Características y tratamiento de los pacientes nonagenarios con enfermedad vascular ingresados en los servicios de medicina interna. Registro NONAVASC-2 / Characteristics and treatment of nonagenarian patients with vascular disease admitted to internal medicine services. NONAVASC-2 registry

Introducción La enfermedad vascular es la causa más frecuente de morbimortalidad, y su prevalencia incrementa con la edad. Los pacientes muy añosos no se encuentran incluidos en los estudios sobre enfermedad vascular, desconociéndose sus características y tratamientos. Objetivo Conocer las características clínicas de los pacientes nonagenarios hospitalizados en servicios de medicina interna con diagnóstico de EV establecida y la adecuación de su manejo farmacológico. Material y métodos El Registro NONAVASC-2 es un estudio observacional, prospectivo y multicéntrico. Se incluyeron pacientes hospitalizados por cualquier causa. La recogida de datos se realizó a través de una base anonimizada online con parámetros sociodemográficos, clínicos, analíticos, terapéuticos y evolutivos. Resultados Se incluyeron 1.049 pacientes con una edad media de 93,14 años (57,8% mujeres). La prevalencia de los factores de riesgo fue muy elevada: hipertensión (84,9%), dislipemia (50,9%) y diabetes mellitus (29,4%). El 33,4% presentaba dependencia grave/total. El 82,9% recibía tratamiento antitrombótico (53,7% antiagregantes, 25,4% anticoagulación y 3,8% doble terapia). Solo el 38,2% recibía estatinas. El porcentaje de dependencia (39,2 vs. 24,1%; p=0,00) y deterioro cognitivo grave (30,8 vs. 13,8%; p=0,00) era significativamente mayor entre los pacientes que no las recibían. El 19% falleció durante el ingreso. Conclusión Los pacientes nonagenarios con EV presentan una elevada comorbilidad, dependencia y mortalidad. A pesar de estar en prevención secundaria, el 17% de ellos no recibía antitrombóticos y solo el 38% estatinas. Esta infraprescripción está condicionada por la situación funcional, entre otros factores, por lo que es necesario realizar más estudios para conocer el impacto sobre su pronóstico (AU)

Introduction Vascular disease is the most frequent cause of morbidity and mortality and its prevalence increases with age. Old patients are not included in studies on vascular disease, their characteristics and treatments being unknown. Objective Know the clinical characteristics of nonagenarian patients hospitalized in Internal Medicine services with a diagnosis of established VD and the adequacy of their pharmacological management. Material and methods The NONAVASC-2 registry is an observational, prospective, multicentre study. Hospitalized patients for any cause were included. Data collection was carried out through an anonymous online database with sociodemographic, clinical, analytical, therapeutic and evolutionary parameters. Results One thousand forty-nine patients with a mean age of 93.14 years (57.8% women) were included. The prevalence of risk factors and VD was high: hypertension (84.9%), dyslipidemia (50.9%) and diabetes mellitus (29.4%). 33.4% presented severe-total dependency. 82.9% received antithrombotic treatment (53.7% antiplatelets, 25.4% anticoagulation and 3.8% double therapy). Only 38.2% received statins. The percentage of severe dependence (39.2% vs 24.1%; p=0.00) and severe cognitive impairment (30.8% vs 13.8%; p=0.00) was significantly higher among patients who did not receive them. 19% died during admission. Conclusions Nonagenarian patients with VD present high comorbidity, dependence and mortality. Despite being in secondary prevention, 17% did not receive antithrombotics and only 38% received statins. The underprescription is conditioned, among other factors, by the functional status. More studies are necessary to determine the impact of this issue on their prognosis (AU)

Characteristics and treatment of nonagenarian patients with vascular disease admitted to internal medicine services. NONAVASC-2 registry.

