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Introducción: Las universidades médicas cubanas enfrentan el desafío, en medio de la pandemia de COVID-19, de mantener la formación de recursos humanos y apoyar directamente las acciones para evitar el contagio de la población. Objetivo: describir el desarrollo del proceso enseñanza aprendizaje durante la integración de estudiantes de las carreras de la Salud a la Atención Primaria en la lucha contra la COVID-19 Método: Se desarrolló un estudio descriptivo transversal de marzo a julio del 2020 en el municipio Santo Domingo, provincia Villa Clara. Además se recopiló información de archivos del Departamento Docente y de Higiene Municipal. Fueron insertados 305 estudiantes y 15 profesores en 44 consultorios, previo análisis contextual se implementó modificaciones en las estrategias didácticas para la educación a distancia a través de uso de datos en telefonía móvil empleando aplicación whatsapp. Resultados: Con predominio de métodos asincrónicos, se logró mantener un flujo informativo y actualizado de las estadísticas necesarias para los partes diarios, la información sobre diferentes asignaturas, estado de salud de los alumnos y profesores, se fortaleció el trabajo político ideológico al contrarrestar la infodemia y otras campañas de desinformación al establecer vínculos con otros grupos administrados por la FEU y el grupo de comunicadores de la Universidad. Conclusiones: La respuesta dinámica del claustro con inserción de las Tecnologías de la Información y las Comunicaciones durante la labor de pesquisa permitió dar continuidad al proceso enseñanza aprendizaje y la obtención de resultados exitosos en el primer enfrentamiento a la pandemia en el municipio.

Se ha demostrado que la vivencia de soledad social está estrechamente relacionada con el estado de salud de la población. La presente investigación evaluó la prevalencia de la hipertensión arterial y su asociación con la percepción de la soledad social en los habitantes mayores de 55 años de la comunidad Antonio Carrillo. En tal sentido se realizó un estudio analítico de prevalencia, utilizando como población 92 personas, se procedió a interrogar sobre el diagnóstico previo de hipertensión arterial, medición de la presión arterial y posteriormente se aplicó el instrumento de recolección de datos ESTE II. Se obtuvo como resultados que, en los habitantes mayores de 55 años de la comunidad, según las características generales de la población, predominó el sexo femenino (63%); asimismo, la mayoría de los habitantes se encontraron entre un rango de edad de 55 a 64 años (52,2%); según estado civil, prevalecieron los habitantes casados (41,3%); con respecto al nivel educativo la mayoría de los habitantes cursó primaria incompleta (24%) y en relación a la situación laboral predominó la inactividad (76,1%). A su vez, se comprobó que 65,2% de la población era hipertensa y en cuanto a la percepción de soledad social, se evidenció que 68,5% de la población presentaba la misma. Se pudo observar que no hubo asociación estadísticamente significativa entre la presión arterial y la percepción de soledad social (p > 0.05), sin embargo, 68,3% de los habitantes con percepción de soledad social fueron hipertensos.

El Doctor Oswaldo Valdivia nació en 1937 en Arequipa, Perú. En 1958 inició sus estudios en la carrera de medicina, en el primer año nació el amor por la neuroanatomía y en el cuarto año inicio su labor como docente. Se graduó como médico en 1964 y de anatomopatólogo en 1966. Contrajo matrimonio en 1962 con Zulema Valdivia con quien tuvo 3 hijos, en 1968 fue becado para entrenamiento de microscopia electrónica en la universidad de Wisconsin. En 1973 decidió emigrar hacia Venezuela debido a la situación del Perú y por una oferta laboral de parte de la Universidad Centroccidental Lisandro Alvarado (UCLA), donde se desenvolvió como profesor de anatomía microscópica a dedicación exclusiva, realizando numerosas contribuciones como la incorporación de la microscopia electrónica en la región centroccidental del país en 1980. Realizó diversos trabajos de investigación, principalmente sobre leishmaniasis, fue profesor jubilado activo de la UCLA hasta sus últimos días. En su ejercicio profesional dio grandes aportes a la salud pública, participando en jornadas de pesquisa de patológica cervical y formando profesionales de la salud. Fallece el 18 de noviembre de 2019 en Perú, su tierra natal.

