[Characteristics, diseases and mortality of people admitted to nursing homes for dependent seniors during the first quarter of 2013 in France]. / Caractéristiques, pathologies et mortalité des résidents en établissements d'hébergement pour personnes âgées dépendantes (Ehpad) admis au cours du premier trimestre 2013 en France.

BACKGROUND: To describe the state of health, through healthcare consumption and mortality, of people admitted to nursing homes (Ehpad) in France. METHODS: People over the age of 65 years admitted to an Ehpad institution during the first quarter of 2013, beneficiaries of the national health insurance general scheme (69% of the population of this age), were identified from the Resid-Ehpad database and their reimbursed health care was extracted from the SNIIRAM database, identifying 56 disease groups by means of algorithms (long-term disease diagnoses and hospitalisations, medicinal products, specific procedures). Disease prevalences were compared to those of other beneficiaries by age- and sex-standardized morbidity/mortality ratios (SMR). RESULTS: A total of 25,534 people were admitted (mean age: 86 years, 71% women). Before admission, these people presented a marker for cardiovascular or neurovascular disease (48% of cases), dementia (34%), cancer (18%), and psychiatric disorders (14%). Compared to non-residents, new residents more frequently presented dementia (SMR=3-40 according to age and sex), psychiatric disorders (SMR=2.5-12, including psychotic disorders SMR=18-21 in the 65-74 year age-group), neurological disorders (SMR=2-12, including epilepsy SMR=14 in the 65-74 year age-group), and cardiovascular and neurovascular disease (SMR=1.2-3). Overall mortality in 2013 was 22%, with a maximum excess between the ages of 65-74 years (males, SMR=8.8, females, SMR=15.9). CONCLUSION: Medical and administrative data derived from linking the Resid-Ehpad/Sniiram databases reveal a severely impaired state of health, considering healthcare use of institutionalized dependent elderly people, and a high prevalence of diseases responsible for severe dependence and excess mortality, especially among the younger residents.

A candidate prostate cancer susceptibility gene at chromosome 17p.

It is difficult to identify genes that predispose to prostate cancer due to late age at diagnosis, presence of phenocopies within high-risk pedigrees and genetic complexity. A genome-wide scan of large, high-risk pedigrees from Utah has provided evidence for linkage to a locus on chromosome 17p. We carried out positional cloning and mutation screening within the refined interval, identifying a gene, ELAC2, harboring mutations (including a frameshift and a nonconservative missense change) that segregate with prostate cancer in two pedigrees. In addition, two common missense variants in the gene are associated with the occurrence of prostate cancer. ELAC2 is a member of an uncharacterized gene family predicted to encode a metal-dependent hydrolase domain that is conserved among eukaryotes, archaebacteria and eubacteria. The gene product bears amino acid sequence similarity to two better understood protein families, namely the PSO2 (SNM1) DNA interstrand crosslink repair proteins and the 73-kD subunit of mRNA 3' end cleavage and polyadenylation specificity factor (CPSF73).

G117C MelB, a mutant melibiose permease with a changed conformational equilibrium.

Replacement of the glycine at position 117 by a cysteine in the melibiose permease creates an interesting phenotype: while the mutant transporter shows still transport activity comparable to the wild type its pre steady-state kinetic properties are drastically altered. The transient charge displacements after substrate concentration jumps are strongly reduced and the fluorescence changes disappear. Together with its maintained transport activity this indicates that substrate translocation in G117C melibiose permease is not impaired but that the initial conformation of the mutant transporter differs from that of the wild type permease. A kinetic model for the G117C melibiose permease based on a rapid dynamic equilibrium of the substrate free transporter is proposed. Implications of the kinetic model for the transport mechanism of the wild type permease are discussed.

Validation study of the s classification for melanoma patients with positive sentinel nodes: the Montreal experience.

