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BACKGROUND: In the global program for the elimination of lymphatic filariasis (LF) longitudinal assessment of the prevalence of microfilaremia and antigenemia is recommended to monitor the effect of mass treatment on transmission. Additional monitoring tools such as entomologic and antibody methods may be useful in identifying residual foci of infection. In this study, we characterized serologic markers of infection and exposure spatially both before and after mass treatment, in an area of initial low Wuchereria bancrofti infection prevalence. METHODS: Consenting persons in the sentinel community were tested for circulating microfilaria and antigen (by immunochromatographic test) before and after the 1st annual mass drug administration of diethylcarbamazine and albendazole. A cohort of 161 persons provided serum specimens both years that were tested for antifilarial IgG (1 and 4) antibody. Every house was mapped using a differential Global Positioning System; this information was linked to the serologic data. W. bancrofti infection in the mosquito vector was assessed with year-round collection. Multiple linear regression was used to investigate the influence of antigen-positive persons on the antifilarial antibody responses of antigen-negative neighbors. RESULTS: After mass treatment, decreases were observed in the sentinel site in the overall prevalence of antigen (10.4% to 6.3%) and microfilaremia (0.9 to 0.4%). Of the persons in the cohort that provided serum specimens both years, 79% received treatment. Antigen prevalence decreased from 15.0% to 8.7%. Among 126 persons who received treatment, antigen and antifilarial IgG1 prevalence decreased significantly (p = 0.002 and 0.001, respectively). Among 34 persons who did not receive treatment, antifilarial IgG1 prevalence increased significantly (p = 0.003). Average antifilarial IgG1 levels decreased in households with high treatment coverage and increased in households that refused treatment. Each 10-meter increase in distance from the residence of a person who was antigen-positive in 2000 was associated a 4.68 unit decrease in antifilarial IgG1 level in 2001, controlling for other factors (p = 0.04). DISCUSSION: Antifilarial antibody assays can be used as a measure of filarial exposure. Our results suggest that micro-scale spatial heterogeneity exists in LF exposure and infection. Treatment appeared to be associated with reduced exposure at the sub-community level, suggesting the need to achieve high and homogeneous coverage. Public health messages should note the benefits of having one's neighbors receive treatment with antifilarial drugs.

We conducted a school-based assessment of the geographic distribution of Wuchereria bancrofti infection in Leogane Commune, Haiti, using the immunochromatographic test. In multivariate analyses performed using generalized linear mixed models, children attending schools in the foothills and plains were 3.95 (95% confidence interval [CI] = 1.28-12.23) and 23.56 (95% CI = 8.99-61.79) times as likely to be infected, respectively, as children attending mountain schools. Infection prevalence decreased with increasing altitude, but some local foci of infection were detected at higher altitudes. Higher school tuition, a marker of socioeconomic status (SES), was not associated with decreased infection prevalence. Our results indicate that although the force of infection in Leogane Commune is greatest below 70 meters above sea level, higher altitude communities are not exempt from infection. Lymphatic filariasis (LF) elimination programs should consider extending infection mapping activities to presumed non-LF altitudes. In addition, higher SES does not confer protection against W. bancrofti infection.

In the global effort to eliminate lymphatic filariasis, annual mass treatments are conducted with diethylcarbamazine (DEC) or ivermectin, combined with albendazole. The success of this strategy depends on achieving high levels of drug coverage, which reduces the number of persons with circulating microfilariae so that transmission of the parasite is interrupted. Because resources are often limited, a simple, inexpensive, and reliable method to estimate drug coverage is needed. During the period December 2000 to February 2001, three methods were used to assess drug coverage in Leogane Commune, Haiti: a probability survey using a cluster sample design (n = 1421 persons); a distribution-point survey based on a convenience sample of houses near the distribution points (n = 4341 persons); and a survey based on a convenience sample of primary schools (n = 5036 children). The coverage estimations were 71.3% (95% CI 66.7-75.9), 73.6% (95% CI 70.1-77.0), and 77.8% (95% CI 73.5-82.1), respectively. Survey costs for the probability, distribution point, and school surveys were US$2217, US$979, and US$312, respectively. The 2 convenience sampling methods provided point estimates of drug coverage that were similar to those of the probability survey. These methods may have a role for monitoring drug treatment coverage between less frequent, but more costly, probability sample surveys.

