F9)beta that was consistent with an NO adduct. However, for reasons discussed in this paper, we now believe that this adduct may be the Hb-S-N.-O-H radical intermediate and not Hb-S-N=O as previously suggested.

Secretion of pre-folded extracellular proteins across the outer membrane of Gram-negative bacteria is mainly assisted by the type II secretion machinery composed of 12-15 proteins. Here, the crystallization and preliminary analysis of one of the essential components of Xanthomonas campestris secretion machinery, the 21 kDa N-terminal domain of XpsE protein (XpsE(N)), are reported. XpsE(N) has been crystallized at 277 K using PEG 400 as precipitant. These crystals belong to the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 56.1, c = 102.7 A. A 98.5% complete native data set from a frozen crystal has been collected to 2.0 A resolution at 100 K with an overall R(merge) of 5.0%. The presence of one subunit of XpsE(N) per asymmetric unit gives a crystal volume per protein weight (V(M)) of 1.92 A(3) Da(-1) and a solvent content of 36.1%.

Bis(maleidophenyl)-PEG2000 (Bis-Mal-PEG2000), a new bifunctional protein cross-linker targeted to sulfhydryl groups, introduces intra-tetrameric cross-links into oxy-HbA in nearly quantitative yields. Structural as well as crystallographic analyses of the cross-linked species, Bis-Mal-PEG2000 HbA, identified Cys-93(beta) as the site of intramolecular cross-linking. The cross-bridging had only a limited influence on the O(2) affinity and cooperativity of HbA in 50 mM BisTris acetate, pH 7.4. However, the Bohr effect was reduced by approximately 60%. Bis-Mal-PEG2000 HbA retained sensitivity to the presence of allosteric effectors 2, 3-diphosphoglycerate, IHP, and chloride, albeit to a lesser degree compared with HbA. Crystallographic analysis revealed the overall structure of deoxy-Bis-Mal-PEG2000 HbA to be similar to deoxy-HbA but for the loss of the salt bridge between Asp-94(beta) and His-146(beta). The large influence of the cross-bridging on the alkaline Bohr effect of HbA is consistent with the loss of this salt bridge. Unlike the "central cavity cross-bridges" described previously, the cross-link introduced by Bis-Mal-PEG2000 into HbA is an "outside the central cavity cross-bridge." In view of its oxy-conformational specificity and limited influence on O(2) affinity, this new cross-linking strategy holds promise for the stabilization of new designer low O(2) affinity Hbs generated by recombinant DNA technology for applications as Hb based therapeutics.

Although numerous reports have documented that the S-nitrosylation of cysteine residues by NO alters the activities of a wide variety of proteins, the direct visualization and the structural consequences of this reversible modification have not yet been reported for any protein. Here we describe the crystal structure of S-nitroso-nitrosylhemoglobin determined at a resolution of 1.8 A. The specific reaction of NO with Cys93beta is confirmed in this structure, and a large S-nitrosylation-induced change in the tertiary structure of the COOH-terminal dipeptides of the beta subunits provides additional insight into the stereochemical mechanism by which blood flow is regulated by the interaction of NO with hemoglobin.