[Study impacto: Descriptive analyzis of pharmacist's clinical practice in onco-hematology]. / Étude impacto : analyse descriptive des pratiques de pharmacie clinique en cancérologie.

Pharmaceutical analyses of chemotherapy prescriptions by hospital pharmacists are activities codified by regulation and rules (bon usage). The involvement of the pharmacists in clinical pharmacy activities in the oncology setting is not clearly identified, justifying the development of a mapping of these activities from a questionnaire addressed to the professionals. One hundred and seven centers have participated to this study at the national level (overall participation rate of 32.4%). More than 95% of them used a computerized ordering system and three quarter of them submit the introduction of new compounds to an analysis by the drug therapeutic committee. Prescription analysis allowed detecting around 2% of errors from the current prescription. Clinical pharmacist participates to tumor boards of onco-hematology (RCP) at a level of 46% for senior pharmacist and 42% for junior pharmacist. This involvement in the RCP allowed anticipating protocol's modification and temporary used authorization. Ninety-two percent of the senior pharmacists estimate that they highlight the risk of no reimbursement for prescription out of the guideline during RCP, resulting to a modification of the prescription for 40% of them. This level of intervention is lower with respectively 64% and 10% for the juniors. This study underlines the expert value of the clinical pharmacist dedicated to oncology setting in pre and post analysis prescriptions. It could be targeted by a prospective analysis of both clinical and pharmacoeconomics impact of these interventions.

The anti-diabetic drug metformin does not affect bone mass in vivo or fracture healing.

SUMMARY: The present study shows no adverse effects of the anti-diabetic drug metformin on bone mass and fracture healing in rodents but demonstrates that metformin is not osteogenic in vivo, as previously proposed. INTRODUCTION: In view of the increased incidence of fractures in patients with type 2 diabetes mellitus (T2DM), we investigated the effects of metformin, a widely used T2DM therapy, on bone mass and fracture healing in vivo using two different rodent models and modes of metformin administration. METHODS: We first subjected 12-week-old female C57BL/6 mice to ovariectomy (OVX). Four weeks after OVX, mice received either saline or metformin administered by gavage (100 mg/kg/daily). After 4 weeks of treatment, bone micro-architecture and cellular activity were determined in tibia by micro-CT and bone histomorphometry. In another experiment, female Wistar rats aged 3 months were given only water or metformin for 8 weeks via the drinking water (2 mg/ml). After 4 weeks of treatment, a mid-diaphyseal osteotomy was performed in the left femur. Rats were sacrificed 4 weeks after osteotomy and bone architecture analysed by micro-CT in the right tibia while fracture healing and callus volume were determined in the left femur by X-ray analysis and micro-CT, respectively. RESULTS: In both models, our results show no significant differences in cortical and trabecular bone architecture in metformin-treated rodents compared to saline. Metformin had no effect on bone resorption but reduced bone formation rate in trabecular bone. Mean X-ray scores assessed on control and metformin fractures showed no significant differences of healing between the groups. Fracture callus volume and mineral content after 4 weeks were similar in both groups. CONCLUSIONS: Our results indicate that metformin has no effect on bone mass in vivo or fracture healing in rodents.

Guidelines for the practical stability studies of anticancer drugs: a European consensus conference.

