Dietary patterns and risk of Parkinson's disease: a case-control study in Japan.

BACKGROUND: Nearly all epidemiologic studies examining the association between the risk of Parkinson's disease (PD) and diet have focused on single foods and specific nutrients. However, epidemiologic evidence for the association of dietary pattern with PD, namely the measurement of overall diet by considering the cumulative effects of nutrients is extremely limited. We conducted a hospital-based case-control study in Japan to examine the relationship between dietary patterns and the risk of PD. METHODS: Patients with PD diagnosed using the UK PD Society Brain Bank criteria (n = 249) and controls without neurodegenerative diseases (n = 368) were recruited. At the time of recruitment, dietary intake during the preceding 1 month was assessed using a validated, self-administered diet history questionnaire. Dietary patterns from 33 predefined food groups (energy-adjusted food g/day) were extracted by factor analysis. RESULTS: Three dietary patterns were identified: 'Healthy', 'Western' and 'Light meal' patterns. After adjustment for potential non-dietary confounding factors, the Healthy pattern, characterized by a high intake of vegetables, seaweed, pulses, mushrooms, fruits and fish, was inversely associated with the risk of PD with a border-line significance (P for trend = 0.06). Multivariate Odds ratio (95% confidence intervals) for PD in the highest quartile of the Healthy pattern was 0.54 (0.32-0.92) compared with the lowest quartile. No associations with PD were detected for the other two dietary patterns. CONCLUSION: In this case-control study in Japan, a dietary pattern consisting of high intakes of vegetables, fruits and fish may be associated with a decreased risk of PD.

Dietary intake of antioxidant vitamins and risk of Parkinson's disease: a case-control study in Japan.

BACKGROUND: antioxidant vitamins are expected to protect cells from oxidative damage by neutralizing the effects of reactive oxygen species. However, epidemiological evidence regarding the associations between antioxidant vitamin intake and Parkinson's disease (PD) is limited and inconsistent. We investigated the relationship between dietary intake of selected antioxidant vitamins, vegetables and fruit and the risk of PD in Japan using data from a multicenter hospital-based case-control study. METHODS: included were 249 patients within 6 years of onset of PD. Controls were 368 inpatients and outpatients without a neurodegenerative disease. Information on dietary factors was collected using a validated self-administered diet history questionnaire. Adjustment was made for sex, age, region of residence, pack-years of smoking, years of education, body mass index, dietary intake of cholesterol, alcohol, total dairy products, and coffee and the dietary glycemic index. RESULTS: higher consumption of vitamin E and ß-carotene was significantly associated with a reduced risk of PD after adjustment for confounders under study: the adjusted odds ratio in the highest quartile was 0.45 (95% confidence interval [CI]: 0.25-0.79, P for trend = 0.009) for vitamin E and 0.56 (95% CI: 0.33-0.97, P for trend = 0.03) for ß-carotene. Stratified by sex, such inverse associations were significant only in women. No material relationships were shown between intake of vitamin C, α-carotene, cryptoxanthin, green and yellow vegetables, other vegetables, or fruit and the risk of PD. CONCLUSIONS: higher intake of vitamin E and ß-carotene may be associated with a decreased risk of PD.

Lack of association of dairy food, calcium, and vitamin D intake with the risk of Parkinson's disease: a case-control study in Japan.

