Statistical study of the influence of fungicide treatments (mancozeb, zoxamide and copper oxychloride) on heavy metal concentrations in Sicilian red wine.

The aim was to assess the influence of mancozeb, zoxamide and copper oxychloride fungicide treatments on Mn, Zn, Cu, Cd and Pb concentrations in Sicilian red wines, grapes, marcs and grape stalks. The experimentation was carried out over two crop years: 2003 and 2004. Trace metals analysis was performed by derivative stripping chronopotentiometry, which allowed detection of concentrations lower than 1 ng g(-1). The data obtained gave evidence that the levels of Mn and Zn in wines from plots treated with zoxamide-mancozeb were about threefold higher than those observed in the control. Wines treated with Cu oxychloride had a significant increase in Cu(II) concentrations with respect to the control; in particular, samples from 2004 showed a 50% increase in Cu levels. Furthermore, as shown in a previous paper, the fungicides treatments studied led to a moderate increase in Pb(II) and Cd(II) levels in treated samples with respect to the control. Wines from 2004 had higher Cu and Pb amounts than wines from 2003; but the concentrations of all the other metals were similar. Statistical analysis of the data by linear discriminant analysis (LDA) and the Kruskal-Wallis test confirmed that both zoxamide-mancozeb treatments and copper oxychloride treatments exerted a significant influence on Mn(II), Zn(II) Cu(II), Pb(II) and Cd(II) concentrations in wines, grapes, marcs and grape stalks samples from both the studied vintages.

In vitro antiretroviral activity and in vitro toxicity profile of SPD754, a new deoxycytidine nucleoside reverse transcriptase inhibitor for treatment of human immunodeficiency virus infection.

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates, its activity in combination with other antiretrovirals, and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L, T215Y/F, plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V, Y115F, and M184V plus one other NAM) or stavudine (V75T/M, M41L, T215F/Y, and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold, respectively (these changes gave values comparable to or less than the corresponding values for zidovudine, lamivudine, abacavir, and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades, including A/G, B, C, D, A(E), D/F, F, and H. SPD754 showed additive effects in combination with other NRTIs, tenofovir, nevirapine, or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 microM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro, SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity.

Aminoglycoside binding to human and bacterial A-Site rRNA decoding region constructs.

The 16S bacterial ribosomal A-site decoding rRNA region is thought to be the pharmacological target for the aminoglycoside antibiotics. The clinical utility of aminoglycosides could possibly depend on the preferential binding of these drugs to the prokaryotic A-site versus the corresponding A-site from eukaryotes. However, quantitative aminoglycoside binding experiments reported here on prokaryotic and eukaryotic A-site RNA constructs show that there is little in the way of differential binding affinities of aminoglycosides for the two targets. The largest difference in affinity is 4-fold in the case of neomycin, with the prokaryotic A-site construct exhibiting the higher binding affinity. Mutational studies revealed that decoding region constructs retaining elements of non-Watson-Crick (WC) base pairing, specifically bound aminoglycosides with affinities in the muM range. These studies are consistent with the idea that aminoglycoside antibiotics can specifically bind to RNA molecules as long as the latter have non-A form structural elements allowing access of aminoglycosides to the narrow major groove.

Chemosensitization of bladder carcinoma cells by bcl-xL antisense oligonucleotides.

PURPOSE: We investigated antisense inhibition of anti-apoptotic bcl-xL and bcl-2 proteins to increase chemosensitization in the T24 and 5637 bladder carcinoma cell lines. MATERIALS AND METHODS: A T24 bladder carcinoma cell line stably over expressing bcl-xL protein was constructed. Apoptosis by cytotoxic agents was estimated by cell cycle analysis and Annexin V binding. To eliminate bcl-xL expression T24 and 5637 cells were treated with C5-propynylated and 2'-O-methylribo-oligonucleotides. Levels of protein and messenger RNA were measured by Western and Northern blot analysis. Cell viability after combined treatment with oligonucleotides and various cytotoxic agents was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and evaluated statistically by Student's 2-sample t test. RESULTS: Forced over expression of bcl-xL protein desensitized the T24 bladder carcinoma cell line to cytotoxic agents. C5-propynylated and 2'-O-methylribo-oligonucleotides down-regulated bcl-xL protein expression in the T24 and 5637 cell lines, and increased their sensitivity to cytotoxic agents. The efficiency of antisense down-regulation of bcl-xL protein expression depended on the type of delivery agent. CONCLUSIONS: Antisense down-regulation of bcl-xL protein sensitizes bladder carcinoma cells to cytotoxic agents. However, it is possible that cellular chemosensitization results from a combination of effects, including nonsequence specificity, irrelevant cleavage and effects of the carriers combined with the specific antisense effects.