Stability studies performed by the pharmaceutical industry are only designed to fulfill licensing requirements. Thus, post-dilution or -reconstitution stability data are frequently limited to 24h only for bacteriological reasons regardless of the true chemical stability which could, in many cases, be longer. In practice, the pharmacy-based centralized preparation may require infusions to be made several days in advance to provide, for example, the filling of ambulatory devices for continuous infusions or batch preparations for dose banding. Furthermore, a non-justified limited stability for expensive products is obviously very costly. Thus, there is a compelling need for additional stability data covering practical uses of anticancer drugs. A European conference consensus was held in France, May 2010, under the auspices of the French Society of Oncology Pharmacy (SFPO) to propose adapted rules on stability in practical situations and guidelines to perform corresponding stability studies. For each anticancer drug, considering their therapeutic index, the pharmacokinetics/pharmacodynamics (PK/PD) variability, specific clinical use and risks related to degradation products, the classical limit of 10% of degradation can be inappropriate. Therefore, acceptance limits must be clinically relevant and should be defined for each drug individually. Design of stability studies has to reflect the different needs of the clinical practice (preparation for the week-ends, outpatient transportations, implantable devices, dose banding…). It is essential to use validated stability-indicating methods, separating degradation products being formed in the practical use of the drug. Sequential temperature designs should be encouraged to replicate problems seen in daily practice such as rupture of the cold-chain or temperature-cycling between refrigerated storage and ambient in-use conditions. Stressed conditions are recommended to evaluate not only the role of classical variables (pH, temperature, light) but also the mechanical stress. Physical stability such as particles' formation should be systematically evaluated. The consensus conference focused on the need to perform more studies on the stability of biotherapies, including a minimum of three complementary separating methods and a careful evaluation of submicron aggregates. The determination of the biological activity of proteins could be also useful. A guideline on the practical stability of anticancer drugs is proposed to cover current clinical and pharmaceutical practice. It should contribute to improved security of use, optimization of centralized handling and reduced costs. Finally, we have attempted to establish a new drug stability paradigm based on practical clinical needs, to complement regulatory guidelines which are essentially orientated to the stability of manufactured drugs.

The localization of protein carboxyl-methylase in sperm tails.

Protein carboxyl-methylase (PCM), an enzyme known to be involved in exocytotic secretion and chemotaxis, has been studied in rat and rabbit spermatozoa. PCM activity and its substrate methyl acceptor protein(s) (MAP) were demonstrated in the supernate after solubilization of the sperm cell membrane by detergent (Triton X-100). A protein methylesterase that hydrolyzes methyl ester bonds created by PCM was demonstrated in rabbit but not in rat spermatozoa. This enzyme was not solubilized by nonionic detergent. The specific activities of PCM in rat spermatozoa from caput and cauda epididymis were similar and lower than that found in testis. By contrast, MAP substrates were low in testis and increased in parallel with sperm maturation in the epididymis. Multiple MAP were demonstrated in spermatozoa by polyacrylamide gel electrophoresis. The pattern of these proteins was similar in spermatozoa from different portions of the reproductive tract. Fractionation of heads and tails of rat spermatozoa on sucrose gradients indicated that PCM was found exclusively in the tail fraction, whereas MAP was detected both in head and tail fractions. The presence of all the components of the protein carboxyl-methylation system in spermatozoa and the localization of PCM and some of its substrates in the sperm tail are consistent with their involvement in sperm cell motility.

Testosterone: a major determinant of extragenital sexual dimorphism.

Sexual dimorphism in selected extragenital tissues is described with emphasis on the molecular basis of the differences. Testosterone rather than 5 alpha-dihydrotestosterone appears to be the major intracellular androgen in organs other than skin and reproductive tract, but other steroid metabolites and their receptors are required to produce the diverse tissue differences observed in males and females. There is also evidence that multiple hormones from several endocrine glands are required to act in concert with androgens to produce and maintain their effects. Although many of the consequences of sexual dimorphism, such as body size and strength, have been evident for centuries, other differences between males and females such as disease incidence, response to drugs and toxins, and the metabolism and assimilation of dietary constituents have only recently been discovered.

Pseudohermaphrodite rat: end organ insensitivity to testosterone.

The pseudohermaphrodite rat is characterized by lack of androgen-dependent differentiation. Treatment of these rats with testosterone failed to produce the expected changes in preputial and adrenal gland weights and hexobarbital metabolism. This insitivity of the end organ to testosterone could not be explained by defective formation of dihydrotestosterone.

Testosterone and androstenedione blood production rates in normal women and women with idiopathic hirsutism or polycystic ovaries.