INTRODUCTION: Vascular disease (VD) is the most frequent cause of morbidity and mortality and its prevalence increases with age. Old patients are not included in studies on VD, their characteristics and treatments being unknown. OBJECTIVE: Know the clinical characteristics of nonagenarian patients hospitalized in Internal Medicine services with a diagnosis of established VD and the adequacy of their pharmacological management. MATERIAL AND METHODS: The NONAVASC-2 registry is an observational, prospective, multicentre study. Hospitalized patients for any cause were included. Data collection was carried out through an anonymous online database with sociodemographic, clinical, analytical, therapeutic and evolutionary parameters. RESULTS: One thousand forty-nine patients with a mean age of 93.14 years (57.8% women) were included. The prevalence of risk factors and VD was high: hypertension (84.9%), dyslipidemia (50.9%) and diabetes mellitus (29.4%). 33.4% presented severe-total dependency. 82.9% received antithrombotic treatment (53.7% antiplatelets, 25.4% anticoagulation and 3.8% double therapy). Only 38.2% received statins. The percentage of severe dependence (39.2% vs 24.1%; p = 0.00) and severe cognitive impairment (30.8% vs 13.8%; p = 0.00) was significantly higher among patients who did not receive them. 19% died during admission. CONCLUSIONS: Nonagenarian patients with VD present high comorbidity, dependence and mortality. Despite being in secondary prevention, 17% did not receive antithrombotics and only 38% received statins. The underprescription is conditioned, among other factors, by the functional status. More studies are necessary to determine the impact of this issue on their prognosis.

Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots.

The objective of this work was to ascertain whether there is a general pattern of carbon allocation and utilisation in plants following herbicide supply, independent of the site of application: sprayed on leaves or supplied to nutrient solution. The herbicides studied were the amino acid biosynthesis-inhibiting herbicides (ABIH): glyphosate, an inhibitor of aromatic amino acid biosynthesis, and imazamox, an inhibitor of branched-chain amino acid biosynthesis. All treated plants showed impaired carbon metabolism; carbohydrate accumulation was detected in both leaves and roots of the treated plants. The accumulation in roots was due to lack of use of available sugars as growth was arrested, which elicited soluble carbohydrate accumulation in the leaves due to a decrease in sink strength. Under aerobic conditions, ethanol fermentative metabolism was enhanced in roots of the treated plants. This fermentative response was not related to a change in total respiration rates or cytochrome respiratory capacity, but an increase in alternative oxidase capacity was detected. Pyruvate accumulation was detected after most of the herbicide treatments. These results demonstrate that both ABIH induce the less-efficient, ATP-producing pathways, namely fermentation and alternative respiration, by increasing the key metabolite, pyruvate. The plant response was similar not only for the two ABIH but also after foliar or residual application.

Profile of NF-κBp(65/NFκBp50) among prostate specific antigen sera levels in prostatic pathologies.

AIM: The aim of this work was to characterise the immunoexpression of NF-κB (p50/p65) in human prostatic pathologies and to study its profiles of activation among sera prostate specific antigen antigen (PSA) according the three groups: 0-4ng/mL, 4-20ng/mL and >20ng/mL. PATIENTS AND METHODS: Twenty-four men with benign prostate hyperplasia (BPH); 19 men with prostate cancer (PC) and five men with normal prostates (NP). Immunohistochemical and western blot analysis was performed. Serum levels of PSA were assayed by immulite autoanalyser. RESULTS: In BPH and PC samples, immunoexpressions were observed for NF-κBp65 and NF-κBp50; while in NP samples, only were detected NF-κBp50. PC samples showed immunoreactions to NF-κBp65 and NF-κBp50 more intense (respectively 24.18±0.67 and 28.23±2.01) than that observed in BPH samples (respectively18.46±2.04 and 18.66±1.59) with special localisation in the nucleus. Different profiles of NF-κBp65 immunoexpressions were observed and BPH patients with sera PSA levels between 0-4ng/mL presented a significant weak percentage compared to BPH patients with sera PSA levels between 4-20ng/mL and >20ng/mL. No immunoreactions to NF-κBp65 were observed in PC patients with sera PSA levels between 4-20ng/mL. CONCLUSION: The sensibility of both NF-κB and PSA to inflammation allowed confirming the relationship between these two molecules and its involvement in prostatic diseases progression (inflammatory and neoplasic).