Three previous cohort studies in the USA reported that dairy product consumption was significantly associated with an increased risk of Parkinson's disease (PD) in men, but not in women. We examined the relationship between consumption of dairy products, calcium, and vitamin D and the risk of PD using data from a multicenter hospital-based case-control study in Japan. Included were 249 cases within 6 years of onset of PD based on the UK PD Society Brain Bank clinical diagnostic criteria. Controls were 368 inpatients and outpatients without a neurodegenerative disease. Information on dietary factors was collected using a validated self-administered diet history questionnaire. Adjustment was made for sex, age, region of residence, pack-years of smoking, years of education, body mass index, and dietary factors including cholesterol, dietary glycemic index, vitamin E, ß-carotene, vitamin B(6), caffeine, iron, and alcohol. Total dairy product consumption was not materially associated with the risk of PD (P for trend = 0.62). No evident relationships were observed between intake of milk, yogurt, cheese, or ice cream and the risk of PD (P for trend = 0.75, 0.63, 0.59, and 0.35, respectively). There were no measurable associations between consumption of calcium or vitamin D and PD (P for trend = 0.37 and 0.69, respectively). No significant interactions were observed between the dietary exposures and sex regarding PD. Our results suggest that intake of dairy products, calcium, and vitamin D was not related to PD, regardless of sex. However, such null relationships might be a consequence of PD.

GST polymorphisms, interaction with smoking and pesticide use, and risk for Parkinson's disease in a Japanese population.

Patients with idiopathic Parkinson's disease (PD) appear to have reduced capacity for detoxification of certain environmental compounds. The glutathione S-transferases (GSTs) are candidate genes for PD because they are involved in the metabolism of pesticides and cigarette smoke. We investigated the relationship of the seven GST polymorphisms (GSTM1 deletion, GSTT1 deletion, GSTP1 rs1695, GSTO1 rs4925, GSTO1 rs11191972, GSTO2 rs156697 and GSTO2 rs2297235) and PD risk with special reference to the interaction with pesticide use or cigarette smoking among 238 patients with PD cases and 370 controls in a Japanese population. None of the GST polymorphisms were associated with PD. GSTO1 rs4925 and GSTO2 rs2297235 were found to be in strong linkage disequilibrium (D' = 0.98). Cigarette smoking was significantly associated with decreased risk of PD. However, no interaction of smoking with any of the GST polymorphisms was observed. Self-reported pesticide use was not associated with increased risk of PD. There was no evidence of interaction between self-reported pesticide use and either GST polymorphism. Our results suggest that the tested GST polymorphisms did not play an important role in PD susceptibility in our Japanese population. Our study does not give evidence of interaction between the GST polymorphisms and smoking may although this study provided sufficient statistical power to detect modest interaction. As for interaction between GSTP polymorphisms and pesticide use, the power of this study to detect an interactive effect was low due to a small number of pesticide users. Future studies involving larger control and case populations and better pesticide exposure histories will undoubtedly lead to a more thorough understanding of the role of the GST polymorphisms in PD development.

Active and passive smoking and risk of Parkinson's disease.

OBJECTIVE: To assess the association between active and passive smoking and the risk of Parkinson's disease (PD), a case-control study with 249 PD patients and 369 controls was carried out in Japan. METHODS: Information on smoking was obtained through a self-administered questionnaire. Adjustment was made for age, sex, region of residence, educational level, and occupational exposure. RESULTS: Ever having smoked cigarettes was associated with a reduced risk of PD [adjusted odds ratio = 0.38; 95% confidence interval (CI): 0.24-0.59]. Risk for former smokers was intermediate between the high risk for never smokers and the low risk for current smokers. Adjusted odds ratios for former and current smokers were 0.51 (95% CI: 0.32-0.82) and 0.12 (95% CI: 0.05-0.26), respectively. There was an inverse dose-response gradient with pack-years smoked. No significant association was detected for passive smoking exposure. CONCLUSION: Our results appear to confirm data from previous epidemiological studies.

Diagnostic accuracy of cardiac metaiodobenzylguanidine scintigraphy in Parkinson disease.