The average plasma testosterone concentration of women with either hirsutism or polycystic ovaries and hirsutism was higher (p < 0.01) than that of normal women although the ranges overlapped. Testosterone blood production rates averaged 830 +/- 120 SE and 1,180 +/- 310 SE mug per day in the two groups of hirsute women and 230 +/- 33 SE mug per day in normal women. The ranges did not overlap. The testosterone metabolic clearance rates of hirsute women (1,090 +/- 140 SE L per day) and of men (1,240 +/- 136 SE L per day) were significantly higher than those of normal women (590 +/- 44 SE L per day). These differences persisted when the metabolic clearance rates were corrected for surface area. We suggest that testosterone metabolic clearance rates vary directly with some function of testosterone production. The mean plasma androstenedione levels (2.8 +/- 0.35 SE and 2.8 +/- 0.30 SE mug per L) and production rates (6,060 +/- 450 SE and 7,360 +/- 345 SE mug per day) of the women with hirsutism or polycystic ovaries, respectively, were significantly higher than those of normal women (1.5 +/- 0.22 SE mug per L; 3,300 +/- 830 SE mug per day). The androstenedione metabolic clearance rates were the same in each group. Plasma androstenedione was the precursor of 49% of plasma testosterone in normal women and of 26% of plasma testosterone in hirsute women. Thus, 74% of the plasma testosterone in these subjects must have been either secreted or derived from a precursor that did not enter the plasma androstenedione pool.

Episodic luteinizing hormone secretion in man. Pulse analysis, clinical interpretation, physiologic mechanisms.

The demonstration that luteinizing hormone (LH) release from the pituitary is episodic rather than constant raises fundamental questions regarding the physiologic control of pulsatile LH secretion and its possible alteration in patients with gonadal disorders. To evaluate this mode of LH secretion, quantitative means of analyzing LH pulse amplitude, frequency, shape, and area were established and utilized to study normal subjects and patients with disorders of gonadotropin secretion. Similar patterns of LH secretion were observed in normal men, in women during the follicular phase of the menstrual cycle, and in patients with hyper- and hypogonadotropism, hirsuitism, and amenorrhea (mean pulse amplitude 39-179% from nadir to peak, frequency 2.7-3.9 secretory spikes/6 h). These observations suggested that the pattern of LH secretion is similar in both normal individuals and in those with a variety of pathologic conditions. By contrast, the pattern of pulsatile secretion appeared to differ in the following conditions. LH pulses of higher amplitude (333+/-170%) and lower frequency (1.6+/-0.24 SEM/6 h) characterized the secretory patterns of women during the luteal phase of the menstrual cycle, suggesting that gonadal steroids may modulate LH pulses. LH pulses of low amplitude (26+/-2.1%) and frequency (1.3+/-0.36/6 h) were observed in women with anorexia nervosa. Either integrated LH levels or a mean LH level determined from multiple samples provided a more accurate reflection of gonadotropin secretion than the use of single LH measurements. With multiple sampling over 6 h, it was possible to reduce the 95% confidence limit of LH estimates from +/-50-90 to +/-12%. This allowed normal subjects to be distinguished from patients with low or moderately elevated LH levels in whom gonadotropin levels in single samples were often in the "normal range."Several aspects of the physiologic control of pulsatile LH secretion were studied. The concordance of follicle-stimulating hormone (FSH) with LH pulses progressively increased as LH pulse height increased (P < 0.01) suggesting possible hypothalamic mediation of gonadotropin pulses. Measurement of the "apparent half-life" of LH after secretory spikes revealed half times of 34-233 min. It is likely that this variability was attributable to at least two phenomena: (a) constant low level LH secretion that continued after certain secretory episodes but not others; (b) variable mixing of newly secreted LH into at least two pools. The alpha adrenergic-blocking agents, chlorpromazine and phentolamine, failed to block LH secretory spikes at doses sufficient to result in a 30 mm drop in systolic blood pressure in normal men.

A study of estrogen metabolic clearance rates and transfer factors.