[Prostate specific antigen and NF-kB in prostatic disease: relation with malignancy]. / Antígeno prostático específico y NF-kB en patología prostática: relación con la malignidad.

INTRODUCTION: NF-kB (p50/p65) is a transcription factor involved in TNF-α-induced cell death resistance by promoting several antiapoptotic genes. We intend to relate the expression of NF-kB (p50 and p65) with serum levels of prostate-specific antigen (PSA), both in normal males and in those with pathologic conditions of the prostate. MATERIALS AND METHODS: this study was carried out in 5 normal, 24 benign prostatic hyperplastic (BPH) and 19 patients with prostate cancer (PC). Immunohistochemical and Western blot analyses were performed on tissue and serum PSA was assayed by PSA DPC Immulite assays (Diagnostics Products Corporation, Los Angeles, CA). RESULTS: in controls, p65 NF-kB was not found and p50 was scantly detected in 60% normal samples in the cytoplasm of epithelial cells. Both p50 and p65 were expressed in 62.5% of the samples with BPH and in 63.2% of those with PC. Both increased its frequency of expression with higher PSA serum levels. CONCLUSIONS: Activation of NF-kB revealed by its nuclear translocation in prostate cancer could be related to cancer progression and elevated seric PSA levels. A better understanding of the biologic mechanism by which circulating PSA levels increase and its relation with NF-kB expression is needed. Possibly, NF-kB blockage could be used as a therapeutic target to counteract proliferation in prostate cancer.

Antígeno prostático específico y NF-kB en patología prostática: relación con la malignidad / Prostate specific antigen and NF-kB in prostatic disease: relation with malignancy

Introducción: NF-kB (p50/p65) es un factor de transcripción implicado en la resistencia a muerte celular provocada por TNF-α que promueve diferentes genes antiapoptóticos. Pretendemos relacionar la expresión de NF-kB con los niveles de antígeno prostático específico (PSA) en suero, tanto en varones sanos como en los que padecen condiciones patológicas de la glándula próstatica. Métodos: el estudio se realizó en 5 varones sanos (controles), 24 pacientes con hiperplasia benigna de próstata (HBP) y 19 pacientes con cáncer de próstata (CP). Se llevó a cabo Western blot e inmunocitoquímica en tejido y se evaluó el PSA sérico mediante PSA DPC immulite assays (Diagnostics Products Corporation, Los Ángeles, CA). Resultados: en los controles no se detectó el componente p65 de NF-kB y el p50 se detectó débilmente en el 60% de las muestras en el citoplasma de células epiteliales. Tanto p50 como p65 se expresaron en el 62,5% de las muestras de HPB y en el 63,2% de los pacientes con CP. Ambos aumentaron su frecuencia de expresión a mayor nivel de PSA. Conclusiones: la activación de NF-kB puesta en evidencia por translocación nuclear en CP parece estar estrechamente relacionada con la progresión de la enfermedad y con los niveles séricos de PSA. Se necesita un mejor conocimiento del mecanismo biológico de la elevación del PSA circulante y de su relación con la expresión de NF-kB. Tal vez el bloqueo de NF-kB podría emplearse como diana terapéutica para frenar la proliferación del cáncer de próstata (AU)