BACKGROUND: Standard of care is to perform a complete lymph node dissection (CLND) in melanoma patients with positive sentinel lymph nodes (SLNs). However, less than 20% will have metastases in non-SLNs. The S classification was described to predict the non-SLN status, hoping to identify a subset of patients who can be spared the CLND. We tried to validate the feasibility and usefulness of this classification. MATERIALS AND METHODS: We performed a retrospective chart review. All melanoma cases between 1996 and 2006 were included, and 359 patients with SLN biopsies were identified. All pathology slides were reviewed with an emphasis on the S classification. RESULTS: There were 365 SLN biopsies performed. A total of 82 patients (22.8%) had positive SLNs, while 277 patients (77.2%) had negative SLNs. There were 22 patients classified as SI, 18 as SII, 37 as SIII, and 5 were unclassified. On CLND, only 10 patients (12.2%) had positive non-SLNs. None of these were classified as SI while 2 patients (11%) were classified as SII and 8 (22%) as SIII. The S category was found to be a predictor of non-SLN status, and this reached statistical significance (P = 0.044). On univariate analysis, only an increasing Breslow depth and ulceration were predictive of a non-SI status. CONCLUSION: Our results suggest that the S classification is easily feasible and predicts the status of non-SLNs. No patient with SI status was found to have additional non-SLN positive nodes. A larger-scale, prospective trial should be done to confirm these results and possibly spare patients the morbidity of CLND with a positive SLN.

Tumour response 3 months after neoadjuvant single-fraction radiotherapy for low-risk breast cancer.

Introduction: Standard treatment for early-stage invasive breast cancer (bca) consists of breast-conserving surgery and several weeks of adjuvant radiotherapy (rt). Neoadjuvant single-fraction rt is a novel approach for early-stage bca. We sought to investigate the effect of delaying surgery after neoadjuvant rt with respect to the rate of pathologic response (pr). Methods: Women 65 years of age or older with a new diagnosis of stage i luminal A bca were eligible for inclusion. A single 20 Gy dose to the primary breast tumour was given, followed by breast-conserving surgery 3 months later. The primary endpoint was the pr rate assessed by microscopic evaluation using the Miller-Payne system. Results: To date, 10 patients have been successfully treated. Median age of the patients was 72 years (range: 65-84 years). In 8 patients, neoadjuvant rt resulted in a tumour pr with median residual cellularity of 3%. No immediate rt complications other than mild dermatitis were noted. Conclusions: This study demonstrates a method for delivering single-fraction rt that can lead to a high level of pr in most patients. Continued accrual to this study and subsequent trials are needed to determine the feasibility, safety, and role of this novel technique in the management of early-stage bca.

Setting up pharmacovigilance based on available endTB Project data for bedaquiline.

SETTING: Active pharmacovigilance (PV) is recommended for TB programmes, notably for multidrug-resistant TB (MDR-TB) patients treated with new drugs. Launched with the support of UNITAID in April 2015, endTB (Expand New Drug markets for TB) facilitated treatment with bedaquiline (BDQ) and/or delamanid of >2600 patients in 17 countries, and contributed to the creation of a central PV unit (PVU).OBJECTIVE: To explain the endTB PVU process by describing the serious adverse events (SAEs) experienced by patients who received BDQ-containing regimens.DESIGN: The overall PV strategy was in line with the 'advanced´ WHO active TB drug safety monitoring and management (aDSM) system. All adverse events (AEs) of clinical significance were followed up; the PVU focused on signal detection from SAEs.RESULTS and CONCLUSION: Between 1 April 2015 and 31 March 2019, the PVU received and assessed 626 SAEs experienced by 417 BDQ patients. A board of MDR-TB/PV experts reviewed unexpected and possibly drug-related SAEs to detect safety signals. The experts communicated on clusters of risks factors, notably polypharmacy and off-label drug use, encouraging a patient-centred approach of care. Organising advanced PV in routine care is possible but demanding. It is reasonable to expect local/national programmes to focus on clinical management, and to limit reporting to aDSM systems to key data, such as the SAEs.

Predictive modeling of a mixture of thyroid hormone disrupting chemicals that affect production and clearance of thyroxine.