In October 2000, 71,187 persons were treated for lymphatic filariasis using albendazole and diethylcarbamazine (DEC) or DEC alone in Leogane, Haiti. We documented the frequency of adverse reactions, severity and cost of treatment. Adverse reactions were classified as minor, moderate, or severe. Overall, 24% (17,421) of the treated persons reported one or more adverse reactions. There were 15,916 (91%) minor and 1502 (9%) moderate adverse reaction reports. Men outnumbered women 2:1 in reporting moderate problems. Three patients, representing roughly one in 25,000 persons treated, were hospitalized with severe adverse reactions judged to be treatment-associated by physician review. The cost per person treated for adverse reactions was more than twice the cost per person treated for lymphatic filariasis (dollar 1.60 versus dollar 0.71). Severe adverse reactions to lymphatic filariasis treatment using DEC with or without albendazole are uncommon. Minor and moderate reactions are more commonly reported and their management represents a challenge to lymphatic filariasis elimination programs.

There is an increasing demand for diagnostic testing for Giardia intestinalis (G. lamblia) and Cryptosporidium parvum, with a priority being placed on obtaining diagnostic results in an efficient and timely manner. Several commercial companies have developed rapid diagnostic tests that are simple to perform and can be completed in less time than traditional methods for detecting Giardia and Cryptosporidium: We compared one of these rapid tests, the ImmunoCard STAT! (Meridian Bioscience, Inc.) lateral-flow immunoassay, with the MERIFLUOR direct fluorescent-antibody (DFA) test, the ProSpecT EZ microplate assay for Giardia and the ProSpecT microplate assay for Cryptosporidium, and modified Kinyoun's acid-fast stained smears for the detection of Cryptosporidium using 246 specimens. The MERIFLUOR DFA (Meridian Bioscience, Inc.) test detected the largest number of cases (32 Giardia and 37 Cryptosporidium) infections and was used to calculate the sensitivity and specificity of the other tests. For Giardia, the sensitivities of the ImmunoCard STAT! and the ProSpecT Giardia EZ microplate assay (Alexon-Trend, Inc.) were 81 and 91%, respectively. For detection of Cryptosporidium, the sensitivities of the ImmunoCard STAT!, the ProSpecT Cryptosporidium microplate assay (Alexon-Trend, Inc.), and modified Kinyoun's acid-fast stained smears were 68, 70, and 78%, respectively. Test specificities were equal to or greater than 99%. Specimens with very small numbers of organisms were not detected by the ImmunoCard STAT!, the ProSpecT microplate assay or modified Kinyoun's acid-fast stained smears.

In October 2001, two inhalational anthrax and four cutaneous anthrax cases, resulting from the processing of Bacillus anthracis-containing envelopes at a New Jersey mail facility, were identified. Subsequently, we initiated stimulated passive hospital-based and enhanced passive surveillance for anthrax-compatible syndromes. From October 24 to December 17, 2001, hospitals reported 240,160 visits and 7,109 intensive-care unit admissions in the surveillance area (population 6.7 million persons). Following a change of reporting criteria on November 8, the average of possible inhalational anthrax reports decreased 83% from 18 to 3 per day; the proportion of reports requiring follow-up increased from 37% (105/286) to 41% (47/116). Clinical follow-up was conducted on 214 of 464 possible inhalational anthrax patients and 98 possible cutaneous anthrax patients; 49 had additional laboratory testing. No additional cases were identified. To verify the limited scope of the outbreak, surveillance was essential, though labor-intensive. The flexibility of the system allowed interim evaluation, thus improving surveillance efficiency.

At least four Bacillus anthracis-containing envelopes destined for New York City and Washington, D.C. were processed at the Trenton Processing and Distribution Center (PDC) on September 18 and October 9, 2001. When cutaneous anthrax was confirmed in a Trenton postal worker, the PDC was closed. Four cutaneous and two inhalational anthrax cases were identified. Five patients were hospitalized; none died. Four were PDC employees; the others handled or received mail processed there. Onset dates occurred in two clusters following envelope processing at the PDC. The attack rate among the 170 employees present when the B. anthracis-containing letters were sorted on October 9 was 1.2%. Of 137 PDC environmental samples, 57 (42%) were positive. Five (10%) of 50 local post offices each yielded one positive sample. Cutaneous or inhalational anthrax developed in four postal employees at a facility where B. anthracis-containing letters were processed. Cross-contaminated mail or equipment was the likely source of infection in two other case-patients with cutaneous anthrax.