Reduced levels of retinyl esters and vitamin A in human renal cancers.

Clinical and preclinical studies suggest that retinoids can inhibit the growth of a small percentage of human renal cancers (RCs), although the majority of RCs both in vitro and in vivo are retinoid resistant. Our recent studies indicate that the metabolism of retinol to retinyl esters is greatly reduced in human carcinoma cell lines of the oral cavity, skin, and breast as compared with their normal epithelial counterparts, suggesting that human carcinoma cells are retinoid deficient relative to normal epithelial cells. We considered whether retinoid resistance in RCs was related to an abnormality in retinoid metabolism. The metabolism of [3H]retinol and of [3H]retinoic acid (RA) was examined in RC cell lines and normal human kidney (NK) epithelial cells cultured in media, in RA, or in RA plus IFN-alpha. The expression of LRAT (lecithin:retinol acyltransferase) was assessed by Northern and Western analysis. Retinol and retinyl ester levels were determined in tissue samples of normal human kidney and renal cell carcinoma. NK cells esterified all of the 50 nM [3H]retinol in which they were cultured. In contrast, six of the seven RC cell lines metabolized only trace amounts of [3H]retinol to [3H]retinyl esters. Consistent with this relative lack of [3H]retinol esterification by the tumor cells, the tumor cells exhibited LRAT transcripts of aberrantly low sizes relative to those in normal epithelial cells. Moreover, the NK cells expressed abundant levels of LRAT protein by Western analysis, whereas the RC cells did not express LRAT protein. When samples of human kidney tumor tissue were compared with samples of normal kidney tissue from patients who had undergone surgery for primary RC, the normal kidney tissues contained much higher levels of retinol and retinyl esters (approximately 0.5-2 microg/gram wet weight) than the tumor tissues in all seven patients examined. Culture of the RC lines in IFN-alpha plus all-trans-RA, a combination therapy used clinically, resulted in higher intracellular levels of [3H]retinol and [3H]retinyl esters. The metabolism of [3H]RA was also examined in these RC lines versus NK cells. Although the NK epithelial cells metabolized [3H]RA, the majority of the RC lines metabolized [3H]RA at a much slower rate. Most of the RC lines metabolized only 10-30% of the 50 nM [3H]RA over 6 h of culture. These data indicate that RCs both in vitro and in vivo are retinol and retinyl ester deficient relative to the normal human kidney, and they suggest that the aberrant differentiation of the neoplastic renal cells results in part from a defect in retinoid metabolism.

Cohort mortality study of North American industrial sand workers. I. Mortality from lung cancer, silicosis and other causes.

BACKGROUND: In 1997 a Working Group of the International Agency for Research on Cancer changed an earlier classification of crystalline silica as a human carcinogen from Group 2A to Group 1, though commenting that the carcinogenicity might vary with industrial circumstances and depend on additional factors affecting biological activity, including the distribution of its polymorphs. OBJECTIVE: We aimed to determine whether pure quartz exposure uncomplicated by the presence of other contaminating carcinogens, as experienced by workers in the production of high-grade industrial sand, was causally related to an increased risk of lung cancer. METHODS: A cohort of 2670 men employed before 1980 for 3 years or more in one of nine North American sand-producing plants and a large associated office complex was selected for study. Of the cohort, 2644 (99%) were traced through 1994, and certificated cause of death ascertained for 1025 (99%) of the 1039 men known to have died. Standardised mortality ratios (SMRs) were calculated for the main causes of death, using both US and state or provincial male mortality rates for reference. FINDINGS: The main analyses of deaths, 20 or more years after first employment against regional rates, gave the following SMRs: all causes 109, lung cancer 139, other malignancies 98, non-malignant respiratory disease 161, and nephritis/nephrosis 244. There were, in total, 37 deaths from silicosis or silico-tuberculosis, with one or more death at least in all nine production plants. Analyses failed to show any relation between lung cancer risk and duration of employment. The increased SMR for lung cancer was wholly due to high rates in four plants in two states, whereas no increase was found in the remainder of the cohort. CONCLUSION: In the absence of information on smoking histories and risk in relation to estimated exposure, the increased SMR for lung cancer (139), although statistically significant, cannot be attributed confidently to crystalline silica. An answer to the question of attributability must await the findings of the nested case-control study, in which level of exposure and smoking habits were ascertained for cases and matched controls. The strong indication in this cohort of excess mortality from non-malignant renal disease deserves further investigation.