We have attempted to measure the metabolic clearance rates (MCR) and the transfer factors of estradiol (E(2)) and estrone (E(1)) during 2-hr and 12-hr infusions. When estradiol-(3)H was infused for 2 hr, apparent equilibrium was reached at 70 min; the 12-hr infusions showed that plasma estradiol-(3)H levels increased slowly throughout the infusion. When estrone-(3)H was infused, constancy of estrone-(3)H levels was not attained in either the 2-hr infusions or in the two 12-hr infusions. The tritium level in the metabolite of the infused estrogen did not become constant in 50% of the short infusions and increased during all the long infusions. Thus, the conversion ratios C(E1E2) and C(E2E1) continually changed and transfer factors could not be calculated. The apparent "MCR'S" calculated on the basis of the 2-hr studies expressed as liters/24 hr per m(2) +/-SD were: "MCR(E1)" (women) 980 +/-94, (men) 1170 +/-95; "MCR(E2)" (women) 615 +/-17, (men) 830 +/-30. The estradiol "MCR's" differed significantly between men and women. "MCR(E2)" was the same using either estradiol-(14)C or -(3)H and was unchanged by the infusion of 170 mug of estradiol daily. Postmenopausal women had estrogen "MCR's" in the same range as premenopausal women. Excess glucocorticoids increased the "MCR(E2)."

The metabolic clearance rate and origin of plasma dihydrotestosterone in man and its conversion to the 5-alpha-androstanediols.

Dihydrotestosterone metabolism was studied with a constant infusion technique in three men, three women, five hirsute women, and four estrogen-treated hirsute women. The mean dihydrotestosterone metabolic clearance rate was higher in men (336 liters/24 hr per m(2) [range, 239-448]) than in women (153 liters/24 hr per m(2) [range, 108-184]). The metabolic clearance rates in hirsute patients were intermediate between those men and women and were decreased by estrogen treatment. These observations demonstrate similarities in the metabolic rates of testosterone and dihydrotestosterone. The conversion of plasma testosterone and androstenedione to dihydrotestosterone was studied in men and hirsute women. Approximately 4 and 2% of plasma testosterone and androstenedione, respectively, were converted to plasma dihydrotestosterone in both groups. From these observations it was determined that a major fraction of plasma dihydrotestosterone was derived from these plasma precursors rather than from glandular secretion. Both 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstan-3beta,17beta-diol (3beta-diol) were identified in plasma during dihydrotestosterone and testosterone infusions. The conversion ratio of dihydrotestosterone to 3alpha-diol (C(BB) (DHT-3alpha)) was greater than the conversion ratio to the 3beta-isomer (C(BB) (DTH-3beta)) in all the patients studied. Both C(BB) (DHT-3alpha) and C(BB) (DHT-3beta) were higher in men (mean values of 0.151 [range, 0.110-0.222] and 0..031 [range, 0.022-0.042]) than in women (means of 0.044 [range, 0.037-0.048] and 0.012 [range 0.010-0.013]). A smaller fraction of testosterone was converted to 3alpha-diol and 3beta-diol.

Studies of the pituitary-Leydig cell axis in young men with hypogonadotropic hypogonadism and hyposmia: comparison with normal men, prepuberal boys, and hypopituitary patients.

Pituitary and gonadal function was studied in seven chromatin-negative men, ages 15-27 yr, with retarded sexual and somatic development, skeletal anomalies, and hyposmia. These hyposmic patients were compared with normal men, prepuberal boys and hypogonadal patients with hypopituitarism. The urinary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels of hyposmic subjects were the same as those of normal boys and hypopituitary patients but significantly lower than those of normal men. Clomiphene citrate did not cause an increase in plasma FSH and LH levels in either hypogonadal group as it does in normal men. In contrast to hypopituitary patients, thyroid and adrenocortical function and release of growth hormone in the hyposmic subjects were normal. The plasma testosterone levels were equally low in prepuberal, hypopituitary, and hyposmic patients but were increased to a greater extent by human chorionic gonadotropin (HCG) treatment in prepuberal and hypopituitary subjects than in the hyposmic patients. Prolonged treatment with HCG has failed to return plasma testosterone levels to normal in two hyposmic patients. These observations suggest that there are defects of both pituitary and Leydig cell function in men with the syndrome of hypogonadism, skeletal anomalies, and hyposmia. They have impaired secretion of FSH and LH and a Leydig cell insensitivity to gonadotropin.

Early androgen action in kidney of normal and androgen-insensitive (tfm/y) mice. Changes in RNA polymerase and chromatin template activities.