Introduction: NF-kB (p50/p65) is a transcription factor involved in TNF-α-induced cell death resistance by promoting several antiapoptotic genes. We intend to relate the expression of NF-kB (p50 and p65) with serum levels of prostate-specific antigen (PSA), both in normal males and in those with pathologic conditions of the prostate. Materials and methods: this study was carried out in 5 normal, 24 benign prostatic hyperplastic (BPH) and 19 patients with prostate cancer (PC). Immunohistochemical and Western blot analyses were performed on tissue and serum PSA was assayed by PSA DPC Immulite assays (Diagnostics Products Corporation, Los Angeles, CA). Results: in controls, p65 NF-kB was not found and p50 was scantly detected in 60% normal samples in the cytoplasm of epithelial cells. Both p50 and p65 were expressed in 62.5% of the samples with BPH and in 63.2% of those with PC. Both increased its frequency of expression with higher PSA serum levels. Conclusions: Activation of NF-kB revealed by its nuclear translocation in prostate cancer could be related to cancer progression and elevated seric PSA levels. A better understanding of the biologic mechanism by which circulating PSA levels increase and its relation with NF-kB expression is needed. Possibly, NF-kB blockage could be used as a therapeutic target to counteract proliferation in prostate cancer (AU)

TNF/IL-1/NIK/NF-kappa B transduction pathway: a comparative study in normal and pathological human prostate (benign hyperplasia and carcinoma).

AIMS: Tumour necrosis factor (TNF)-alpha induces death or cell proliferation by activation of nuclear factor (NF)-kappaB, also activated by interleukin (IL)-1 alpha. The aim was to investigate upstream and downstream components of NIK transduction pathway in normal (NP), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostatic carcinoma (PC). METHODS AND RESULTS: Immunohistochemistry and Western blotting were performed. In NP, the cytoplasm of epithelial cells was intensely immunoreactive to IL-1 receptor-associated kinase (IRAK), TNF receptor-associated factor (TRAF)-6, NF-kappaB inducing kinase (NIK), I kappa kappa alpha/beta, I kappaB alpha and p-I kappaB; weakly to NF-kappaB-p50; and negative to NF-kappaB-p65. BPH samples were intensely immunoreactive to IRAK, TRAF-6, NIK, I kappa kappa alpha/beta, I kappaB alpha, p-I kappaB; weakly to NF-kappaB-p50 and NF-kappaB-p65. Whereas low-grade PIN showed intermediate results between NP and BPH, results in high-grade PIN were similar to those found in PC (low Gleason). In PC, immunoreactivity was intense for IRAK, TRAF-6, NIK, I kappa kappa alpha/beta (increasing with Gleason), I kappaB alpha, p-I kappaB (decreasing with Gleason); weak for NF-kappaB-p50 and NF-kappaB-p65 (decreasing with Gleason). Nuclear NF-kappaB was observed in PC. CONCLUSIONS: NF-kappaB enhances cell proliferation, but also ATF-2 or Elk-1. Since IL-1 and TNF-alpha are related to inflammation and their immunoexpression increases in PC, inhibition of these cytokines might be a possible target for PC treatment, because they decrease the activity of all transduction pathway members that activate transcription factors such as NF-kappaB, Elk-1 or ATF-2.

TNF-alpha/IL-1/NF-kappaB transduction pathway in human cancer prostate.

TNFalpha exerts apoptosis throughout an intracellular transduction pathway that involves the kinase proteins TRAF-2 (integration point of apoptotic and survival signals), ASK1 (pro-apoptotic protein), MEK-4 (p38 activator and metastasis suppressor gene), JNK (stress mitogen activated protein kinase) and the transcription factor AP-1. TNFalpha also exerts proliferation by p38 activation, or when TRAF-2 simultaneously induces the transcription factor NF-kappaB by NIK. NIK and p38 may also be activated by IL-1. P38 activated several transcription factors such as Elk-1, ATF-2 and NF-kappaB. NIK also may activate NF-kappaB. The aim of the present article was to evaluate the different components of this TNFalpha/IL-1 transduction pathway in human prostate carcinoma (PC) in comparison with normal human prostate. In prostate cancer, pro-apoptotic TNFalpha/AP-1 pathway is probably inactivated by different factors such as p21 (at ASK-1 level) and bcl-2 (at JNK level), or diverted towards p38 or NIK activation. IL-1alpha enhances proliferation through IL-1RI that activates either NIK or p38 transduction pathway. P38 and NIK activate different transcription factors related with cell proliferation and survival such as ATF-2, Elk-1 or NF-kappaB. In order to search a possible target to cancer prostate treatment we proposed that inhibition of several proinflamatory cytokines such as IL-1 and TNFalpha might be a possible target for PC treatment, because decrease the activity of all transduction pathway members that activate transcription factors as NF-kappaB, Elk-1 or ATF-2.

