Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone.

The endocrine system displays highly complex interactions among its components. Excesses or deficiencies of hormone production in one gland may alter the production of hormones by others. Several physiological functions are affected by a balance among hormones acting either together or in sequence. For example, FSH secretion has been demonstrated to be affected by hypothalamic influences upon the anterior pituitary through a specific releasing factor, the decapeptide LRF. This decapeptide stimulates the release of both LH and FSH by the pituitary, and these gonadotropins cause the production of steroids by the testes and the ovaries. Gonadal steroids in the blood act directly upon the anterior pituitary to regulate the output of gonadotropins as originally proposed by Moore and Price in 1932 (3), or act indirectly upon the hypothalamus to adjust the output of pituitary hormones in accordance with the needs of the reproductive system. However, such a simple negative feedback of steroids on the hypothalamic-hypophysial axis cannot account for the differential secretion of FSH observed during the estrus cycle. Therefore, the concept that a gonadal protein, inhibin, specifically regulates FSH secretion was proposed. This concept has now been validated by the isolation and characterization of two forms of inhibin that exert their effects on the pituitary to suppress FSH secretion both in vitro and probably in vivo. Furthermore, the production of inhibin is stimulated by FSH, thus establishing a reciprocal relationship between the release of FSH and inhibin. Since hormones in the body are controlled through interlocking complexes of factors, a variety of secondary factors, in one way or another, may also exert influence on the regulation of FSH secretion. As an example, TGF beta, a protein growth factor found in all tissues, promotes the basal secretion of FSH by the pituitary and enhances FSH-mediated estrogen production by the granulosa cells. It is therefore not surprising that two forms of a novel protein, activin and activin A, isolated from the same FF from which inhibins were isolated, show bioactivities similar to those of TGF beta. These activins are formed as dimers of the two beta-subunits of inhibin, probably as a result of the rearrangement of the gene products. This novel observation that different arrangements of gene products can result in opposite biological activities may thus reflect a wholly different level of control of FSH secretion. If such a phenomenon occurs in other biosystems, it would represent an important form of homeostatic mechanism for controlling biologically active substances.(ABSTRACT TRUNCATED AT 400 WORDS)

Decompression not escharotomy in acute burns.

The concept of escharotomy has long been associated with acute burns care. Nevertheless the practice of escharotomy is frequently flawed and there is considerable diversity in the teaching of the procedure. It is proposed that there should be a fundamental change in the teaching of acute burn management and the concept of decompression should be promoted. The justification for this change comes from a review of the present knowledge base using indexed, library and web-based information sources and also a review of a series of patients transferred to a regional burns unit over a five-year period which revealed that 37% of patients who required surgical decompression had not been appropriately treated prior to transfer. Based on relevant compartmental anatomy a change in the surgical decompression of limbs is proposed to allow safer and more effective management.

Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins.

Two forms of inhibin (A and B), gonadal polypeptide hormones that selectively suppress the secretion of FSH from the anterior pituitary, have been characterized from the porcine and human species, each being composed of a common alpha-chain and one of two distinct, but homologous beta-chains, i.e. alpha beta A and alpha beta B. Using cDNAs encoding the porcine inhibin subunits we have cloned and sequenced the cDNAs encoding the alpha, beta A, and beta B chains of rat ovarian inhibin. Northern analyses of rat testicular RNA with rat ovarian cDNA probes show the presence of mRNAs encoding alpha and beta B chains, but no detectable mRNA encoding the beta A chain under our experimental conditions. This suggests that there may be specific and distinct physiological roles for inhibins A and B. In addition, if there is no extratesticular source of beta A mRNA, then the male rat may be devoid of the stimulators of the secretion of FSH, i.e. activin (beta A beta B) and homoactivin A (beta A beta A), which are derived from the beta subunits of the two inhibins.

D-RNAi (messenger RNA-antisense DNA interference) as a novel defense system against cancer and viral infections.

D-RNAi (Messenger RNA-antisense DNA interference), a novel posttranscriptional phenomenon of silencing gene expression by transfection of mRNA-aDNA hybrids, was originally observed in the effects of bcl-2 on phorbol ester-induced apoptosis in human prostate cancer LNCaP cells. This phenomenon was also demonstrated in chicken embryos and a human CD4(+) T cell line, H9. The in vivo transduction of beta-catenin D-RNAi was shown to knock out more than 99% endogenous beta-catenin gene expression, while the in cell transfection of HIV-1 D-RNAi homolog rejected viral gene replication completely. D-RNAi was found to have long-term gene knockout effects resulting from a posttranscriptional gene silencing mechanism that may involve the homologous recombination between intracellular mRNA and the mRNA components of a D-RNAi construct. These findings provide a potential intracellular defense system against cancer and viral infections.