Anthelmintic treatment of sick preschool-age children at health facilities is a potentially effective strategy for intestinal helminth control in this age-group. We conducted a study from July 1998 to February 1999 in western Kenya to determine whether the Integrated Management of Childhood Illness (IMCI) guidelines' clinical assessment can be used to identify helminth-infected children, and to evaluate the nutritional benefit of treating sick children without pallor with an anthelmintic (mebendazole is already part of IMCI treatment for sick children aged 2-4 years with palmar pallor in areas where hookworm and Trichuris trichiura infections are endemic). Sick children aged 2-4 years seen at 3 rural health facilities were clinically evaluated and tested for haemoglobin concentration, malaria parasites, and intestinal helminths. Children without pallor were randomly assigned to receive a single dose of 500 mg of mebendazole or a placebo and re-examined 6 months later. Among the 574 children enrolled, 11% had one or more intestinal helminths. Most infections were of light intensity. Selected clinical signs and symptoms available from the IMCI assessment, including palmar pallor and low weight-for-age, were not associated with helminth infection. Six months after enrollment, no differences in growth of children without pallor were observed between the mebendazole (n = 166) and placebo (n = 181) groups. However, there was a significantly greater mean increase in weight, height, and weight-for-age Z score among the helminth-infected children in the mebendazole group (n = 22) as compared with helminth-infected children in the placebo group (n = 20). We conclude that even lightly infected preschool-age children without palmar pallor benefit from anthelmintic treatment; however, in this study setting of low helminth prevalence and intensity, helminth-infected children could not be identified using the IMCI guidelines. Cost-effectiveness studies are needed to help define helminth prevalence thresholds for routine anthelmintic treatment of sick preschool-age children seen at first-level health facilities.

In this study we documented unexpected moderate-to-severe iodine deficiency in Haitian schoolchildren although they live in a coastal community where presumably they have access to iodine-containing seafood. This fact combined with the lack of an iodized salt supply and endemic lymphatic filariasis makes community distribution of diethylcarbamazine-fortified, iodized salt an attractive strategy for elimination of lymphatic filariasis and iodine deficiency disorders in this area of Haiti. Combining lymphatic filariasis elimination with other public health interventions is one strategy to increase its public health benefit and maximize the impact of limited public health resources.

This review of the safety of the co-administration regimens to be used in programmes to eliminate lymphatic filariasis (albendazole + ivermectin or albendazole + diethylcarbamazine [DEC]) is based on 17 studies conducted in Sri Lanka, India, Haiti, Ghana, Tanzania, Kenya, Ecuador, the Philippines, Gabon, Papua New Guinea, and Bangladesh. The total data set comprises 90,635 subject exposures and includes individuals of all ages and both genders. Results are presented for hospital-based studies, laboratory studies, active surveillance of microfilaria-positive and microfilaria-negative individuals, and passive monitoring in both community-based studies and mass treatment programmes of individuals treated with albendazole (n = 1538), ivermectin (9822), DEC (576), albendazole + ivermectin (7470), albendazole + DEC (69,020), or placebo (1144). The most rigorous monitoring, which includes haematological and biochemical laboratory parameters pre- and post-treatment, provides no evidence that consistent changes are induced by any treatment; the majority of abnormalities appear to be sporadic, and the addition of albendazole to either ivermectin or DEC does not increase the frequency of abnormalities. Both DEC and ivermectin show, as expected, an adverse event profile compatible with the destruction of microfilariae. The addition of albendazole to either single-drug treatment regimen does not appear to increase the frequency or intensity of events seen with these microfilaricidal drugs when used alone. Direct observations indicated that the level of adverse events, both frequency and intensity, was correlated with the level of microfilaraemia. In non microfilaraemic individuals, who form 80-90% of the 'at risk' populations to be treated in most national public health programmes to eliminate lymphatic filariasis (LF), the event profile with the compounds alone or in combination does not differ significantly from that of placebo. Data on the use of ivermectin + albendazole in areas either of double infection (onchocerciasis and LF), or of loiais (with or without concurrent LF) are still inadequate and further studies are needed. Additional data are also recommended for populations infected with Brugia malayi, since most data thus far derive from populations infected with Wuchereria bancrofti.