OBJETIVOS: - Conocer la percepción de enfermedad en los pacientes crónicos polimedicados desde Atención Primaria. - Determinar si existe una mayor percepción de enfermedad en el grupo que recibe la intervención enfermera. - Determinar si existe una mayor adhesión al tratamiento en el grupo que recibe la intervención enfermera.

Abstract Background Emigrants are often a selected sample and in good health, but migration can have deleterious effects on health. Many immigrant groups report poor health and increased use of health services, and it is often claimed that they tend to use emergency primary health care (EPHC) services for non-urgent purposes. The aim of the present study was to analyse immigrants’ use of EPHC, and to analyse variations according to country of origin, reason for immigration, and length of stay in Norway. Methods We conducted a registry based study of all immigrants to Norway, and a subsample of immigrants from Poland, Germany, Iraq and Somalia, and compared them with native Norwegians. The material comprised all electronic compensation claims for EPHC in Norway during 2008. We calculated total contact rates, contact rates for selected diagnostic groups and for services given during consultations. Adjustments for a series of socio-demographic and socio-economic variables were done by multiple logistic regression analyses. Results Immigrants as a whole had a lower contact rate than native Norwegians (23.7% versus 27.4%). Total contact rates for Polish and German immigrants (mostly work immigrants) were 11.9% and 7.0%, but for Somalis and Iraqis (mostly asylum seekers) 31.8% and 33.6%. Half of all contacts for Somalis and Iraqis were for non-specific pain, and they had relatively more of their contacts during night than other groups. Immigrants’ rates of psychiatric diagnoses were low, but increased with length of stay in Norway. Work immigrants suffered less from respiratory and gastrointestinal infections, but had more injuries and higher need for sickness certification. All immigrant groups, except Germans, were more often given a sickness certificate than native Norwegians. Use of interpreter was reduced with increasing length of stay. All immigrant groups had an increased need for long consultations, while laboratory tests were most often used for Somalis and Iraqis. Conclusions Immigrants use EPHC services less than native Norwegians, but there are large variations among immigrant groups. Work immigrants from Germany and Poland use EPHC considerably less, while asylum seekers from Somalia and Iraq use these services more than native Norwegians.

Abstract Background There is evidence that exposure to passive smoking in general, and in babies in particular, is an important cause of morbimortality. Passive smoking is related to an increased risk of pediatric diseases such as sudden death syndrome, acute respiratory diseases, worsening of asthma, acute-chronic middle ear disease and slowing of lung growth. The objective of this article is to describe the BIBE study protocol. The BIBE study aims to determine the effectiveness of a brief intervention within the context of Primary Care, directed to mothers and fathers that smoke, in order to reduce the exposure of babies to passive smoking (ETS). Methods/Design Cluster randomized field trial (control and intervention group), multicentric and open. Subject: Fathers and/or mothers who are smokers and their babies (under 18 months) that attend pediatric services in Primary Care in Catalonia. The measurements will be taken at three points in time, in each of the fathers and/or mothers who respond to a questionnaire regarding their baby's clinical background and characteristics of the baby's exposure, together with variables related to the parents' tobacco consumption. A hair sample of the baby will be taken at the beginning of the study and at six months after the initial visit (biological determination of nicotine). The intervention group will apply a brief intervention in passive smoking after specific training and the control group will apply the habitual care. Discussion Exposure to ETS is an avoidable factor related to infant morbimortality. Interventions to reduce exposure to ETS in babies are potentially beneficial for their health. The BIBE study evaluates an intervention to reduce exposure to ETS that takes advantage of pediatric visits. Interventions in the form of advice, conducted by pediatric professionals, are an excellent opportunity for prevention and protection of infants against the harmful effects of ETS. Trial Registration Clinical Trials.gov Identifier: NCT00788996.