Thyroid hormone (TH) disrupting compounds interfere with both thyroidal and extrathyroidal mechanisms to decrease circulating thyroxine (T(4)). This research tested the hypothesis that serum T(4) concentrations of rodents exposed to a mixture of both TH synthesis inhibitors (pesticides) and stimulators of T(4) clearance in the liver (polyhalogenated aromatic hydrocarbons, PHAHs) could be best predicted by an integrated addition model. Female Long-Evans rats, 23 days of age, were dosed with dilutions of a mixture of 18 PHAHs (2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin like PCBs) and a mixture of 3 pesticides (thiram, pronamide, and mancozeb) for four consecutive days. Serum was collected 24 hours after the last exposure and T(4) concentrations were measured by radioimmunoassay. Animals exposed to the highest dose of the mixture experienced a 45% decrease in serum T(4). Three additivity model predictions (dose addition, effect addition, and integrated addition) were generated based on single chemical data, and the results were compared. Effect addition overestimated the effect produced by the combination of all 21 chemicals. The results of the dose- and integrated-addition models were similar, and both provided better predictions than the effect-addition model. These results support the use of dose- and integrated additivity models in predicting the effects of complex mixtures.

Nitrous oxide, methane and carbon dioxide patterns and dynamics from an experimental pig mass grave.

The objective of the three-year study was to examine spatial and temporal patterns of fluxes and soil pore air concentrations of methane (CH4), carbon dioxide (CO2) and nitrous oxide (N2O) from an experimental mass grave located in a temperate environment. The mass grave (5×10m) contained twenty pig carcasses at a depth of approximately 1m was compared to a plot of the same dimensions containing only disturbed soil, as well as an undisturbed plot. Soil pore air CH4 concentrations were sub-ambient (<1.8ppm) except at 75 and 100cm depths at the mass grave in years 1 and 2 but decreased in year 3. The consumption of CH4 within the aerobic soil resulted in small negative fluxes at the soil surface. Soil pore air CO2 concentration showed an increase with depth in all three plots, with the largest increase (>100,000ppm at 1m) in the mass grave, though there was a marked decrease from years 1 to 3. Surface fluxes of CO2 showed strong seasonal variations, peaking in summer. Soil pore air N2O concentration showed major increases in the mass grave, compared to the other two plots with the pattern maintained over the three years, resulting in larger surface fluxes of N2O. To establish the role of the carcasses in N2O dynamics, we incubated a soil sample containing carcass material which resulted in fast rates of N2O production and consumption. The maintenance of elevated pore air concentration and surface flux of N2O throughout the 3 years suggests that this is a long-term pattern and likely the best of the three gases to use to detect graves. Thus, we suggest that measurement of soil pore air concentrations, especially of N2O, could be a simple and effective approach to help determine the location of clandestine graves.

Mechanisms of cyclophosphamide action on hepatic P-450 expression.

Cyclophosphamide was administered to adult male rats (130 mg/kg, single i.p. injection) and its effects on the P-450 enzymes that contribute to the activation of this drug in rat liver were then assessed. P-450-mediated cyclophosphamide 4-hydroxylase activity in isolated rat liver microsomes decreased by approximately 70% over a 9-day period following drug treatment. This decrease was due to the loss of cytochrome P-450 form 2c (IIC11), a major contributor to cyclophosphamide 4-hydroxylation in untreated male rat liver, while the other major hepatic cyclophosphamide 4-hydroxylase, P-450 PB-1 (IIC6), was largely unaffected. The loss of P-450 2c activity did not result from a decrease in P-450 reductase or from direct inactivation of the P-450 protein by cyclophosphamide or its metabolites, but rather was due to a reduction in hepatic P-450 2c protein and mRNA levels. Hepatic P-450 2a (IIIA2) and P-450 RLM2 (IIA2) were also suppressed by cyclophosphamide treatment. Serum testosterone, which contributes to the expression of P-450s 2c, 2a, and RLM2, was severely depleted in the cyclophosphamide-treated rats; however, this loss was not the direct cause of the decreases in these hepatic P-450s, since the decreases were not reversed upon restoration of normal testosterone levels by human chorionic gonadotropin stimulation of testicular androgen production. In contrast to the suppression of these testosterone-dependent P-450s, P-450 3 (IIA1), P-450j (IIE1), and the P-450-independent microsomal enzyme steroid 5 alpha-reductase were each elevated in rat liver following cyclophosphamide administration. In contrast to P-450 3 and steroid 5 alpha-reductase, however, the elevation of P-450j protein was transient and was not accompanied by an increase in P-450j-associated hepatic microsomal aniline hydroxylase activity. In vitro experiments revealed that P-450j was severalfold more susceptible to inactivation by the cyclophosphamide metabolite acrolein as compared with P-450 3. These observations suggest that P-450j protein is induced by cyclophosphamide treatment but that the protein is inactivated by the cyclophosphamide metabolite acrolein. These findings establish that cyclophosphamide treatment can modulate hepatic P-450 activities through multiple mechanisms and in a manner that may alter P-450 metabolism of cyclophosphamide and perhaps other anticancer drugs that undergo bioactivation in the liver.