P38 MAPK protects against TNF-alpha-provoked apoptosis in LNCaP prostatic cancer cells.

PURPOSE: One of the most relevant aspects in cell death regulation is the signalling of apoptosis by the serine/threonine kinases MAPKs. The aim of this study was to investigate the effects of TNF-alpha stimulation on MAPK activation, and the pro- or anti-apoptotic role of these kinases in LNCaP and PC3 cells. MATERIAL AND METHODS: Treatments were carried out using several TNF-alpha concentrations, as well as specific pharmacological inhibitors of MAPKs. Apoptosis rates were evaluated by DAPI staining and flow cytometry. MAPK phosphorylation/activation was measured by Western blot. RESULTS: TNF-alpha induced apoptosis in a dose-dependent manner in LNCaP but not in PC3 cells. The MAPK inhibitors revealed that the apoptotic rate in LNCaP cells increased significantly following p38 inhibition. The kinase inhibitors failed to cause changes in apoptosis in PC3 cells. CONCLUSIONS: The potentiation of apoptosis by p38 inhibition points to this kinase as a possible target for the treatment of androgen-dependent prostatic cancer.

Cell cycle control related proteins (p53, p21, and Rb) and transforming growth factor beta (TGFbeta) in benign and carcinomatous (in situ and infiltrating) human breast: implications in malignant transformations.

A comparative study of the products of the cell cycle control genes p53 (mutated form), p21, Rb (nonphosphorylated and phosphorylated form) and TGFbeta was performed by immunohistochemistry and Western blot, in benign breast disorders and breast cancer (in situ and infiltrating tumors). For the five proteins studied, the relative numbers of positively stained cells were higher in in situ carcinoma than in benign breast diseases. In infiltrating breast tumors, the relative numbers of positively stained cells were even higher than in in situ tumors except for the percentage of pRb immunostained cells, which decreased slightly in infiltrative tumors. For the other four proteins, the percentages of positively stained cases were similar to those found in in situ tumors. In the three groups of patients, TGFbeta immunoreaction appeared in the cytoplasm while immunoreactions to p53, p21, Rb, and pRb were found always in the nucleus except for p21 in in situ tumors, which showed cytoplasmic immunoreaction. Present results suggest that accumulation of mutated p53, cytoplasmic p21, and pRb in breast gland epithelium might be a crucial point in the development of in situ adenocarcinoma. In the infiltrating tumors, the expression of p21 in the nuclei and the decrease in pRb expression suggest an insufficient attempt to hinder cell proliferation.

The p38 transduction pathway in prostatic neoplasia.