BACKGROUND AND PURPOSE: To estimate the diagnostic accuracy of cardiac (123)I-metaiodobenzylguanidine (MIBG) scintigram for detection of Parkinson disease. METHODS: A cross-sectional study with index test of MIBG scintigram and reference standard of U.K. Parkinson's Disease Brain Bank Criteria was performed in 403 patients. Ratio of cardiac-to-mediastinum MIBG accumulation was determined at 20 min (early H/M) and 4 h (late H/M). Area under the receiver-operator characteristic (ROC) curve, sensitivity and specificity in detecting Parkinson disease were analyzed. Accuracy was analyzed in a subgroup of patients with disease duration of 3 years or less. RESULTS: Area under the ROC curve was 0.89 using either early or late H/M as a diagnostic marker (95% CI 0.85-0.92 for early H/M and 0.86-0.93 for late H/M). Sensitivity and specificity were 81.3% (76.1-85.8%) and 85.0% (77.7-90.6%) for early H/M and 84.3% (79.3-88.4%) and 89.5% (83.01-94.1%) for late H/M. In the subgroup with duration of 3 years or less, the ROC curve area, sensitivity, and specificity were 0.86 (0.79-0.92), 76.0% (64.8-85.1%), and 83.9% (71.7-92.4%) for early H/M and 0.85 (0.78-0.92), 73.3% (61.9-82.9%), and 87.5% (75.9-94.8%) for late H/M. CONCLUSION: Although diagnostic accuracy of cardiac MIBG scintigram is high, it is limited because of insufficient sensitivity in patients with short duration.

Oxidative stress causes abnormal accumulation of familial amyotrophic lateral sclerosis-related mutant SOD1 in transgenic Caenorhabditis elegans.

Mutations in the Cu/Zn superoxide dismutase (SOD1) genes are present in approximately 20% of families suffering from familial amyotrophic lateral sclerosis (FALS). Results from several transgenic studies in which FALS-related SOD1 mutations have been expressed have suggested that mutant SOD1 proteins induce cytotoxicity through a toxic gain of function, although the specific mechanism of this has not been fully clarified. To investigate the mechanism of toxicity induced by the mutant SOD1 associated with FALS, we generated transgenic Caenorhabditis elegans strains that contain wild-type and mutant human A4V, G37R and G93A SOD1 recombinant plasmids. The transgenic strains expressing mutant human SOD1 showed greater vulnerability to oxidative stress induced by 0.2 mM paraquat than a control that contained the wild-type human SOD1. In the absence of oxidative stress, mutant human SOD1 proteins were degraded more rapidly than the wild-type human SOD1 protein in C.elegans. In the presence of oxidative stress, however, this rapid degradation was inhibited, and the transgenic C.elegans co-expressing mutant human SOD1 and green fluorescent proteins (GFPs) in muscle tissues demonstrated discrete aggregates in the adult stage. These results suggest that oxidative damage inhibits the degradation of FALS-related mutant human SOD1 proteins, resulting in an aberrant accumulation of mutant proteins that might contribute to the cytotoxicity.

[The usefulness of urinary FDP in the diagnosis of bladder cancer: comparison with NMP22, BTA and cytology].

PURPOSE: We assessed the utility of urine fibrin/fibrinogen degradation products (FDP) as the screening test for bladder cancer. MATERIALS AND METHODS: Single voided specimens were obtained from 87 consecutive patients (61 men and 26 women, mean age 70.7) on cystoscopy, and FDP, NMP22, BTA and cytology test were performed for the same specimens. Final diagnosis of bladder cancer was made by histological examination, which were compared with the results of above four screening methods. RESULTS: Histologically confirmed bladder cancer was found in 14 cases. Overall sensitivity of urinary FDP, NMP22, BTA and cytology were 79, 64, 36 and 36%, respectively. While the sensitivity of FDP was significantly higher than that of BTA and cytology, no significant difference was found between FDP and NMP22. Overall specificity of these four methods were 69, 78, 92 and 90%, respectively. The specificity of FDP and NMP22 were significantly lower than that of BTA and cytology, but satisfactory as a screening test. The sensitivity of the four methods for low-grade and non-invasive tumors were 70, 50, 30 and 10% (G1 or G2, n = 10), and 75, 58, 33 and 25% (Ta or T1, n = 12), respectively. FDP might have a high sensitivity for even low-grade and non-invasive tumors. CONCLUSIONS: FDP in voided urine is a good screening method for bladder cancer because of its high sensitivity for low-grade and non-invasive tumors, and its diagnostic ability could be superior to NMP22.