Reversal of multidrug resistance in vivo by dietary administration of the phytochemical indole-3-carbinol.

A major obstacle to successful chemotherapy is the development of multidrug resistance (MDR) by cancer cells. MDR is characterized by enhanced cellular efflux of many structurally and functionally diverse compounds, including many anticancer drugs, due to overexpression of the MDR-1 gene product, P-glycoprotein. We hypothesized that the phytochemical, indole-3-carbinol (I3C), and some of its acid-condensation derivatives may inhibit P-glycoprotein-mediated transport due to their aromatic and nitrogen components, thus increasing the accumulation and efficacy of anticancer drugs and acting as a dietary adjuvant to conventional chemotherapy. I3C was subjected to acid conditions similar to those occurring in the stomach following ingestion and three acid-condensation products; a dimer, a noncyclic trimer, and a cyclic trimer were isolated and purified by high-performance liquid chromatography. The ability of I3C and its acid-condensation derivatives to reverse MDR was investigated using murine B16 melanoma cells that were transfected with the human MDR-1 gene (B16/hMDR-1 cells) and were cross-resistant to vinblastine and doxorubicin. The I3C acid-condensation product mixture, but not I3C, sensitized B16/hMDR-1 transfectants to the toxicity of vinblastine and doxorubicin. All three I3C acid-condensation products also increased the accumulation of the P-glycoprotein substrate, doxorubicin, in B16/hMDR-1 transfectants to levels comparable to parental B16 cells. The I3C acid-condensation product mixture competed with azidopine for binding to P-glycoprotein, suggesting that the observed MDR-reversing effect of the acid-condensation products was due to direct interaction with P-glycoprotein. The ability of p.o. administered I3C to reverse MDR was also tested in vivo. The resistance of B16/hMDR-1 transfectants to vinblastine and doxorubicin was preserved after i.p. injection and growth in nude mice. Tumor mass in mice that were provided with 333 or 500 mg/kg mouse/day I3C in their diet and injected s.c. with the anticancer drugs doxorubicin or vinblastine was significantly reduced as compared to tumor mass in mice provided with standard diet and injected with these anticancer drugs or mice provided with 500 mg/kg mouse/day I3C and not injected with anticancer compound. The concentrations of I3C used had no effect on survival or the general appearance and behavior of the mice. Collectively, these results indicate that ingestion of the common dietary constituent I3C results in its conversion to acid-condensation derivatives that sensitized MDR tumors to chemotherapeutic drugs without eliciting direct toxicity to the host.

Platinum anticancer drugs modulate P-450 mRNA levels and differentially alter hepatic drug and steroid hormone metabolism in male and female rats.