Gonadocrinins: peptides in ovarian follicular fluid stimulating the secretion of pituitary gonadotropins.

A newly discovered small peptide purified from rat follicular fluid stimulates the pituitary to release FSH and LH in vitro as well as in vivo. Dialysates of crude acid extracts of ovarian follicular tissue and fluid from rats pretreated with PMS gonadotropin stimulate the secretion of both LH and FSH, but not PRL, GH, or TSH, in a pituitary monolayer culture system. This stimulating factor, named gonadocrinin for operational facility, is smaller than 3500 daltons; its biological activity disappears after treatment with trypsin. Gonadocrinin is not recognized by two-antisera binding the decapeptide LRF even though D-Phe2,D-Trp6-LR, an LRF analog antagonist, competitively inhibits the activity of ovarian gonadocrinin. Cultured rat granulosa cells also secret substances with gonadocrinin activity in vitro, indicating that the granulosa cells probably are in vivo the source of gonadocrinin. A crude preparation of gonadocrinin given iv to rats on the second day of diestrus induced secretion of LH comparable to that produced by a 250-ng LRF injection. Gonadocrinin has chemical characteristics different from those of LRF. When purified gonadocrinin or LRF was applied to an identical isocratic high pressure liquid chromatography system, LRF was eluted at a position different from that of gonadocrinin, indicating that, chemically, gonadocrinin is not identical to the hypothalamic decapeptide, LRF.

Multiple stimulation of the adenohypophysis by combinations of hypothalamic releasing factors.

We have investigated the in vitro and in vivo interactions of the four hypothalamic releasing factors, LHRH, corticotropin-releasing factor, TRH, and GH-releasing factor on anterior pituitary hormone secretions, using a 2 X 2 X 2 X 2 factorial experimental design. This experimental design allows for the evaluation of both the main treatment effects of the hypothalamic releasing factors as well as all of the possible interactions between them. Significant main treatment effects were: LHRH on LH and FSH, corticotropin-releasing factor on ACTH and beta-endorphin, TRH on TSH, and GH-releasing factor on GH. These results confirm the specificity of the four releasing factors on their respective target cells. There were no significant interactions between any of the releasing factors on anterior pituitary hormone secretions. These results suggest that the changes in pituitary secretion that are observed under physiological conditions are not due to interactions between the hypothalamic releasing factors at the level of the pituitary, but rather to other secondary interactions that modify pituitary activation or response. These results also indicate that the clinical pituitary reserve tests can be expanded to include all four hypothalamic releasing factors, since any lack of response will reflect a specific pituitary defect and not a failure to respond owing to interaction of the secretagogues administered.

Dried Staphylococcus aureus as a rapid immunological separating agent in radioimmunoassays.

A rapid (less than or equal to 60 seconds) immunological separation of antigen-antibody complexes from free antigen has been developed in radioimmunoassays (RIAs) of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL) and beta-endorphin by using suspensions of dried Staphylococcus aureus rich in protein-A. In the systems tested parallel dose-response curves were obtained for protein-A and second antibody precipitations. The sensitivity of the protein-A method is equal to or higher than that of second antibody method. Tissue culture medium and serum hormone levels measured with RIAs using protein-A are similar to those detected with double antibody methods. The technique may be of general use in all RIAs utilizing antisera from species whose IgG are known to be bound by protein-A.

High-performance subtractive hybridization of cDNAs by covalent bonding between specific complementary nucleotides.

We have developed an improved subtractive hybridization method that provides a fast, simple and reliable isolation of desired different sequences from two compared DNA libraries, one of which contains all unwanted homologues (subtracter) and another contains certain desired heterologues (tester). The DNA library can be made from either mRNA or genomic DNA. An excess amount of modified subtracter DNA from control cells was generated by chemical carboxylation of the pyrimidines to provide covalent affinity to the purines of a natural tester DNA. Hybridization of the control subtracter and the experimental tester DNA was performed with a heat-melting and then cool-reassociation technique. The desired different sequences remained in the form of hydrogen-bonded, homologous sequences of both libraries covalently bonded to each other, resulting in no separation during PCR and cloning. Consequently, the DNA sequences obtained from the covalent homology subtraction represent the nucleotide sequences abundant in the tester but rare in the subtracter library.