Intranuclear accumulation of testosterone was compared with early changes in transcriptional events in kidneys from normal female and androgen-insensitive (tfm/y) mice. Following a subcutaneous injection of [3H] testosterone, total nuclear uptake of the steroid was maximal at 30 min and declined to about 40% of the peak value by 4 h after hormone administration. After a single subcutaneous dose of testosterone, RNA polymerase activity assayed in intact nuclei in the presence of Mg2+ and alpha-amanitin (nucleolar RNA polymerase I), as well as the enzyme activity sensitive to low concentration of the toxin (nucleoplasmic RNA polymerase II), increased within 15 min and attained peak values at 2 and 1 h, respectively. The activity of both polymerases declined almost to the control level by 4 h and then increased again with a second peak at 20 and 12 h for RNA polymerase I and II, respectively. Similarly, the template capacity of mouse kidney chromatin, as measured with mammalian RNA polymerase II, increased by 15 min, reached a peak at 1 h and returned to control level by 4 h following hormone treatment. A second dose of testosterone given at the nadir (4 h) was not capable of stimulating renal chromatin template activity significantly as compared to the effect observed after the initial hormone treatment. Contrary to the testosterone-stimulated changes in transcriptional events observed in normal female mice, androgens elicited no response in androgen-insensitive tfm/y mice, animals lacking cytosol androgen receptors. These results strongly support the contention that hormone-specific receptors are obligatory to steroid-mediated modifications in gene transcription.

Progestins can mimic, inhibit and potentiate the actions of androgens.

There is an extensive background on the androgen responsiveness of the mouse kidney which can be demonstrated histologically by hypertrophy of the Bowman's capsule and the proximal convoluted tubule. Although androgens increase many renal proteins, beta-glucuronidase and ODC are distinguished by exquisite genetic regulation of the magnitude of the response induced by testosterone. Both the qualitative and quantitative expression of the genes for these enzymes are strain specific, and are dependent upon regulatory alleles. Ornithine decarboxylase is of particular interest since the response of this enzyme is rapid compared to that of beta-glucuronidase. Recent studies using a newly developed androgen receptor assay have demonstrated that the duration of retention of the androgen receptor complex in the nucleus correlates with the magnitude of the androgenic response. Progestins can mimic, inhibit, or potentiate the action of androgens. These responses have been termed the androgenic, antiandrogenic and synandrogenic actions of progestins, respectively. The androgenic and antiandrogenic action of this class of steroids are manifest on many tissues and on many endpoints within a given organ. These effects are believed to involve an early step(s) of androgen action which is common to all sensitive tissues. Results to date suggests that this early step involves the androgen receptor. By contrast, the synandrogenic action of progestins is limited in that it is not observed on all tissues, and not even on all endpoints within a single organ. In the mouse kidney, the synandrogenic actions of progestins have been most extensively studied on beta-glucuronidase. With this enzyme this unusual response to progestins can be demonstrated only in mice which carry the Gus-ra allele. This observation suggests that the potentiating action of progestins on beta-glucuronidase is manifest directly on the Gus gene complex. It is not certain at this time whether a similar mechanism is involved in the potentiation of androgen action on other organs such as the prostate. The androgenic action of progestins is believed to be similar to that of other androgens. Androgenic progestins such as MPA bind to the androgen receptors and translocate them to nuclei. This is followed by a dose dependent increase of proteins similar to what is observed after testosterone administration. In addition, the regulatory genes which modulate androgen action have the same effect on the androgenic effect of progestins. The fact that the potency of progestins such as MPA is less than that of testosterone is believed to relate in part to their lower affinity for the androgen receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic variation in androgen regulation of ornithine decarboxylase gene expression in inbred strains of mice.