Cohort mortality study of North American industrial sand workers. II. Case-referent analysis of lung cancer and silicosis deaths.

BACKGROUND: A cohort mortality study of 2670 men in nine North American industrial sand plants resulted in 83 deaths from lung cancer 20 or more years after hire (standardized mortality ratio 139) and 37 deaths from silicosis (including seven from silico-tuberculosis). The lung cancer excess was unrelated to duration of employment and not found in all plants. OBJECTIVES: The primary aim was to determine whether lung cancer risk among these employees was related to quantitative estimates of crystalline silica exposure, after allowance for cigarette smoking. A secondary aim was to do the same for silicosis mortality, partly as a means of validating the estimated levels of exposure. METHODS: A nested case-referent study was undertaken with cases matched with up to two controls on plant, age and date of first employment from men who survived the case. Exposures were estimated by linking work histories to a job-exposure matrix, undertaken separately. Cigarette smoking information was obtained from medical records and other sources, blind as to case-control status. Matched statistical analyses were conducted using conditional logistic regression. FINDINGS: Odds ratios for silicosis mortality were significantly related to cumulative silica exposures and tended to a relationship with category of average crystalline silica concentration, but inconsistently with length of employment. After accounting for a strong effect of cigarette smoking, odds ratios for lung cancer were related to cumulative crystalline silica exposure and to average silica concentration, but not to length of employment. CONCLUSION: These findings support a causal relationship between lung cancer and quartz exposure after allowance for cigarette smoking, in the absence of cristobalite or other known occupational carcinogens.

Cohort mortality study of North American industrial sand workers. III. Estimation of past and present exposures to respirable crystalline silica.

BACKGROUND: Lung cancer and silicosis mortality were examined longitudinally and by a case-referent analysis in a cohort of workers selected from the North American industrial sand industry. Date of hire in the case-referent sub-cohort extended as far back as the second decade of the twentieth century. OBJECTIVE: The aim of this study component was to develop estimates of average and cumulative exposure to respirable crystalline silica for the 342 selected cases and referents. METHODS: Process and dust control histories were developed for each plant, and quantitative exposure data obtained from each of them and from a trade organization. An algorithm was developed to convert historical exposures reported in particle count concentrations to modern measures of mass concentration of respirable crystalline silica. Personal exposures were adjusted for use of protective equipment based on frequency of use and type of protection. FINDINGS: Between 1974 and 1998, a total of 14249 exposure measurements had been taken using a cyclone and membrane filter and gave an overall geometric mean of 42 microg/m3. The only exposure data identified earlier were based on approximately 500 samples collected across the industry between 1947 and 1955 using the Greenburg-Smith impinger, with analysis by microscopy. These data were converted to modern measures using a factor of 1 mppcf = 276 microg/m3 respirable dust and then adjusting for percentage silica. In general, the highest exposures occurred in bagging and bulk-loading operations and the lowest in wet processing of sand. CONCLUSIONS: There has been a substantial decline in exposure levels in this industry over time. The decline was rapid between the 1940s and 1970s and current exposures are, on average, less than 50 microg/m3. The use of personal protective equipment was judged to have had little impact on exposure before the 1970s.

Two histidine residues are essential for catalysis by lecithin retinol acyl transferase.