Expression of activins and activin receptors in human retinoblastoma cell line Y-79.

Activin is a member of TGF beta family and is known to inhibit neuronal differentiation in certain tumor cell lines. In this study, the messenger RNA expression of activin subunits and activin receptors was characterized in retinoblastoma cell line Y-79 using the reverse transcription-polymerase chain reaction as well as in situ hybridization. The identity of the RT-PCR products was confirmed by DNA sequencing of PCR products. The activin protein production was determined by immunocytochemistry. We found that Y-79 cells transcribe mRNAs coding activin subunits and activin receptors and produce activin proteins. Our results imply that activin may have autocrine functions in these cells.

Inhibin and Activin Modulate Transforming Growth Factor-beta-Induced Immunosuppression.

Inhibin, activin, and transforming growth factor beta (TGFbeta) inhibited lipopolysaccharide (LPS)-induced lymphocyte proliferation in a dose-dependent fashion. These induced suppressions were neutralized by coincubation of a preparation of antibodies to inhibin and TGFbeta, respectively. Inhibin and activin also facilitated TGFbeta-mediated immunosuppression of LPS-induced proliferation of splenocytes. These gonadal proteins showed no effect on phytohemagglutinin- or concanavalin A (Con-A)-induced proliferation of lymphocytes. However, inhibin facilitated and activin inhibited the TGFbeta-mediated immunosuppression in thymocytes stimulated by Con-A. These findings suggest that inhibin or activin by itself, and/or together with TGFbeta, may play an important role in immune response. Copyright 1995 S. Karger AG, Basel

Localization of Activin and Activin Receptors in Rat Spinal Motoneurons.

The purpose of this experiment was to determine whether rat spinal motoneurons (a) produce activin protein and (b) transcribe mRNAs coding for the betaA-subunit of activin and activin receptors II and IIB. The production of activin was determined by immunocytochemistry. The expression and localization of the mRNAs were elucidated by the reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization techniques. We have observed that activin A protein was produced and mRNAs encoding activin betaA-subunit and activin receptors II and IIB were expressed by motoneurons of the rat spinal cord. Furthermore, the identity of RT-PCR products was confirmed by DNA sequencing. It is concluded that activin may have a functional role in the maintenance of rat spinal motoneurons. Copyright 1996 S. Karger AG, Basel

Inhibin production and secretion in human placental cells cultured in vitro.

In this study, human trophoblast cells were isolated from term placentas by trypsin-DNase digestion and Percoll gradient centrifugation. After the cells were cultured in vitro, bioactive inhibin and immunoreactive inhibin were measured in the culture medium and cellular lysate by an ovine pituitary cell culture system and an immunoenzymatic assay. The trophoblast cells were capable of producing inhibin, as indicated by the observation that enhancement of inhibin content and secretion was dependent upon the cell number and time in culture. High levels of inhibin were observed in the culture medium and the cellular lysate with cell numbers of over 1 x 10(6) cells/well, whereas the inhibin levels decreased until they were nearly undetectable when the cell number was less than 0.25 x 10(6) cells/well. In the culture medium, a sharp increase of inhibin levels was observed after 2 days of culture. Bio-inhibin and immuno-inhibin concentrations in the culture medium increased tenfold and fourfold, respectively, from day 2 to day 4. The peak level of bio-inhibin (3.5 U/mL) occurred on day 4 and that of immuno-inhibin (3.55 U/mL) on day 6. In contrast, the maximal level of bio-inhibin (4.45 U/mL) and immuno-inhibin (4.25 U/mL) in the cellular lysate was observed on day 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of human thyrotropin-releasing hormone (TRH) from an endocrine pancreatic tumor.

An extract of a pancreatic carcinoid tumor obtained at autopsy from a patient who had suffered from Cushing's syndrome was found to have the ability to release thyrotropin (but not any other pituitary hormones) from cultured rat anterior pituitary cells, and to bind to a specific thyrotropin-releasing hormone (TRH) antiserum. The tumor contained 2.2 and 3.9 nmol/g of TRH bio- and immunoreactivity, respectively. The active material was purified and its amino acid composition and chromatographic properties were found to be identical with those of synthetic ovine/porcine TRH. This represents the first isolation of human TRH and the first established case of a 'TRHoma', a TRH-producing tumor.