Severe hyponatremia caused by hypothalamic adrenal insufficiency.

A 60-year-old woman was admitted with severe hyponatremia. Basal values of adrenocorticotropic hormone (ACTH), thyroid hormone and cortisol were normal on admission. Impairment of water diuresis was observed by water loading test. Initially, we diagnosed her condition as the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). By provocation test, we finally confirmed that the hyponatremia was caused by hypothalamic adrenal insufficiency. The basal values of ACTH and cortisol might not be sufficient to exclude the possibility of adrenal insufficiency. Therefore, it is necessary to evaluate adrenal function by provocation test or to re-evaluate it after recovery from hyponatremia.

Reactive oxygen species influence the acrosome reaction but not acrosin activity in human spermatozoa.

It is now widely accepted that the higher levels of reactive oxygen species (ROS) produced by damaged or deficient spermatozoa are associated with a loss of motility and a decreased capacity for sperm-oocyte fusion. Furthermore, earlier studies show, under physiological conditions, that some ROS may be involved in capacitation and hyperactivation of human spermatozoa. We measured ROS levels, acrosome reaction (AR) and acrosin activity (AA) in semen samples from suspected subfertile men to reveal the influence of ROS on AR and AA of human spermatozoa. Semen samples were obtained from 60 patients. Samples with > or = 1 x 10(6) leukocytes/mL were excluded from the study. ROS production was determined using a chemiluminescence technique. AR was determined using a triple stain technique. The percentage of acrosome-reacted spermatozoa after low temperature induction of the AR (test value), and the inducibility of AR (= the difference between the test value and the control), were calculated. The AA was analysed by determining the proteolytic potential of spermatozoa on gelatin plates. The mean halo diameter and percentage of halo formation in each sample were measured as AA parameters. Scatter plots of ROS levels and AR parameters showed that the percentage of acrosome reacted spermatozoa and AR inducibility were better in samples with low rather than high ROS levels. On the other hand, there were no apparent similarities between ROS and the AA parameters. Therefore, the percentage of acrosome-reacted spermatozoa and AR inducibility were significantly higher in the low than in the high ROS group (p = 0.028, p = 0.0001, respectively). In addition, there was no significant difference in AA parameters between groups. These findings suggest that lower ROS in semen may have a role in AR but excessive ROS may exert a negative influence on AR, while ROS in semen has no relationship to AA.

Activation of cyclin-dependent kinase 2 (Cdk2) in growth-stimulated rat astrocytes. Geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E gene expression.

The role of the mevalonate cascade in the control of cell cycle progression in astrocytes has been investigated. Serum stimulation of rat astrocytes in primary culture induces the expression of cyclin E followed by the activation of cyclin-dependent kinase 2 (Cdk2) during G1/S transition. The expression of p27, cyclin D1, and the activities of Cdk4 and Cdk-activating kinase (CAK), composed of Cdk7 and cyclin H, were not affected. Serum did, however, stimulate the expression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA at mid-G1 phase. Moreover, an inhibitor of HMG-CoA reductase, pravastatin, reduced cyclin E expression and Cdk2 activation and caused G1 arrest in the astrocytes. In contrast, mevalonate and its metabolite, geranylgeranylpyrophosphate (GGPP) but not farnesylpyrophosphate (FPP), reversed the inhibitory effects of pravastatin on cyclin E expression and Cdk2 activation and allowed G1/S transition. Rho small GTPase(s) were geranylgeranylated and translocated to membranes in the presence of GGPP during G1/S transition. The effect of GGPP on cyclin E expression was abolished by botulinum C3 exoenzyme, which specifically inactivates Rho. These data indicate that geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E expression, Cdk2 activation, and G1/S transition in rat astrocytes.