Lecithin retinol acyl transferase (LRAT) is a novel membrane bound enzyme that catalyzes the formation of retinyl esters from vitamin A and lecithin. The enzyme is both essential for vision and for the general mobilization of vitamin A. The sequence of LRAT defines it as a novel enzyme unrelated to any other protein of known function. LRAT possesses a catalytically essential active site cysteine residue. The enzyme also contains six histidine residues. It is shown here that two of these residues (H57 and H163) are essential for catalysis. A mechanistic hypothesis is presented to account for these observations.

Esterification of all-trans-retinol in normal human epithelial cell strains and carcinoma lines from oral cavity, skin and breast: reduced expression of lecithin:retinol acyltransferase in carcinoma lines.

When exogenous [(3)H]retinol (vitamin A) was added to culture medium, normal human epithelial cells from the oral cavity, skin, lung and breast took up and esterified essentially all of the [(3)H]retinol within a few hours. As shown by [(3)H]retinol pulse-chase experiments, normal epithelial cells then slowly hydrolyzed the [(3)H]retinyl esters to [(3)H]retinol, some of which was then oxidized to [(3)H]retinoic acid (RA) over a period of several days. In contrast, cultured normal human fibroblasts and human umbilical vein endothelial cells (HUVEC) did not esterify significant amounts of [(3)H]retinol; this lack of [(3)H]retinol esterification was correlated with a lack of expression of lecithin:retinol acyltransferase (LRAT) transcripts in normal fibroblast and HUVEC strains. These results indicate that normal, differentiated cell types differ in their ability to esterify retinol. Human carcinoma cells (neoplastically transformed epithelial cells) of the oral cavity, skin and breast did not esterify much [(3)H]retinol and showed greatly reduced LRAT expression. Transcripts of the neutral, bile salt-independent retinyl ester hydrolase and the bile salt-dependent retinyl ester hydrolase were undetectable in all of the normal cell types, including the epithelial cells. These experiments suggest that retinoid-deficiency in the tumor cells could develop because of the lack of retinyl esters, a storage form of retinol.

Parallel synthesis of isatin-based serine protease inhibitors.

The synthesis of N-functionalised isatins using parallel, solution synthesis is described. Functionalised polymers were employed as stoichiometric and catalytic reagents as well as purification media in the exercise, and the derivatives were screened against a panel of serine proteases; high percentage inhibition was observed in several cases.

Bcl-xL in prostate cancer cells: effects of overexpression and down-regulation on chemosensitivity.

Both Bcl-xL and Bcl-2, antiapoptotic members of the Bcl family, are found in prostate cancer cell lines. Although these proteins may have similar antiapoptotic functions, it is not clear to what extent each serves as an antiapoptotic effector in prostate cancer cells. We engineered LNCaP and PC-3 cells to overexpress Bcl-xL protein and demonstrated that this desensitized them to the effects of cytotoxic chemotherapy. We then used two "antisense" strategies to down-regulate Bcl-xL protein expression in the parental lines. The first strategy used CS-propynylated phosphorothioate-phosphodiester oligonucleotides and co-down-regulated both Bcl-xL and Bcl-2; the second strategy used isosequential "gap-mer" phosphorothioate oligonucleotides containing 2'-O-methyl oligoribonucleotides at the 3' and 5' termini. In this case, only Bcl-xL protein expression was affected. The most active oligonucleotides of both types decreased the level of Bcl-xL protein expression to 5-30% of the control level. Multiple controls were inactive. Experiments combining oligonucleotide treatment with cytotoxic chemotherapeutic agents (paclitaxel, docetaxel, etoposide, vinblastine, carboplatin, and mitoxantrone) demonstrated a marked increase in the sensitivity of these prostate cancer cells. However, the increase in chemosensitivity in PC-3 cells was statistically identical (except mitoxantrone) for both "antisense" strategies, indicating that basal expression of Bcl-2, in contrast to that of Bcl-xL, may play little cytoprotective role in these cells.

Drug resistance and drug combination features of the human immunodeficiency virus inhibitor, BCH-10652 [(+/-)-2'-deoxy-3'-oxa-4'-thiocytidine, dOTC].