Effects of interleukin-1 beta on secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by cultured rat anterior pituitary cells.

Interleukin-1 beta (IL-1 beta) at doses of 0.15 and 1.5 nM significantly inhibited FSH secretion and stimulated LH secretion by cultured rat pituitary cells after 24-72 hr incubation whereas 15 pM of IL-1 beta was not effective. Treatment with IL-1 beta for 12-48 hr did not affect intracellular content of FSH. However, treatment with 0.15 and 1.5 nM of IL-1 beta for 72 hr significantly suppressed intracellular content of FSH whereas various doses of IL-1 beta incubated with the cells for 12-72 hr showed no effect on the intracellular content of LH. Pretreatment with IL-1 beta for 48 hr inhibited both GnRH-mediated LH and FSH secretions by the pituitary. The secretion of FSH and LH mediated by an activator of protein kinase C, phorbol 12-myristate 13-acetate, was also significantly suppressed by pretreatment with IL-1 beta for 48 hr. These results suggest that (a) IL-1 beta has opposite effects on the secretion of LH and FSH and (b) pretreatment with IL-1 beta suppresses GnRH-mediated stimulation of LH and FSH by the pituitary and this suppressive effect of IL-1 beta may involve the suppression of a protein kinase C-dependent mechanism.

Activin and activin receptors in the normal urinary bladder: immunohistochemistry, in situ hybridization, and RT-PCR.

To determine whether rat urinary bladder produces activin-A, a multifunctional growth factor in the transforming growth factor beta superfamily (TGF beta), we have conducted immunohistochemistry using specific antibodies for activin beta A-subunit which were raised against a synthetic cyclic fragment of the beta A-subunit of activin. The mature activin-A molecule was identified at transitional epithelial cells, smooth muscle, and endothelial cells. To determine if messenger RNAs for the beta A-subunits of activin-A and activin receptors are expressed in these cells, both in situ hybridization with specific probes and the reverse transcription-polymerase chain reaction (RT-PCR) technique with primers specific for the beta A-subunit of activin and activin receptors, respectively, were used. Messenger RNA expression of the beta A-subunit of activin-A and activin receptors were detected by RT-PCR and localized in the transitional epithelial, smooth muscle, and endothelial cells as determined by in situ hybridization. In addition, the identity of the cDNA product of RT-PCR was verified with DNA sequencing. The localization of mature activin-A protein and its corresponding message as well as that of activin receptors to urinary bladder cells suggest that activin-A may have an autocrine function in the urinary bladder, perhaps in the transitional epithelial, smooth muscle, and endothelial cells themselves.

Expression and localization of inhibin alpha-subunit in rat retinal photoreceptor cells.

To determine whether rat retinal photoreceptor cells produce inhibin, a molecule closely related to activin, a multifunctional growth factor in the transforming growth factor beta superfamily (TGF beta), we have conducted immunohistochemistry using specific antibodies for inhibin which were raised against a synthetic N-terminal fragment of the alpha-subunit of inhibin. The mature inhibin molecule was identified at both the inner and outer segments of photoreceptor cells. To determine if messenger RNA for the alpha-subunit of inhibin is expressed in the retinal cells, both in situ hybridization with a specific probe and the reverse transcription-polymerase chain reaction (RT-PCR) technique with primers specific for the alpha-subunit of inhibin were used. Messenger RNA expression of the alpha-subunit of inhibin was detected by RT-PCR and localized in the photoreceptor cells as determined by in situ hybridization. In addition, the identity of the cDNA product of RT-PCR was verified with Southern analysis and DNA sequencing. The localization of mature inhibin protein and its corresponding message to photoreceptor cells suggest that inhibin may have a paracrine function in the retina, perhaps in the photoreceptor cells themselves.

Expression and localization of inhibin/activin subunits and activin receptors in the normal rat prostate.

Activin, a member of transforming growth factor beta (TGF beta), plays an important role during embryonic development, and defects of this growth factor results in degenerative disorders as demonstrated by gene knock out studies. TGF beta has been shown to have dual effects on the regulation of growth of prostate cancer cells. Recently, we have reported that activin was localized and messenger RNAs encoding activin and its receptors were expressed in human prostate cancer cells. To determine whether normal prostate cells produce inhibin and/or activin, immunohistochemistry was conducted on rat prostate glands using specific antibodies for inhibin and activin. The inhibin and activin were present in the cytoplasm and nuclei of epithelial cells whereas stromal cells were not stained. The expression of mRNA for the inhibin/activin subunits was determined using both in situ hybridization and the reverse transcription-polymerase chain reaction (RT-PCR) technique. In addition, the identity of the cDNA product of RT-PCR was verified with DNA sequencing. These findings suggest that inhibin is only produced and mRNA encoding the alpha-subunit for inhibin is only expressed in the normal rat prostate but activin and its receptors are produced and expressed in both normal rat prostate as well as human prostate cancer cells.