We examined the effects of chlorine on oocyst viability, under the conditions of controlled pH and elevated calcium concentrations required for most community swimming pools. We found that fecal material may alter the Ct values (chlorine concentration in mg/L, multiplied by time in minutes) needed to disinfect swimming pools or other recreational water for Cryptosporidium parvum.

This randomized, placebo-controlled trial investigated the efficacy and nutritional benefit of combining chemotherapeutic treatment for intestinal helminths (albendazole) and lymphatic filariasis (ivermectin). Children were infected with Ascaris (29.2%), Trichuris (42.2%), and hookworm (6.9%), with 54.7% of children having one or more of these parasites. Wuchereria bancrofti microfilaria were found in 13.3% of the children. Children were randomly assigned to treatment with placebo, albendazole, ivermectin, or combined therapy. Combination treatment reduced the prevalence of Trichuris infections significantly more than either drug alone. Combination therapy also significantly reduced the prevalence and density of W. bancrofti microfilaremia compared with placebo or ivermectin alone. Only combination therapy resulted in significantly greater gains in height (hookworm-infected children) or weight (Trichuris-infected children) compared with the placebo group. Combined albendazole and ivermectin was a more efficacious treatment for intestinal helminth and W. bancrofti infections in children and resulted in nutritional benefits not found with either drug alone.

Background: In the spring of 1996, an outbreak of cyclosporiasis associated with fresh Guatemalan raspberries occurred in the United States and Canada. Another multistate outbreak of cyclosporiasis occurred in North America in the spring of 1997., Objective: To identify the vehicle of the outbreak that occurred in the spring of 1997., Design: Retrospective cohort studies of clusters of cases associated with events (such as banquets) and traceback investigations of sources of implicated produce., Setting: United States and Canada., Patients: Persons who attended events associated with clusters of cases of cyclosporiasis., Measurements: Identification of clinically defined or laboratory-confirmed cases of cyclosporiasis and risk factors for infection., Results: 41 clusters of cases were reported in association with events held from 1 April through 26 May in 13 U.S. states, the District of Columbia, and 1 Canadian province. The clusters comprised 762 cases of cyclosporiasis, 192 (25.2%) of which were laboratory confirmed. In addition, 250 laboratory-confirmed sporadic cases were reported in persons who developed gastrointestinal symptoms from April through 15 June, for a total of 1012 cases. Fresh raspberries were the only food common to all 41 events and were the only type of berry served at 9 events (22.0%). Statistically significant associations between consumption of raspberry-containing items and cyclosporiasis were documented for 15 events (40.5% of 37). For 31 of the 33 events with well-documented traceback data, the raspberries either definitely came from Guatemala (8 events) or could have come from Guatemala (23 events). The mode of contamination of the raspberries remains unknown. The outbreak ended shortly after the exportation of fresh raspberries from Guatemala was voluntarily suspended at the end of May 1997., Conclusions: Similar multistate, multicluster outbreaks of cyclosporiasis associated with consumption of Guatemalan raspberries have occurred in consecutive years. These outbreaks highlight the need for better understanding of the biology and epidemiology of Cyclospora cayetanensis and for stronger prevention and control measures to ensure the safety of produce eaten raw.