Influence of urogenital infections on sperm functions.

Many studies have examined the impact of genital tract infections on male fertility; however, the effect of bacteriospermia on sperm quality is still controversial. Bacterial infections are more frequently found in semen samples from asymptomatic infertile patients than in those from fertile men. Bacteriospermia is also a common problem of male partners from couples undergoing IVF. Therefore, the effects of microorganisms on human sperm acrosome reaction of oocytes have been studied in vitro and in vivo. Incubation of spermatozoa with Escherichia coli or Mycoplasma hominis in vitro resulted in reduced sperm motility and inducibility of acrosome reaction (delta AR) after exposure to calcium ionophore A23187. To show possible effects of E. coli and mycoplasma species on sperm functions in vivo, data from 488 patients were evaluated, in whose ejaculates microbiological examinations and determinations of acrosome reaction after exposure to low temperature had been performed. U. urealyticum and E. coli were found in semen samples from 52 and 31 men, respectively. M. hominis was only present in a minor number of samples and was not included in this study. Semen concentrations of E. coli and U. urealyticum ranged between 500-100,000 cfu x ml-1 and 100-80,000 cfu x ml-1. No correlation was found between delta AR and concentration of bacteria (Spearman rank correlation coefficient, E. coli: r-0.081, P = 0.6644; U. urealyticum: r = -0.081, P = 0.5698). In 69% of cases with U. urealyticum infection and reduced inducibility of acrosome reaction, this sperm function was normal after antibiotic therapy. However, improvement of acrosomal function may only be due to intra-individual variations of acrosome reaction. While E. coli and mycoplasma species affect sperm functions in vitro, the present data and a review of the literature fail to demonstrate similar effects in vivo.

[Significance of rectal contractions during multichannel urodynamic study].

BACKGROUND: The influence of rectal contractions on urination was examined using multichannel urodynamic study. METHODS: We reviewed a total of 246 consecutive urodynamic studies. Each study consisted of a uroflow measurement and multi-channel urodynamic study, evaluating total vesical pressure, abdominal (rectal) pressure, subtracted detrusor pressure and perianal electromyography. Rectal contractions were defined as periodic fluctuations over 5 cmH2O in abdominal pressure detected by a rectal balloon catheter. No relationship of these contractions with cough and breathing was observed. RESULTS: Of the 246 patients, 17 (6.9%) had a positive study for rectal contractions. The patients, who had positive rectal contractions, averaged 70-year-old were older than negative subjects averaged 62-year-old. In multichannel urodynamics, the flow rate was significantly decreased, and electromyographic activity was increased at the moment of each rectal contractions. CONCLUSIONS: The rectal contractions are not artifactual and may be regarded as one of causes responsible for urinary difficulty in the elderly.

Scavenging effect of N-acetyl-L-cysteine against reactive oxygen species in human semen: a possible therapeutic modality for male factor infertility?

A new approach to reduce the level of reactive oxygen species (ROS) in human semen by using N-acetyl-L-cysteine (NAC) was evaluated. Semen samples were incubated with or without NAC (1.0 mg ml-1) at room temperature. The chemiluminescent signal of the oxidation of luminol was detected by means of an MTP reader after 0, 20, 40, 60 and 120 min, respectively, using 200 microM luminol. In addition, the dose-dependent action of NAC (0.1, 1.0 and 5.0 mg ml-1) and the influence of NAC on functional sperm parameters (motility and acrosome reaction) were studied. ROS levels decreased significantly after 20 min incubation with NAC. This reduction was greater in the high ROS group (> 30000 counts/10(7) viable sperm at t = 0) than in the low ROS group (< 30000). In addition, a marked dose-dependence of NAC was observed. Concerning sperm function, total sperm motility improved after incubation with NAC, but no significant change was observed with respect to the acrosome reaction. NAC (at concentrations of 1.0 mg ml-1) significantly reduced ROS in human semen and showed the possibility of improving impaired sperm function. After further testing NAC might be useful for the treatment of male infertility patients.