The heterosubstituted nucleoside analogue dOTC [( )-2'-deoxy-3'-oxa-4'-thiocytidine, BCH-10652] is a racemic compound structurally related to 3TC (lamivudine), but has the oxygen and sulphur in the furanosyl ring transposed. Both the enantiomers (-)dOTC (BCH-10618) and (+)dOTC (BCH-10619) had equivalent activity against wild-type strains of HIV-1 in C8166 T-cells (EC50 1.0-10.0 microM) and in PBMCs (EC50 0.1-3.0 microM). Investigation of the activity of dOTC and its enantiomers against laboratory strains of HIV-1 with defined resistance to 3TC, AZT (zidovudine), ddl (didanosine), PMEA (adefovir), nevirapine and saquinavir indicated that sensitivity was maintained (<3-fold change in EC50) in all cases, with the exception of HIV-1RF 3TC-resistant viruses. The degree of resistance recorded for dOTC (four- to sevenfold), (-)dOTC (five- to eightfold) and (+)dOTC (five- to >18-fold) against these M1841 or M184V mutants, was significantly less than that recorded for 3TC (>100-fold). In addition, the inhibitory effect of the compounds against clinical isolates of HIV-1 recovered from patients with suspected resistance to 3TC and AZT was investigated. Clinical isolates were genotyped using the Murex Line Probe Assay (LiPA) and subgrouped into wild-type, 3TC-resistant and dual 3TC/AZT-resistant, as well as undefined or mixed genotype populations. Compared with the mean EC50 values obtained with genotypically and phenotypically wild-type clinical isolates, the mean EC50 values calculated for isolates phenotypically resistant to 3TC or 3TC and AZT were only 2.6-, 1.6- and 8.2-fold higher for dOTC, (-)dOTC and (+)dOTC, respectively. When the rate of emergence of virus resistant to dOTC and its enantiomers in vitro was investigated, virus resistant to (+)dOTC was readily selected for (<10 passages), and a methionine (ATG) to isoleucine (ATA) amino acid change at codon 184 was identified. In contrast, virus resistant to dOTC and (-)dOTC took longer to appear (15-20 passages), with a methionine (ATG) to valine (GTG) amino acid change at position 184 identified in both cases. In addition, virus passaged 20 times in the presence of dOTC also had a partial lysine (AAA) to arginine (AGA) exchange at position 65. These viruses showed only low-level resistance to dOTC and its enantiomers, but were highly resistant to 3TC. The antiviral effects of dOTC in combination with the nucleoside RT inhibitors AZT, 3TC, d4T (stavudine) and ddl, the non-nucleoside RT inhibitor nevirapine and the protease inhibitors saquinavir, ritonavir and indinavir was investigated. Two-way drug combination assays were carried out in peripheral blood mononuclear cell (PBMC) cultures by measuring the reduction in p24 viral antigen levels, and data was analysed using the MacSynergy II program. dOTC in combination with 3TC or d4T showed a moderate synergistic effect while all other combinations had an additive interaction.

Determination of carbon monoxide with a modified zeolite sorbent and methanization-gas chromatography.

The purpose of this study was to develop an alternative sorbent sampling technique to concentrate CO from an air sample for subsequent instrumental analysis. Y52 zeolite doped with 9.4 wt % cuprous ions was found to have high capacity, stability to air, and thermal reversibility for CO. The Cu(I)-modified zeolite was packed in glass tubes, preceded by a drying tube containing silica gel. Air was sampled through the tubes at the flow rate of 100 mL/min. Collected CO was thermally desorbed at 300 degrees C and determined by gas chromatography with reduction of CO to methane and flame ionization detection (TD-GC-CH4-FID). Breakthrough capacity of the sorbent was found to be 2.74 mg CO per gram of sorbent. For 2-L air samples containing 12.5 to 100 ppm CO and 50% relative humidity at room temperature, recovery of CO was found to be 96.6% with pooled relative standard deviation of 5.8%. The estimated detection limit for a 2-L sample was 0.2 ppm. Collected CO was stable at room temperature for 1 day and up to 7 days at 4 degrees C if the sorbent tube was flushed with helium before storage. In field testing, the ratio of CO measured by the new technique and by a reference technique was found to be 0.93 with pooled relative standard deviation of 6.3%. This unique new sorbent coupled with TD-GC-CH4-FID shows promise as a sensitive and specific alternative for measurement of CO in air.