Background: Lymphatic filariasis and intestinal helminth infections are important disorders in tropical areas. Periodic treatment with albendazole is now used in many school-based intestinal helminth-control programmes. However, few such programmes exist for lymphatic filariasis, despite evidence that single-dose treatment with ivermectin can greatly reduce the concentration of Wuchereria bancrofti microfilariae in the blood for months to years. We aimed to assess the potential for school-based control of lymphatic filariasis by investigating the efficacy and tolerability or combined ivermectin and albendazole in Haitian schoolchildren., Methods: In January, 1996, we collected 832 20 microL capillary blood samples for inclusion in a randomised controlled study from children aged 5-11 years, and examined them by microscopy for W bancrofti microfilariae. Infected children were randomly assigned treatment with placebo (n = 29), a single 200-400 micrograms/kg dose of ivermectin (mean, 273 micrograms/kg, n = 28), 400 mg albendazole (n = 29), or a combination of 200-400 micrograms/kg ivermectin and 400 mg albendazole (n = 24). Children with high concentrations of microfilariae in the blood were admitted to hospital and adverse reactions were monitored for 3-5 days, otherwise children were examined at school or during a visit to their home. 4 months after treatment, we examined blood samples again for microfilariae., Findings: 113 microfilaraemic children were enrolled (mean age 7.8 years). 4 months after treatment, the proportion of children who remained positive for microfilariae was significantly lower in the ivermectin plus albendazole group (four [17%]), but there were no significant changes in the other three groups (20 [69%] placebo, 22 [76%] albendazole alone, 17 [61%] ivermectin alone remained positive; p = 0.004). Geometric mean microfilarial concentration decreased from 9.3 to 5.3 per 20 microL blood among children who received placebo; from 15.5 to 1.5 per 20 microL blood among those who received ivermectin only (p = 0.032); from 14.1 to 5.1 per 20 microL blood among those who received albendazole alone; and from 13.7 to 0.3 per 20 microL blood among those who received both ivermectin and albendazole (p = 0.0001). Systemic adverse reactions did not differ significantly between children who received ivermectin alone and those who were treated with ivermectin and albendazole [corrected]., Interpretation: For children with W bancrofti microfilaraemia, combined treatment with ivermectin and albendazole was more effective than treatment with ivermectin only, with no measurable increase in severity of adverse reactions.

A fluorescent antibody (FA) assay for hepatitis E virus antigen (HEVAg) in infected liver tissue was used to confirm the presence of virus-specific antigens in hepatocytes during the course of infection. With the cloning of the HEV genome it is now possible to determine which viral antigens are recognized by this FA assay. Recombinant HEV proteins covering the carboxyl half of HEV open reading frame 2 (ORF2) were used in this study to demonstrate that some of the most immunoreactive virus-specific antigens detected by FA are contained within this region of ORF2 (nucleotides 6169-7126).

The molecular features of each of the major viruses of non-A, non-B hepatitis, namely hepatitis C virus (HCV) and hepatitis E virus (HEV) are briefly described. The organization of the genome of each of these viruses is discussed and compared to those of other related or distantly related viruses that contain single-stranded, positive-sense RNA genomes. HCV has been tentatively classified as a separate genus within the Flaviviridae, whereas HEV has been loosely associated with caliciviruses and subsequently assigned to the Caliciviridae, although it does possess unique genetic features not found in other caliciviruses.

Hepatitis C virus (HCV) antigen was detected immunohistochemically using fluorescein isothiocyanate-labeled immunoglobulin G fractions from chimpanzee and human sera strongly reactive with recombinant hepatitis C virus structural and non-structural proteins. The antigen was localized in the cytoplasm of hepatocytes in all 9 chimpanzees with acute hepatitis C, in 5 of 10 chimpanzees with chronic HCV infection, and in 11 of 12 patients with chronic hepatitis C. The specificity of the hepatocellular HCV and FITC-labeled probes for HCV was ascertained by blocking studies with paired serum samples obtained from 8 infected and uninfected chimpanzees or from 14 patients during the acute and chronic phases of HCV infection. Absorption experiments on FITC-labeled probes with selected host proteins (normal liver homogenate, plasma proteins, red blood cells) did not indicate cross reactivity of the probes with these antigens. Direct immunomorphologic evidence for the HCV specificity of hepatocellular HCV antigen deposits and the FITC-labeled polyclonal anti-HCVAg probe was established in absorption experiments using recombinant HCV nonstructural proteins. The putative HCV NS3 protein was the most prominent component of hepatocellular HCV antigen.

The molecular properties of the genomes of both hepatitis C virus (HCV) and hepatitis E virus (HEV), the major etiologic agents of non-A, non-B hepatitis, are briefly described. The organization of the genome of each of these viruses is discussed and compared to those of other related or distantly related viruses that contain single-stranded, positive-sense RNA genomes. In the case of HEV, the reactivity of expressed proteins and genetic divergence of geographically distinct isolates are also described within the context of sequence variation, type-common epitopes and type-specific epitopes.