Geranylgeranylated rho small GTPase(s) are essential for the degradation of p27Kip1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5 cells.

Cyclin-dependent kinase (Cdk) enzymes are activated for entry into the S phase of the cell cycle. Elimination of Cdk inhibitor protein p27Kip1 during the G1 to S phase is required for the activation process. An inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase prevents its elimination and leads to G1 arrest. Mevalonate and its metabolite, geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, restore the inhibitory effect of pravastatin on the degradation of p27 and allow Cdk2 activation. By the addition of geranylgeranyl pyrophosphate, Rho small GTPase(s) are geranylgeranylated and translocated to membranes during G1/S progression. The restoring effect of geranylgeranyl pyrophosphate is abolished with botulinum C3 exoenzyme, which specifically inactivates Rho. These results indicate (i) among mevalonate metabolites, geranylgeranyl pyrophosphate is absolutely required for the elimination of p27 followed by Cdk2 activation; (ii) geranylgeranylated Rho small GTPase(s) promote the degradation of p27 during G1/S transition in FRTL-5 cells.

Geranylgeranylpyrophosphate, a metabolite of mevalonate, regulates the cell cycle progression and DNA synthesis in human lymphocytes.

We investigated the role of the intrinsic mevalonate cascade in DNA synthesis and cell cycle progression in human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). PHA stimulated the expression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA prior to the DNA synthesis (S phase). Pravastatin, an HMG-CoA reductase inhibitor, inhibited DNA synthesis and blocked the entry to S phase in PHA-stimulated PBMC. Mevalonate restored these inhibitory effects. Thus, we examined two major metabolites of mevalonate, geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), using a novel liposome system for uptake into the cells. GGPP, not FPP, restored the pravastatin-induced inhibitions. These data indicated that 1) the intrinsic mevalonate cascade plays critical roles for the entry to S phase and DNA synthesis, and that 2) GGPP is an essential metabolite of mevalonate cascade for cell cycle progression in PBMC stimulated by PHA.

Serum levels of dehydroepiandrosterone sulfate in patients with asymptomatic cortisol producing adrenal adenoma: comparison with adrenal Cushing's syndrome and non-functional adrenal tumor.

The reported number of adrenal incidentalomas has been increasing because of wider application of imaging techniques. Patients with asymptomatic cortisol producing adrenal adenoma (ASCA) which secretes cortisol without clinical evidence of Cushing's syndrome has been more frequently observed than previously assumed, and they have a risk of adrenal insufficiency after adrenalectomy. Therefore patients with incidentalomas should be screened for cortisol overproduction. The aim of this study is to discover an easy screening test to uncover ASCA. We investigated the hormone profiles of 4 patients with ASCA in comparison with 11 patients with non-functional adrenal tumor and 10 patients with adrenal Cushing's syndrome. We also investigated the expression of dehydroepiandrosterone sulfotransferase (DHEA-ST) in surgically removed attached non-neoplastic adrenal tissues by immunostaining, which was considered to represent the degree of suppression of the hypothalamo-pituitary-adrenal axis. Serum dehydroepiandrosterone sulfate (DHEA-S) levels of all the patients with ASCA and adrenal Cushing's syndrome were lower than those of healthy subjects of corresponding age, but they were within the normal range in the patients with non-functional adrenal tumors. The serum DHEA-S level reflects the degree of suppression of the normal adrenal gland by cortisol hypersecretion from adrenal tumors. But the serum level of DHEA-S decreases with age, and because the normal range of serum DHEA-S is low in elderly subjects, we should be careful to evaluate the level of DHEA-S in elderly patients with adrenal Cushing's syndrome or ASCA. The immunohistochemical study showed DHEA-ST expression was noticeably suppressed in the adjacent adrenal cortex in ASCA and adrenal Cushing's syndrome. The decreased expression of DHEA-ST may reflect autonomous neoplastic cortisol secretion and subsequent ACTH suppression in ASCA and adrenal Cushing's syndrome. A single measurement of plasma ACTH or measurement of ACTH response to corticotropin-releasing hormone was not enough to screen for ASCA because of the wide variation among the cases. Dexamethasone suppression test is essential in identifying ASCA and also a single determination of serum DHEA-S is easy and may be useful for the screening of ASCA in adrenal incidentalomas in young and middle aged subjects, and is especially useful for outpatients.