RNA aptamers that specifically bind to a 16S ribosomal RNA decoding region construct.

RNA-RNA recognition is a critical process in controlling many key biological events, such as translation and ribozyme functions. The recognition process governing RNA-RNA interactions can involve complementary Watson-Crick (WC) base pair binding, or can involve binding through tertiary structural interaction. Hence, it is of interest to determine which of the RNA-RNA binding events might emerge through an in vitro selection process. The A-site of the 16S rRNA decoding region was chosen as the target, both because it possesses several different RNA structural motifs, and because it is the rRNA site where codon/anticodon recognition occurs requiring recognition of both mRNA and tRNA. It is shown here that a single family of RNA molecules can be readily selected from two different sizes of RNA library. The tightest binding aptamer to the A-site 16S rRNA construct, 109.2-3, has its consensus sequences confined to a stem-loop region, which contains three nucleotides complementary to three of the four nucleotides in the stem-loop region of the A-site 16S rRNA. Point mutations on each of the three nucleotides on the stem-loop of the aptamer abolish its binding capacity. These studies suggest that the RNA aptamer 109.2-3 interacts with the simple 27 nt A-site decoding region of 16S rRNA through their respective stem-loops. The most probable mode of interaction is through complementary WC base pairing, commonly referred to as a loop-loop 'kissing' motif. High affinity binding to the other structural motifs in the decoding region were not observed.

A novel high throughput screening assay for HCV NS3 helicase activity.

A novel assay for measurement of Hepatitis C virus (HCV) NS3 helicase activity was developed using Flashplate technology. This assay involves the use of a DNA duplex substrate and recombinant HCV NS3 produced in Escherichia coli. The DNA duplex consisted of a pair of oligonucleotides, one biotinylated, the other radiolabeled at their respective 5' termini. This DNA duplex was immobilized, via the biotin molecule, on the surface of a neutravidin-coated SMP103 Flashplate (NEN Life Science Products). Helicase activity results in the release of the radiolabeled oligonucleotide, which translates in signal reduction with respect to control wells. Biochemical characterization of the HCV NS3 helicase activity was performed using this assay. We demonstrated that the NS3-mediated unwinding is proportional to both the amount of DNA substrate in the well, and to the NS3 concentration in the reaction. Most of the NS3-mediated unwinding was achieved in the initial 60 min of incubation. As expected the reactions were ATP-dependent and found to be affected by the concentration of MgCl(2), MnCl(2), KCl, EDTA, and by pH. We found this assay to be highly reproducible since only slight variation was observed when a total of 68 helicase reactions were performed on one plate. Therefore, this Flashplate helicase assay is fast, convenient and reproducible. These criteria make it suitable for high throughput screening of potential NS3 helicase inhibitors.

Lecithin retinol acyltransferase contains cysteine residues essential for catalysis.

Lecithin retinol acyltransferase (LRAT) is an essential enzyme in vitamin A metabolism and mobilization. The membrane-bound enzyme catalyzes the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate retinyl esters. The sequence of LRAT is novel and hence does not suggest a mechanistic class to which the enzyme belongs. However, the activity of the enzyme is exceedingly sensitive to affinity labeling and group-specific reagents directed toward thiol groups. LRAT from human retinal pigment epithelium has cysteine residues at positions 161, 168, 182, and 208. Site-specific mutagenic studies show that C182 and C208 can be converted to alanines with little affect on activity. The activities of the C161A and C168A mutants are virtually nil. Moreover, while C168S is substantially active, C161S possesses only a few percent of the activity of wild-type (WT) LRAT. Also, pH-rate profiles show that C168S has virtually the same profile as WT LRAT, while C161S shows an aberrant profile quite unlike that of WT LRAT. Therefore, LRAT is a thiol acyltransferase and C161 may be the essential nucleophilic residue critical for catalysis.