Comparison of the deduced amino acid sequence from the structural region of the Hutchinson strain of hepatitis C virus (HCV-H) with four other HCV isolates clearly divides the five isolates into two groups based on sequence homology. The first group includes HCV-H, HCV-1, and HC-J1, while the second includes HCV-J1 and HC-J4. Among the five isolates the first 190 residues (putative nucleocapsid) are highly conserved whereas residues 196-513 exhibit significant diversity and include a hypervariable region encompassing residues 386-404. A series of overlapping decapeptides were synthesized by solid-phase pin technology according to sequence from HCV-H (amino acids 1-513), HC-J4 (amino acids 181-513), and regions from the three other isolates which exhibited sequence variation. A modified ELISA was used to measure immunoreactivity of sera from clinical posttransfusion cases and experimentally infected chimpanzees. Comparison of pre- and postinfection samples revealed 16 clusters of immunoreactive peptides within the structural region, none of which was found in the hypervariable region. Only one cluster (amino acids 73-89) was recognized by all human and chimpanzee sera. Clear variation in the immune response was observed between individuals, although no obvious difference in reactivity between acute and chronic cases was observed. Within individual profiles, the reactivity to each peptide cluster and the total number of reactive clusters increased over time.

Liver enzyme levels, viral RNA, and the immune response against both structural and nonstructural hepatitis C virus (HCV) proteins have been studied in experimentally infected chimpanzees in order to further understand the natural history of HCV infection. An ELISA for measuring both IgG and IgM responses to core (c22), 33c (NS3), and c100 (NS4) was employed. The IgG response rates were 5/8 for core, and 8/8 for both 33c and c100. Utilizing this antigen combination, at least one antibody response is measureable at, or within 3 weeks of, the major ALT peak. Although no individual antibody response is universally associated with initial detection of seroconversion, the combination of all three recombinant proteins measures seroconversion an average of 54 days earlier than with c100 alone, in 6/8 of the animals. IgM responses were measureable in 5/8 of the chimpanzees, were of shorter duration, and usually arose concomitantly with IgG responses. IgM appears to be a good indicator of primary infection since neither boosting nor recrudescence of disease during the chronic phase of disease elicited a secondary IgM response. Viral RNA can be measured 4-7 days (average = 9 days) postinfection with the period preceding the ALT peak being characterized by several PCR positive segments interrupted by periods in which no viral RNA can be measured. Following the ALT peak, chronically infected animals with recurring ALT elevations are generally PCR positive with intercedent PCR negative periods. Those animals that appear to have biochemically resolved disease generally have PCR negative profiles, although they still may periodically exhibit PCR positive sera. This indicates that with the recent advent of new screening techniques, a more stringent definition of HCV resolution will be required.

A 1700 base cDNA fragment coding for the putative structural gene(s) of hepatitis E virus (HEV) was inserted into the pATH 10 expression vector. The fusion protein (C2) expressed by this plasmid was found to contain epitopes recognized by anti-HEV antibodies. C2 protein was used in a Western blot format to examine its usefulness in detecting anti-HEV antibodies in well documented human cases of HEV and non-human primates infected with HEV. Both IgM and IgG anti-HEV could be detected in our Western blot assay. This Western blot assay was found not to detect antibodies from acute-phase sera from patients with either HAV or HBV. The C2 protein contains broadly cross-reactive epitopes, and the Western blot assay was able to detect anti-HEV antibodies in patient sera from Asia, Africa, and North America. The optimum serum dilution for the detection of both IgM and IgG was 1:25.

A review of superficial gastric cancer (SGC) has been carried out. Data on the 20 cases who underwent surgery in the 6 years 1982-87 were obtained. The diagnosis of malignancy was established histologically preoperatively in 95% of cases, although more than one endoscopy was required in 50%. In 75%, the primary was confined to the mucosa and in the remainder it had penetrated the submucosa. Lymph node metastases were present in 20%, but distant metastases were not detected. There were no postoperative deaths, and no patient was diagnosed as having recurrent disease during a median follow-up period of 33 months.