It has been proposed that, among other cellular responses, TNF-alpha induces not only cell death, but also cell proliferation by activation of p38. It has also been reported that IL-1-alpha favours cell proliferation by p38 activation. The aim of the present study was to evaluate upstream (alpha-PAK, MEK-6) and downstream (Elk-1 and ATF-2) components of the p38 transduction pathway in normal prostate, benign prostatic hyperplasia (BPH), and prostate carcinoma (PC). Immunohistochemical and western blot analyses were performed in 20 samples of normal prostate, 47 samples of BPH, and 27 samples of PC. In all normal prostates, immunoreactivity for p-Elk-1 and p-ATF-2 was observed in epithelial cell nuclei, but no expression of alpha-PAK or MEK-6. In BPH, there was expression of alpha-PAK (cytoplasm) and MEK-6 (cytoplasm), while the proportions of lesions that were immunoreactive for p-Elk-1 (nucleus and cytoplasm) and p-ATF-2 (nucleus) decreased. In PC, the percentages of cells that were immunoreactive for alpha-PAK (cytoplasm) or MEK-6 (cytoplasm) rose slightly in comparison with BPH, while the percentages of cells that were immunoreactive for p-Elk-1 (nucleus and cytoplasm) or p-ATF-2 (nucleus and cytoplasm) were much higher than in BPH. It is concluded that overexpression of alpha-PAK, MEK-6, p38, p-Elk-1, and p-ATF-2 in BPH, and more intensely in PC, enhances cell proliferation. In BPH, such proliferation is triggered by IL-1 and in part counteracted by the TNF-alpha/AP-1 pathway, which promotes apoptosis. In PC, proliferation is triggered by IL-1 and TNF-alpha (the TNF-alpha/AP-1 pathway is diverted towards p38 activation). Since in a study of the same patients immunoexpression of IL-1alpha and IL-1RI was previously observed to be increased in PC, inhibition of p38 is a possible target for PC treatment, as this inhibition would both decrease IL-1-induced cell proliferation and increase TNF-alpha-induced cell death.

IL-6, its receptors and its relationship with bcl-2 and bax proteins in infiltrating and in situ human breast carcinoma.

AIMS: To characterize the expression pattern of IL-6 and its receptors (IL-6R(alpha) and gp130), to relate this pattern to bcl-2 and bax expression and to elucidate the effects on the proliferation/apoptosis equilibrium in benign conditions and in situ and infiltrating breast cancer. METHODS AND RESULTS: The immunoexpression of IL-6 and its receptors (IL-6R(alpha) and gp130), and their relationship with bcl-2 and bax proteins, were studied in in situ and infiltrating tumours and in benign breast lesions by means of Western blotting and immunohistochemistry. The percentages of samples positive for IL-6, bcl-2 and bax and their immunoreaction densities were higher in in situ carcinomas and infiltrating tumours than in benign lesions; although in in situ lesions were not so high as in infiltrating tumours, except for bax, whose immunoexpression was as weak as in benign conditions, resulting in a bcl-2/bax ratio higher than in infiltrating tumours. CONCLUSIONS: The high expression of IL-6 and its receptors in tumours might be related to the enhanced cell proliferation occurring in breast cancer. IL-6 could act by increasing bcl-2 expression and thus altering the proliferation/apoptosis balance toward neoplastic cell proliferation. The increased bax immunoreactivity observed only in infiltrating tumours, which was not so high as the increase in bcl-2 immunoreactivity, might be interpreted as an attempt to hinder cell proliferation.

Formation of multiple complexes between beta-dystroglycan and dystrophin family products.

Beta-dystroglycan is expressed in a wide variety of tissues and has generally been reported with an Mr of 43 kDa, sometimes accompanied with a 31 kDa protein assumed to be a truncated product. This molecule was recently identified as the anomalous beta-dystroglycan expressed in various carcinoma cell lines. We produced and characterized a G5 polyclonal antibody specific to beta-dystroglycan that is directed against the C-terminal portion of the molecule. We provide evidence that beta-dystroglycan may vary in size and properties by studying different Xenopus tissues. Besides normal beta-dystroglycan with an Mr of 43 kDa in smooth and cardiac muscle and sciatic nerve extracts, we found it in skeletal muscle and brain proteins with an Mr of 38 and 65 kDa, respectively. Glycosylation properties and proteolytic susceptibilities of these different beta-dystroglycans are analysed and compared in this work. Crosslinking experiments with various beta-dystroglycan preparations obtained from skeletal and cardiac muscles and brain gave rise to specific new covalent products with Mr of 125 kDa (doublet band), or 120 and 130 kDa, or 140 and 240 kDa, respectively. We provide evidence, using various similar beta-dystroglycan preparations, that the immunoprecipitation procedure with G5 specific polyclonal antibody allows consistent pelleting of various dystrophin-family isoforms. Skeletal muscles from Xenopus reveals the presence of two distinct beta-dystroglycan complexes, one with dystrophin and another one which involves alpha-dystrobrevin. Cardiac muscle and brain from Xenopus are shown to contain three beta-dystroglycan complexes related to various dystrophin-family isoforms. Dystrophin or alpha-dystrobrevin or Dp71 were found in cardiac muscle and dystrophin or Dp180 or Up71 in brain. This variability in the relationship between beta-dystroglycan and dystrophin-family isoforms suggests that each protein--currently known as dystrophin associated protein--could not be present in each of these complexes.