Treatment of male rats with the anticancer drug cisplatin leads to feminization of the profile of cytochrome P-450 and other microsomal enzymes involved in steroid hormone and drug metabolism (G.A. LeBlanc, and D.J. Waxman, J. Biol. Chem., 263: 15732-15739, 1988). The present study uses the rat model to evaluate the differential effects of cisplatin treatment on liver microsomal enzymes between genders, and also examines whether the modulation of enzyme activities by cisplatin and its analogues involves changes in P-450 gene expression. While cisplatin treatment of male rats caused a severalfold increase in female-predominant hepatic enzymes, including testosterone 5 alpha-reductase and testosterone 7 alpha-hydroxylase (P-450 form 2A1), it partially decreased the expression of these enzymes in females. The reduced expression of these estrogen-dependent enzymes in females may derive from the loss of circulating estradiol that was shown to occur in response to cisplatin treatment. Analysis of mRNA levels of individual P-450 forms revealed that the effects of cisplatin on P-450-catalyzed steroid hydroxylase activities in both male and female rats are primarily operative through the drug's effects on P-450 mRNA expression. P-450-dependent cyclophosphamide activation was significantly compromised in male rats after cisplatin administration; however, this activity was not altered in cisplatin-treated females. This sex-dependent effect of cisplatin was due to its suppression of P-450 form 2C11, a male-specific P-450 that is a major contributor to microsomal cyclophosphamide bioactivation in male rat liver. The clinically active cisplatin analogue iproplatin elicited effects very similar to those of cisplatin, while carboplatin and transplatin did not have significant effects on hepatic P-450 expression. Together, these findings demonstrate that the response of rat liver to cisplatin-induced changes in hepatic P-450 enzyme profiles and cyclophosphamide bioactivation capacity differs between the sexes, and in addition, these effects can be minimized by use of carboplatin in place of cisplatin.

Temperature-dependent relationship between K+ influx, Mg2+-ATPase activity, transmembrane potential and membrane lipid composition in mycoplasma.

The temperature-dependent relationship between K+ active influx, Mg2+-ATPase activity, transmembrane potential (delta psi) and the membrane lipid composition has been investigated in mycoplasma PG3. Native organisms were grown in a medium containing 10 microgram/ml cholesterol and either oleic plus palmitic (chol (+), O + P) or elaidic (chol (+), E) acids. Adapted cells were grown in a medium free of exogenous cholesterol and supplemented with elaidic acid (chol (-), E). Arrhenius plots of 42K+ active influx gave a linear relationship for (chol (+), O + P) cells (EA = -9 kcal). On the other hand, when oleic plus palmitic acids are replaced by elaidic acid, an upward discontinuity appears between 28 and 30 degrees C, which is associated with a large increase in the apparent activation energy of the process (t > 30 degrees C, EA = -24 kcal; t < 30 degrees C, EA = -40 kcal). Finally, a biphasic response with a break at approx. 23 degrees C (EA = -7 kcal, t > 23 degrees C; EA = -44 kcal, t < 23 degrees C) is observed for (chol(-), E) organisms. From the lack of correspondence between these effects on the K+ influx and the temperature dependence of both the Mg2+-ATPase activity and delta psi, it is suggested that changes in the membrane lipid composition affect the K+ transport at the level of the K+ carrier itself. Differential scanning calorimetry, steady-state fluorescence polarization of diphenylhexatriene and freeze-fracture electron microscopy experiments further suggest that the effect is largely due to modifications of the membrane microviscosity and that the K+ carrier is associated with the most fluid lipid species present in the membrane.

Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements.

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K+ concentration (Ki: 200--300 mM) which greatly exceeds that of the growth medium, and a low Na+ concentration (Na+i: 20 mM). Unlike Na+i,K+i varies with cell aging. The K+ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K+ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, K+i decreases and is partially compensated by a gain in Na+. This substitution completely reverses when metabolic substrate is added (K+ reaccumulation process). Kinetic analysis of K+ movement in cells with steady K+ level shows that most of K+ influx is mediated by an autologous K+-K+ exchange mechanism. On the other hand, during K+ reaccumulation by K+-depleted cells, a different mechanism (a K+ uptake mechanism) with higher transport capacity and affinity drives the net K+ influx. Both mechanisms are energy-dependent. Ouabain and anoxia have no effect on K+ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ -dependent ATPase activity.

Active K+ transport in Mycoplasms mycoides var. Capri. Relationships between K+ distribution, electrical potential and ATPase activity.