Abstract Background Indoor air pollution (IAP) from solid fuels is a serious health problem in low-income countries that can be alleviated using improved stoves. Although women are the principal users, few studies have investigated the self-assessed impact of the stoves on their health and lives. Methods This study was conducted in rural highland Guatemala, involving 89 intervention and 80 control Mayan Indian young women (mean 27.8 years, SD 7.2). Outcomes were assessed after approximately 18 months use of the new stove. Our objectives were to compare self-rated health and change in health among women participating in a randomised control trial comparing a chimney stove with an open fire, to describe impacts on women's daily lives and their perceptions of how reduced kitchen smoke affects their own and their children's health. Results On intention-to-treat analysis, 52.8% of intervention women reported improvement in health, compared to 23.8% of control women (p < 0.001). Among 84 intervention women who reported reduced kitchen smoke as an important change, 88% linked this to improvement in their own health, particularly for non-respiratory symptoms (for example eye discomfort, headache); 57% linked reduced smoke to improvement in their children's health, particularly sore eyes. Conclusion Women's perception of their health was improved, but although smoke reduction was valued, this was linked mainly with alleviation of non-respiratory symptoms like eye discomfort and headache. More focus on such symptoms may help in promoting demand for improved stoves and cleaner fuels, but education about more severe consequences of IAP exposure is also required.

Introduction: Acute undifferentiated febrile illnesses (AUFIs) impose a large burden in the tropics. Understanding of AUFI's epidemiology is limited. Insufficient diagnostic capacity hinders the detection of outbreaks. The lack of interconnection in healthcare systems hinders timely response. We describe a protocol to study the epidemiology and aetiologies of AUFI and pathogen discovery in strategic areas of Latin America (LA)., Methods and Analysis: Global Infectious Diseases Network investigators comprising institutions in Colombia, Dominican Republic, México, Perú and the USA, developed a common cohort study protocol. The primary objective is to determine the aetiologies of AUFI at healthcare facilities in high-risk areas. Data collection and laboratory testing for viral, bacterial and parasitic agents are performed in rural and urban healthcare facilities and partner laboratories. Centralised laboratory and data management cores deploy diagnostic tests and data management tools. Subjects >6 years with fever for <8 days without localised infection are included in the cohort. They are evaluated during the acute and convalescent phases of illness. Study personnel collect clinical and epidemiological information. Blood, urine, nasal or pharyngeal swabs and saliva are collected in the acute phase and blood in convalescent phase. Specimens are banked at -80°C. Malaria, dengue and COVID-19 are tested onsite in the acute phase. The acute-phase serum is PCR tested for dengue, chikungunya, Venezuelan equine encephalitis, Mayaro, Oropouche, Zika, and yellow fever viruses. Paired convalescent and acute serum antibody titters are tested for arbovirus, Leptospira spp, and Rickettsia spp. Serum is used for viral cultures and next-generation sequencing for pathogen discovery. Analysis includes variable distributions, risk factors and regression models. Laboratory results are shared with health authorities and network members., Ethics and Dissemination: The protocol was approved by local ethics committees and health authorities. The results will be published in peer-reviewed journals. All study results are shared with local and regional health authorities., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Neuroblastoma, Wilms tumor, and hepatoblastoma are the most common pediatric abdominal malignancies. Management of these diseases is a multidisciplinary process that continues to evolve based on the results of international collaborative trials and advances in understanding of tumor biology. Each of these tumors has unique characteristics and behavior which are reflected in their respective staging systems. It is important for clinicians involved in the care of children with abdominal malignancies to be familiar with current staging guidelines and imaging recommendations. This article reviews the current role of imaging in the management of these common pediatric abdominal malignancies, with emphasis on initial staging., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