Dystrophin and dystrophin-associated protein in muscles and nerves from monkey.

Since all organs (i.e. skeletal, cardiac, smooth muscles and sciatic nerve) are never only taken from a single patient, all these tissues were obtained from one cynomolgus monkey, a model closely resembling humans. This work describes an up-to-date reinvestigation of the dystrophin-glycoprotein complex and related molecules in various monkey tissues such those cited above. We used monoclonal and polyclonal antibodies produced in our laboratory, which are directed against dystrophin, utrophin, short-dystrophin products, alpha-dystrobrevin, beta-dystroglycan, alpha-syntrophin, alpha-, beta-, gamma-, delta-, epsilon-sarcoglycan, and sarcospan. For each molecule, we determined their molecular weight and tissue localization. Regardless of the tissue analyzed, at least one dystrophin or utrophin as full-length molecule and one short-dystrophin product or dystrobrevin as proteins belonging to the dystrophin superfamily were found. Beta-dystroglycan, beta and delta sarcoglycans were always detected, while other sarcoglycans varied from all to only three components. Epsilon sarcoglycan appears to be specific to smooth muscle, which is devoid of alpha sarcoglycan. Sarcospan is only absent from sciatic nerve structures. Among the different muscles investigated in this study, short dystrophin products are only present in cardiac muscle. All of these findings are summarized in one table, which highlight in one single animal the variability of the dystrophin-glycoprotein complex components in relation with the organ studied. This statement is important because any attempt to estimate protein restoration needs in each study the knowledge of the expected components that should be considered normal.

First immunocytochemical study of echinoderm smooth muscle: the Antarctic cushionstar Odontaster validus Koehler (Echinodermata, Asteroidea).

Our immunocytochemical observations reveal that the muscle present in the tips of the arms of the Antarctic cushionstar Odontaster validus contains caldesmon and calponin but not troponin. Thus, the muscle clearly belongs to the smooth muscle category. Distributions of contractile proteins such as actin, myosin (the latter a typical vertebrate muscle filament protein), paramyosin, and miniparamyosin (the latter two being characteristic of thick invertebrate muscle filaments) were also determined immunocytochemically. The results suggest that the thin filaments of the starfish smooth muscle are similar to those of the vertebrate muscle, but that the thick filaments differ from those of vertebrates and possess traits that are also seen in the muscle organization of invertebrates. The absence from the O. validus muscle of titin and nebulin, proteins so far known almost exclusively from the striated vertebrate muscle, comes as no surprise, but immunoreactivity to mini-titin (a protein of the same family as titin and its replacement in invertebrates) was strong and unambiguously recognizable between filaments. Odontaster validus' histochemical characteristics may be a reflection of the phylogenetic position of the echinoderms as deuterostome invertebrates or they may express an adaptation of the muscle to the harsh environmental conditions under which it has to function in the Antarctic water.

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle.

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.

Smooth muscle proteins as intracellular components of the chromatophores of the Antarctic fishes Pagothenia borchgrevinki and Trematomus bernacchii (Nototheniidae).