The addition of 5 . 10(-5) M or less of dicyclohexylcarbodiimide to Mycoplasma mycoides var. Capri preferentially influences K+ influx rather than efflux and reduces by 30--40% the activity of the membrane-bound Mg2+- ATPase. Adding valinomycin to metabolizing cells does not markedly affect K+ distribution but induces a rapid and complete loss of intracellular K+ in non-metabolizing cells. Uncoupling agents such as dinitrophenol, carbonylcyanide p-trifluoromethoxyphenylhydrazone, dissipate the K+ concentration gradient only when combined with valinomycin. Variations in the merocyanine fluorescence intensity indicate that a transmembrane electrical potential (delta psi) is generated on cell energization. This delta psi, not affected by valinomycin or uncouplers when used alone, is collapsed by a mixture of both. No change in fluorescence intensity can be detected when glucose is added to dicyclohexylcarbodiimide treated organisms. These experiments suggest that the membrane-bound Mg-ATPase activity control K+ distribution in these organisms through the generation of a transmembrane electrical potential difference.

Effect of membrane cholesterol on potassium transport in Mycoplasma mycoides var. Capri (PG3).

Relationships between membrane lipid composition and physiological properties, particularly intracellular potassium levels, have been studied at 37 degrees C in Mycoplasma mycoides var. Capri (PG3). Native organisms grown on medium supplemented with either oleic acid plus palmitic acid or elaidic acid have identical growth characteristics, acidification properties and intracellular K content. On the other hand, when the cholesterol normally present in the membrane (20--25% of total lipids) is reduced to less than 2%, we observe: (1) the intracellular K content decreases (20 microgram K/mg cell protein instead of 40) and is independent of the phase of growth; (2) K passive permeability is drastically increased but K distribution remains in equilibrium with the transmembrane potential (delta psi); (3) organisms stop growing at pH 6.5 (instead of 5.2) and acidification is reduced by 40%, suggesting a large increase in H+ permeability, and (4) intracellular Na contents rise from 3 to 9 microgram Na/mg cell protein. Replenishing cholesterol in membranes of depleted cells results in a recovery of the high intracellular K level (35--40 microgram K/mg cell protein) and acidification properties. It is suggested that cholesterol affects the cation content via the increase in proton permeability which in turn controls the value of the delta psi responsible for the value of intracellular K equilibrium. Changes in K passive permeability, although related to the amount of cholesterol present in the plasma membrane, are probably not involved in the control of the intracellular K level.

Androgen metabolism in invertebrates and its modulation by xenoandrogens: a comparative study.

Marisa cornuarietis (Mollusc), Hyalella azteca (Crustacean), and Paracentrotus lividus (Echinoderm) demonstrated the ability to metabolize androgens through different pathways catalyzed by 5alpha-reductases (5alpha-R), hydroxysteroid dehydrogenases (HSD), hydroxylases, sulfotransferases (SULT), and fatty-acid acyl-CoA acyltransferases (ATAT). Interspecies differences and tissue-specific distribution of those enzymatic activities were observed. Xenobiotics, such as triphenyltin, tributyltin, and fenarimol, interfered with some of the pathways studied, namely, testosterone sulfation, testosterone esterification, and 5alpha-R activity. The work evidenced different sensitivity of those pathways to androgenic compounds, together with interphyla differences in androgen metabolism.

Pituitary regulation of the male-specific steroid 6 beta-hydroxylase P-450 2a (gene product IIIA2) in adult rat liver. Suppressive influence of growth hormone and thyroxine acting at a pretranslational leve;.