[The value of F-waves in an early electrodiagnosis of the lower cervical cord infarction].

In a case of infarction in the lower cervical spinal cord, F-waves were lost in the paralyzed hand muscles 48 hours after the onset, in spite of normal compound muscle action potentials (CMAP). Subsequently the amplitude of CMAP decreased markedly in size 14 days later, when needle EMG revealed acute denervation. At 11 days after the onset MRI demonstrated a linear lesion in the ventral portion of the lower cervical spinal cord suggesting ischemia. Thus, the loss of F-waves is useful in early diagnosis of the lower cervical spinal cord infarction, which reflects the decrease in the excitability of the anterior horn cells taking place soon after ischemic insult.

Characterization of guinea pig estrogen sulfotransferase expressed by Chinese hamster ovary cell-K1 stable transfectants.

Estrogen sulfotransferase (EST) activity expressed by Chinese hamster ovary (CHO)-K1 cells stably transfected with a plasmid containing a guinea pig EST complementary DNA insert was subjected to biochemical characterization, and the EST protein was further examined by nondenaturing isoelectric focusing and immunoblot analysis. CHO-K1 cells transfected with the same plasmid without the EST complementary DNA insert as well as untransfected CHO-K1 cells did not demonstrate either EST activity or the presence of an immunologically related protein. The EST expressed by the stably transfected CHO-K1 cells was found to manifest Michaelis-Menten kinetics and would use only estrogenic steroids as substrates, whereas other forms of steroids, such as pregnenolone, dehydroepiandrosterone, cortisol, and testosterone, were not acted on. When 17 beta-estradiol was used as a substrate, sulfonation occurred exclusively at the 3 position; 17-sulfonate was not formed. Thus, the expressed EST acted selectively on the 3-hydroxyl group of phenolic steroids. The apparent Km values for estrone, 17 beta-estradiol, and estriol were 60, 70, and 40 nM, respectively. The maximum velocity (Vmax) determinations for estrone and 17 beta-estradiol were equivalent, whereas the Vmax for estriol was reduced by 33%. Of the three estrogens, only 17 beta-estradiol caused substrate inhibition at a high concentration. Steroid sulfonation requires 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the active sulfonate donor, and the Km value for PAPS was 1.2 microM. In steroid sulfotransferase reactions, two products are formed: the sulfonated steroid product and the desulfonated cofactor, 3'-phosphoadenosine-5'-phosphate (PAP). The sulfonation of 17 beta-estradiol was inhibited by PAP in a dose-dependent manner. In addition, the Km for PAPS was increased by PAP, whereas the Vmax was unaffected, indicating competitive inhibition (Ki, approximately 0.52 microM). The EST protein expressed by the CHO-K1 cell stable transfectants demonstrated a mol wt of 34 kilodaltons, as determined by sodium dodecyl sulfate-gel electrophoresis. Additionally, when the expressed EST protein was subjected to isoelectric focusing, it was found to consist of multiple charge isoforms. These findings are comparable to what has been previously reported for native guinea pig adrenocortical EST. Furthermore, the charge isoform pattern that was demonstrated for the expressed EST was similar to the pattern observed for the native protein.