Melanophores, xanthophores, and iridophores from the skins of the two Antarctic fish species Pagothenia borchgrevinki and Trematomus bernacchii were tested immunocytochemically for the presence of a variety of muscle proteins. Actin, myosin, and calmodulin, not surprisingly, were confirmed for all three chromatophore types of the two fishes, but the presence of caldesmon and calponin, both characteristic proteins of smooth muscle fibers, represents a new discovery. It is not known at this stage whether these proteins occur also in the chromatophores of other fishes and are not restricted to Antarctic species. Since, however, motility control of particles in fish chromatophores and the regulation of smooth muscle tension both involve the sympathetic nervous system, the presence of similar target proteins should not come as a surprise. The fact that none of the chromatophores tested positive for troponin shows that there is no close relationship between pigment cells and striated muscle. The lack of alpha-actinin in iridophores, but its presence in melanophores and xanthrophores, is thought to be a reflection of the considerably greater pigment translocations within the latter two types of chromatophore cells.

Androgen receptor (AR), estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) expression in the testis of the newt, Triturus marmoratus marmoratus during the annual cycle.

Expression of androgen receptor (AR), estrogen receptor alpha (ER-alpha) and estrogen receptor beta (ER-beta) in the testis of the marbled newt (Triturus marmoratus marmoratus) was investigated, with special attention to changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. Primordial germ cells, primary and secondary spermatogonia and spermatocytes showed a positive reaction to the 3 receptor antibodies during the annual reproductive cycle. Follicular cells were positive to AR, ER-alpha and ER-beta during the spermiogenesis and quiescence periods in the glandular tissue. Interstitial cells showed reactivity to AR, ER-alpha and ER-beta in the spermiogenesis and the quiescence periods, and presented no labelling to these receptors in the proliferative period. These findings suggest that, as in mammals, there is an androgen-estrogen regulation of the function and development of the newt testis.

Dystrophin-associated proteins in obliquely striated muscle of the leech Pontobdella muricata (Annelida, Hirudinea).

The distribution of dystrophin-associated proteins (beta-dystroglycan, alpha-, beta-, gamma- and delta-sarcoglycan, alpha-syntrophin and sarcospan) were studied in obliquely striated muscle of the leech Pontobdella muricata. Western blot analysis and immunohistochemical electron microscopy, using various polyclonal antibodies, were employed. Western blot analysis of all of these antibodies showed a single band, with approximately the same molecular weights as similar proteins detected in vertebrate muscles. The immunoelectron microscopy study confirmed specific immunogold labelling in the membrane of muscle cells. Since all dystrophin complex components have similar molecular weights and the same localisation in leech as in vertebrate skeletal muscle, we assume that these proteins have similar properties in leech and vertebrate muscle. The presence of these molecules in annelid muscles, together with a short version of dystrophin (previously described as IDLp-140) is of particular interest since phylogenetic and functional studies on this material could help to shed new light on the role and function of this complex in the muscle membrane.

Comparative evolution of muscular dystrophy in diaphragm, gastrocnemius and masseter muscles from old male mdx mice.

X chromosome-linked muscular dystrophic mdx mouse lacks the sarcolemmal protein dystrophin and represents a genetic homologue of human Duchenne muscular dystrophy (DMD). The present study analysed some aspects of pathological processes such as fibrosis, frequency of centralized nuclei, presence of degenerative or regenerative fibres, expression of utrophin and associated protein complexes, and myosin heavy chain isoforms in three muscles [diaphragm (DIA), gastrocnemius (GTC) and masseter (MAS)] from old male mdx mice. All parameters investigated comparatively in these pathological muscles provided evidence that the MAS mdx muscle presents a slight deterioration pattern in comparison to that of DIA and GTC muscles. Utrophin and associated proteins are present in many cell clusters with continuous membrane labelling in MAS muscle. Respective proportions of myosin heavy chain isoforms, measured by electrophoresis/densitometry, showed only slight change in GTC muscle, significant evolution in DIA muscle but drastic isoform conversions in MAS muscle. These results highlighted the difference in deterioration susceptibility of various muscles to muscular dystrophy. The reason why this occurs in MAS muscles is still obscure and discussed in terms of the comparative developmental origins of these muscles.