Oligonucleotide probes that distinguish between two closely related mRNAs encoding steroid 6 beta-hydroxylases of rat P-450 gene family CYP3A were used to individually assess their responsiveness to pituitary hormone regulation. Northern blot analysis revealed that the elevation of immunoreactive P-450 IIIA2 in livers of hypophysectomized rats reflects an elevation of the constitutive, male-specific P-450 IIIA2 (P-450 2a) and not an induction of the drug-inducible P-450 IIIA1 (P-450p). P-450 IIIA2 mRNA levels in intact adult male rats were found to be markedly reduced by GH administered as a continuous infusion at levels as low as 1 mU/h, indicating that GH acts at a pretranslational step to suppress expression of this P-450 enzyme. In hypophysectomized male rats, however, this same hormone treatment was only partially effective at suppressing P-450 IIIA2 mRNA and protein, suggesting that other pituitary-dependent factors contribute to the suppression observed in the intact rats. Further analysis revealed that T4, but not ACTH or human CG, can act in concert with GH to effect a more complete suppression of hepatic P-450 IIIA2 mRNA and protein in hypophysectomized rats. T4 also suppressed the expression of another GH-regulated, male-specific hepatic enzyme, designated P-450 IIA2 (P-450 RLM2), particularly in hypophysectomized female rats. In contrast, the GH-responsive P-450 IIA1 (P-450 3) was much less affected by T4 treatment. Thus, while T4 can modulate P-450 IIIA2 expression, it does not serve as a universal regulator for hepatic expression of GH-responsive P-450s.

Structure of two in tandem human 17 beta-hydroxysteroid dehydrogenase genes.

Two human 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) genes (h17 beta-HSDI and h17 beta-HSDII) included in tandem within an approximately 13 kilobase pair fragment were isolated from a genomic lambda EMBL3 DNA library using cDNA encoding human 17 beta-HSD (hpE2DH216) as probe. We have determined the complete exon and intron sequences of the two genes as well as their 5' and 3'-flanking regions. Human 17 beta-HSDII contains six exons and five short introns for a total length of 3250 base pairs. The exon sequence of h17 beta-HSDII is identical to the previously reported hpE2DH216 cDNA while the overlapping nucleotide sequences of the corresponding exons and introns of h17 beta-HSDI and h17 beta-HSDII show 89% homology. In addition, we have used the hpE2DH216 cDNA to demonstrate the widespread expression of 17 beta-HSD mRNAs in steroidogenic and peripheral target tissues. These new findings provide the basis for a better understanding of the molecular mechanisms involved in 17 beta-HSD deficiency and peripheral sex steroid metabolism.

Characterization of cDNAs for human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17: evidence of two mRNA species with distinct 5'-termini in human placenta.

Human placenta estradiol 17 beta-dehydrogenase (E2DH) cDNA clones were isolated from a lambda gt11 expression library by screening with 33 mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of E2DH and with polyclonal antibodies raised against the enzyme purified from human placenta. Using 32P-labeled fragments from the coding and 5'-untranslated regions, two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kilobases (kb) while a minor one is found at 2.2 kb. Primer extension analysis identifies the major mRNA as starting 9-10 nucleotides upstream from the in-frame ATG initiating codon while the longer mRNA has at least 814 noncoding nucleotides at its 5'-terminus. Sequence analysis of the longest cDNA clone (2092 base pairs) shows that this clone possesses identical coding and noncoding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5'-noncoding fragment hybridized only to the 2.2 kb band, thus providing additional evidence for the existence of two distinct mRNA species which differ only in their 5'-noncoding regions. Using hpE2DH36 cDNA as a probe for in situ hybridization, the human E2DH gene was localized to the q11-q12 region of chromosome 17. The cloned cDNAs encode E2DH, a 327-amino acid protein having a calculated molecular weight of 34,853. Since E2DH is the enzyme required for the formation of 17 beta-estradiol, the availability of the cDNA encoding the enzyme should permit a detailed investigation of the factors regulating the expression and activity of this crucial enzyme, in both normal and malignant tissues, especially breast cancer.

Full length cDNA structure and deduced amino acid sequence of human 3 beta-hydroxy-5-ene steroid dehydrogenase.

Polyclonal antibodies raised against 3 beta-hydroxysteroid dehydrogenase isolated from human placenta were used to screen a lambda gt11 expression cDNA library from the same tissue. The protein deduced from cDNA sequences contains 372 amino acids with a calculated mol wt of 42,216. Since 3 beta-hydroxysteroid dehydrogenase is the enzyme catalyzing the formation of all classes of hormonal steroids, the availability of the cDNA encoding this enzyme opens new possibilities for a detailed investigation of the factors regulating the expression and activity of this crucial enzyme in adrenal, gonadal as well as peripheral tissues.