Pseudomonas sp. M, isolated from soil by elective culture on R,S-mevalonate as the sole source of carbon, possessed an inducible transport system for mevalonate. This high-affinity system had a pH optimum of 7.0, a temperature optimum of 30 degrees C, a Km for R,S-mevalonate of 88 microM, and a V max of 26 nmol of mevalonate transported per min/mg of cells (dry weight). Transport was energy dependent since azide, cyanide, or m-chlorophenylhydrazone caused complete cessation of transport activity. Transport of mevalonate was highly substrate specific. Of the 16 structural analogs of mevalonate tested, only acetoacetate, mevinolin, and mevaldehyde significantly inhibited transport. Growth of cells on mevalonate induced transport activity by 40- to 65-fold over that observed in cells grown on alternate carbon sources. A biphasic pattern for cell growth, as well as for induction of mevalonate transport activity, was observed when mevalonate was added to a culture actively growing on glucose. The induction of transport activity under these conditions began within 30 min after the addition of mevalonate and reached 60% of maximal activity during phase I. A further increase in mevalonate transport activity occurred during phase II of growth. Glucose was the preferred carbon source for growth during phase I, whereas mevalonate was preferred during phase II. Only one isomer of the R,S-mevalonate mixture appeared to be utilized, since growth ceased after 45 to 50% of the total mevalonate was depleted from the medium. However, nearly 30% of the preferred mevalonate isomer was depleted from the medium during phase I without significant metabolism to CO2. These results suggest that mevalonate or a mevalonate catabolite may accumulate in cells of Pseudomonas sp. M during phase I and that glucose metabolism may inhibit or repress the expression of enzymes further along the mevalonate catabolic pathway.

We have cloned and sequenced a 505-base-pair (bp) segment of DNA situated upstream of mvaA, the structural gene for (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) of Pseudomonas mevalonii. The DNA segment that we characterized includes the promoter region for the mva operon. Nuclease S1 mapping and primer extension analysis showed that mvaA is the promoter-proximal gene of the mva operon. Transcription initiates at -56 bp relative to the first A (+1) of the translation start site. Transcription in vivo was induced by mevalonate. Structural features of the mva promoter region include an 80-bp A + T-rich region, and -12, -24 consensus sequences that resemble sequences of sigma 54 promoters in enteric organisms. The relative amplitudes of catalytic activity, enzyme protein, and mvaA mRNA are consistent with a model of regulation of this operon at the transcriptional level.

Pseudomonas sp. M grown on mevalonate as the sole source of carbon has 200- to 800-fold induced levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The enzyme, which was purified to a homogeneous state in 54% yield (final specific activity, 60.5 mumol of NAD+ reduced per min per mg of protein), converted R-mevalonate (Km = 0.15 mM) to S-HMG-CoA. Activity was sensitive to sulfhydryl modifying reagents. The apparent molecular weight of the holoenzyme was 178,000 and that of the subunit 43,000. The enzyme thus appears to be a tetramer. Comparison of a 23-residue amino-terminal sequence with the cDNA-derived sequence of Chinese hamster ovary cell HMG-CoA reductase showed little homology and antibody raised against the Pseudomonas enzyme did not appear to cross-react with rat liver HMG-CoA reductase. Addition of mevalonate to cells growing on glucose was followed by a rapid and biphasic induction of HMG-CoA reductase activity. During phase I, mevalonate or its catabolites may accumulate in intact cells of Pseudomonas sp. M and acetoacetate, a competitive inhibitor of HMG-CoA reductase (Ki = 3.2 mM), may feedback inhibit the enzyme under these conditions.

We have cloned, determined the primary structure of, and overexpressed in Escherichia coli the gene mvaA, which is the 1,287-base structural gene for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [EC 1.1.1.88] of Pseudomonas mevalonii. The amino acid composition of HMG-CoA reductase agreed with that predicted from the nucleotide sequence of mvaA, and DNA-derived sequences were identical to all experimentally determined peptide sequences. Overexpression of mvaA in E. coli yielded quantities of HMG-CoA reductase over 1,500-fold higher than those present in control cultures. Comparison of the primary structure of the P. mevalonii enzyme with the DNA-derived primary structure for a mammalian HMG-CoA reductase revealed two regions of similarity suggestive of functional relatedness. An open reading frame, ORF1, lies on the 3' side of mvaA, and a potential ribosome-binding site for ORF1 overlaps the termination codon of mvaA.