Activin A inhibits growth and hexamethylene bisacetamide-induced differentiation in human retinoblastoma Y-79 cells.

Activin regulates the growth and differentiation of a variety of cells and is a member of the transforming growth factor-beta (TGF-beta) family. Previously, we found that the retinoblastoma cell line Y-79 expresses both activins and activin receptors, suggesting that activin may have an autocrine function in these cells. In this study, the effects of exogeneous activin A on cultured Y-79 cells were examined. The results demonstrate that activin A inhibits hexamethylene bisacetamide (HMBA) -induced Y-79 cell differentiation in both serum-containing and serum-free medium. Activin A also inhibits Y-79 cell growth in serum-containing medium but not in serum-free medium.

Endoscopic-assisted subcutaneous mastectomy and axillary dissection with immediate mammary prosthesis reconstruction for early breast cancer.

BACKGROUND: Endoscopic surgery has been applied successfully in breast lump excision, breast augmentation, subcutaneous mastectomy for gynecomastia, and axillary dissection. Since subcutaneous mastectomy has been proven to be oncologically safe for early breast cancer, we have sought to develop a reproducible minimally invasive endoscopic-assisted technique to address this condition. METHODS: Between December 1998 and May 1999, endoscopic-assisted subcutaneous mastectomy and axillary dissection with immediate reconstruction using a mammary prosthesis was performed in nine patients with early breast cancer at the Prince of Wales Hospital, Hong Kong. A 5-cm skin incision was made along the line of the lowest axillary skin crease. Dissection was continued down to the lateral border of the pectoralis major muscle. A subpectoral pocket was gently created by an endoscopic breast dissector. The endoscopic breast retractor and 10-mm/30 degrees scope were introduced into the subpectoral pocket, and further dissection was carried out using a 7-in harmonic scalpel under endoscopic vision down to a level 1 cm caudal to the inframammary fold. This subpectoral space was used for the insertion of the mammary prosthesis later on. Endoscopic-assisted subcutaneous mastectomy was performed afterward. Combined level I and level II axillary dissection was carried out via the same incision under direct vision. RESULTS: Apart from minor skin flap bruises in our first two patients, there were no major complications. Histological examination of all the specimens showed clear margins. Postoperative radiotherapy and chemotherapy were given in the usual manner. All patients were satisfied with the reconstructive outcome. CONCLUSIONS: We have described a novel endoscopic technique for subcutaneous mastectomy with immediate mammary prosthesis reconstruction in treating early breast cancer patient. This technique can minimize skin incision, reduce blood loss, and improve reconstructive outcome. It is easy to learn and well accepted by patients.

An epidemiological study of 1063 hospitalized burn patients in a tertiary burns centre in Hong Kong.

A total of 1063 acute burn patients were admitted to the Burns Unit of Prince of Wales Hospital, Hong Kong between March 1993 and February 1999. There were 678 males and 385 females with a male to female ratio of 1.76:1. The median age was 13.1 year-old and the median burn size was 6% total body surface area (TBSA). Pediatric patients under the age of 15 year-old accounted for 550 (51.7%) admissions and 235 (42.7%) of them were toddlers <2 year-old, while adult patients of age above 15 year-old accounted for the other 513 (48.3%) admissions. There was no seasonal variation in admission. Domestic burns resulted in 756 (71.1%) injuries followed by industrial burns that caused 175 (16.5%) admissions. The median hospital stay was 9 days and 54 patients (5.1%) had inhalation injury requiring intubation and ventilatory support. Twenty-four patients died in this series which yielded a mortality rate of 2.3%. The median age for this mortality group was 46.6 year-olds with a median extent of burns of 68% TBSA. There were 16 males and 8 females with a male to female ratio of 2:1. Eighteen (75%) patients had flame burns and 15 (83.3%) of them had inhalation injury. The mortality group had significantly larger burn size (P<0.001), higher incidence of inhalation injury (P<0.001) and older age (P<0.001) compared to the survivors.