Previous studies have indicated that androgen regulation of certain gene products in murine kidney is genetically controlled. In the present work, the expression of renal ornithine decarboxylase (ODC) gene(s) was used as a biological marker to study androgen responsiveness of eight inbred strains of mice (A/J, C57BR/cdJ, 129/J, C57L/J, BALB/cJ, SM/J, RF/J, and C57BL/6J). Kidneys of untreated females from these strains did not have significantly different basal ODC activities or ODC mRNA concentrations. However, renal enzyme concentrations in intact male mice exhibited marked strain-dependent variation; three strains (RF/J, SM/J, and C57BR/cdJ) had 5- to 20-fold higher activities than the other five strains. Renal ODC mRNA content showed similar genetic variability in the male mice; animals with highest enzyme activity had higher mRNA levels than those with low activity. These results could not be explained by differences in either serum testosterone levels or renal nuclear androgen receptor content, suggesting that the animals were differentially sensitive to endogenous androgens. To evaluate further the androgen regulation of ODC gene expression, female mice were treated with testosterone-releasing implants for 5-7 days. The two strains (A/J and C57BL/6J) that had low enzyme activity in response to endogenous testosterone in male mice also showed blunted responses to exogenous androgen administration, as measured by the induction of ODC and its mRNA. The relative distribution of the two mRNA species coding for ODC (2.2 and 2.7 kb in size) exhibited strain-dependent variation that did not, however, correlate with the androgen responsiveness. Studies of the mRNA levels in reciprocal F1 hybrids of C57BR/cdJ and C57BL/6J mice suggested that androgen sensitivity of ODC gene expression, at least in these crosses, was inherited in an autosomal dominant manner.

Expression and regulation of testicular inhibin alpha-subunit gene in vivo and in vitro.

Inhibin, a gonadal peptide, selectively suppresses FSH release from the pituitary. The cDNAs coding for ovarian inhibin have been isolated and characterized. However, little is known about testicular inhibin. In this study we have isolated inhibin alpha-subunit cDNA from human testicular cDNA libraries and determined inhibin alpha-subunit mRNA levels in testes. The longest cDNA isolated from human testis was 1380 nucleotides long and contained a nucleotide sequence identical to that of human placental inhibin alpha-subunit and isolated human inhibin alpha-subunit gene, but different from human ovarian inhibin alpha-subunit in two amino acids in the signal peptide. A single 1.5-kilobase species of inhibin alpha-subunit mRNA was identified in the testes of several species. This mRNA was the same size as those in human ovary and placenta. The regulation of inhibin alpha-subunit mRNA in rat testis was next examined. The concentration of testicular inhibin alpha-subunit mRNA peaked between 20-25 days of age and gradually declined thereafter. Hypophysectomy decreased testicular inhibin alpha-subunit mRNA levels. Supplementation of hypophysectomized animals with FSH restored inhibin alpha-subunit mRNA levels to those in intact controls. By contrast, treatment with testosterone had no effect. Similarly, in Sertoli cell-enriched cultures, FSH, but not testosterone, increased inhibin alpha-subunit mRNA levels. We conclude that 1) human testicular inhibin alpha-subunit mRNA is similar to that of human ovary and placenta; and 2) inhibin alpha-subunit mRNA in Sertoli cells is regulated by FSH, but not testosterone, both in vivo and in vitro.

Characterization and regulation of testicular inhibin beta-subunit mRNA.

To understand the possible structures of testicular inhibin, we have isolated cDNAs coding for inhibin subunits from human testicular cDNA libraries. In this study we report that the nucleotide and predicted amino acid sequences for human testicular inhibin beta-B-subunit are similar to those of human ovary. In rat testis two species of beta-B-subunit mRNA [4.4 and 3.3 kilobases (kb)] appeared to be present in equal concentration, as opposed to rat ovary where a predominant band of 4.4 kb and a minor band of 3.3 kb were observed. One major species of beta-A-subunit mRNA (6.5 kb) was identified in both testis and ovary. The concentration of beta-A-subunit mRNA in the testis was very low, representing only 0.5% of that in rat ovary. The accumulation of beta-B-subunit mRNA peaked at 20 days of age and declined thereafter in a pattern similar to that of the alpha-subunit gene. Hypophysectomy caused a marked increase in the concentration as well as the total content of beta-B-subunit but no change in beta-A-subunit mRNA in rat testis. We have previously reported that FSH markedly increased alpha-subunit mRNA levels both in vivo and in vitro. By contrast, neither FSH nor testosterone has any significant effect on the accumulation of beta-A- or beta-B-subunit mRNAs in hypophysectomized animals or Sertoli cell primary cultures. We conclude that 1) the mRNAs for both beta-subunits are not regulated by FSH; and 2) hypophysectomy does not change and increases, respectively, the mRNAs for the beta-A- and beta-B-subunits. We conclude that the inhibin subunit mRNAs are differentially regulated in rat testis.

Hormonal regulation of rat androgen-binding protein (ABP) messenger ribonucleic acid and homology of human testosterone-estradiol-binding globulin and ABP complementary deoxyribonucleic acids.

Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11. The library was screened immunochemically, using two different antibodies against rABP. The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis. The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis. No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver. The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons. The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination. Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.

A gonadotropin releasing hormone analogue prevents cyclical attacks of porphyria.

Acute intermittent porphyria is a genetic disease in which endogenous hormones affect clinical expression. Premenstrual exacerbations can occur, sometimes often, in women with this disease. Gonadotropin releasing hormone analogues can prevent ovulation by reducing secretion of luteinizing hormone and follicle-stimulating hormone. In six patients with well-documented acute intermittent porphyria and frequent cyclical exacerbations, daily administration of an agonistic gonadotropin releasing hormone analogue, ([ImBzl]-D-His6,Pro9-NET)gonadotropin releasing hormone, intranasally or subcutaneously for as long as 26 months reduced or eliminated premenstrual attacks and caused only minor side effects. Adjustments in dosage or route of administration were sometimes needed. We conclude that endocrine manipulation by treatment with a gonadotropin releasing hormone agonist will prevent neurovisceral attacks of acute intermittent porphyria due to cyclical changes in endogenous hormones and is a safe alternative to exogenous steroids, which may induce attacks of this disease.

New androgen-stimulated proteins in the kidneys of female mice.

Testosterone administration to female mice for 25 days produced a 70% increase in total kidney protein in both A/J and C57BL/6J mice. This is in contrast to the known androgen-responsive proteins, such as beta-glucuronidase and alcohol dehydrogenase, which each represent less than 1% of the total kidney proteins even after maximum stimulation. To investigate this discrepancy, we initiated a study to identify major proteins which increase with androgen treatment. Three new cytoplasmic proteins designated T1, T2, and T3 were found in different subcellular fractions of both A/J and C57BL/6J mice. T1 (43,000 daltons) and T2 (60,000 daltons) were found in the mitochondrial-lysosomal fraction of 25-day androgen-treated mice. T3 (54,000 daltons) was found in the microsomal fraction. Each of these proteins increased several-fold during androgen treatment, so that they were easily identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis. By contrast, no major changes were noted in the soluble proteins. A nonhistone chromosomal protein of 54,000 mol wt (T4) was found in chromatin preparations from androgen-stimulated A/J mice. Additional studies with androgen-insensitive Tfm/Y mice and with various hormones indicated that stimulation of the T proteins was dependent on androgenic steroid and a functional androgen receptor.

The prosence of estrogen receptor in kidneys from normal and androgen-insensitive tfm/y mice.

In vitro and in vivo binding of [3H]estradiol to cytosol proteins and nuclei was studied in kidneys from normal and androgen-insensitive tfm/y mice. The use of tfm/y mice permitted study of the estrogen-receptor complex in the absence of androgen receptor. A high affinity, [3H]estradiol-labeled, 8S molecule was demonstrated in low salt sucrose gradients. This macromolecule sedimented more slowly in gradients containing KC1 (0.5 M). Binding of [3H]estradiol was inhibited by 100-fold excess estrone, estradiol, or diethylstillbestrol but was not affected by testosterone, androstenedione, or progesterone. Studies of equilibrium-binding kinetics for estradiol indicated a Kd of 1.4 X 10(-9) M and 4.4 X 10(-14) mol of binding sites/mg cytosol protein. Furthermore, the binder was an acidic, heat-labile protein (pI = 4.8) which could be precipitated from cytosol with 33% ammonium sulfate, and needed sulfhydryl groups for activity. The demonstration of an estradiol-binding protein in vitro was correlated with specific [3H]estradiol uptake by tfm/y kidney nuclei in vivo. We concluded that the mouse kidney contains an estradiol-binding protein, distinct from that for androgens, which has many of the characteristics of a steroid receptor. The presence of an estrogen receptor in both normal and tfm/y mice indicates that a genetic defect in one receptor does not influence the properties of another. We concluded that androgen and estrogen receptors are under independent genetic control.