N-methyl-D-aspartate receptors (NMDARs) play vital roles in normal brain functions (i.e., learning, memory, and neuronal development) and various neuropathological conditions, such as epilepsy, autism, Parkinson's disease, Alzheimer's disease, and traumatic brain injury. Endogenous neuroactive steroids such as 24(S)-hydroxycholesterol (24(S)-HC) have been shown to influence NMDAR activity, and positive allosteric modulators (PAMs) derived from 24(S)-hydroxycholesterol scaffold can also enhance NMDAR function. This study describes the structural determinants and mechanism of action for 24(S)-hydroxycholesterol and two novel synthetic analogs (SGE-550 and SGE-301) on NMDAR function. We also show that these agents can mitigate the altered function caused by a set of loss-of-function missense variants in NMDAR GluN subunit-encoding GRIN genes associated with neurological and neuropsychiatric disorders. We anticipate that the evaluation of novel neuroactive steroid NMDAR PAMs may catalyze the development of new treatment strategies for GRIN-related neuropsychiatric conditions., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Determining the amount of a drug transferred into breast milk is critical for benefit-risk analysis of breastfeeding when a lactating mother takes medications. In this study, we developed a human mammary epithelial cell (MEC)-based permeability assay to assess drug permeability across the mammary epithelium. Human MEC cell MCF10F formed tight junctions when cultured on Transwells with culture medium containing insulin, hydrocortisone and epidermal growth factor (EGF). Formation of integral cell barrier and morphology of the cells were confirmed by assessing trans-epithelial electrical resistance (TEER), flux of fluorescent tracers and imaging with transmission electron microscopy (TEM). MCF10F cells showed consistent P-glycoprotein (P-gp) transporter expression when culturing on Transwell inserts versus on petri dish. A few P-gp transporter drug substrates were used to estimate the permeability from this assay. Human plasma and breast milk were used as incubation medium in basolateral and apical chambers respectively to mimic physiological conditions. The predicted milk to plasma (M/P) ratios were reasonably good. The current effort to develop the MEC-based permeability assay to facilitate M/P ratio prediction showed promising results. This assay may have a potential to be developed as a useful in vitro technique for determining the transfer of small-molecule therapeutic drugs into breast milk., Competing Interests: All authors declare no conflicts of interest relevant to the content of this article., (© 2022 The Authors. Published by Elsevier B.V.)

Background: Focal liver lesions are common in children and adults after Fontan procedures., Objective: To explore the relationship between liver shear stiffness, using magnetic resonance (MR) elastography, and the presence of focal liver lesions in patients after Fontan procedures (total cavopulmonary anastomosis)., Materials and Methods: The retrospective study was approved by the institutional review board and the requirement for informed consent was waived. By searching institutional electronic medical records, we identified all patients with a history of Fontan palliation of congenital heart disease who had undergone same-day liver MR elastography and liver MRI without and with intravenous contrast material between January 2012 and December 2017. Using imaging reports, patients were placed into two groups: 1) no focal liver lesions and 2) one or more focal liver lesions. Patient age, sex, mean liver shear stiffness (kPa) and maximum single anatomical level liver shear stiffness (kPa) were recorded. The Mann-Whitney U test was used to compare age and liver stiffness between groups, while the Fisher exact test was used to assess the impact of gender on liver lesions., Results: Forty-eight patients met study inclusion criteria; 33 (69%) had one or more focal liver lesions. The median age was 20.0 years (IQR [interquartile range]: 10.8-29.1 years) for patients without liver lesions and 19.9 years (IQR: 17.2-27.0 years) for patients with liver lesions (P=0.49). Eleven of 21 male patients (52.4%) had liver lesions compared to 22 of 27 female patients (81.5%) (P=0.058). Mean (4.62 kPa [IQR: 4.10-5.59 kPa] vs. 4.10 kPa [IQR: 3.44-4.80 kPa]; P=0.02) and maximum (5.53 kPa [IQR: 4.64-6.56 kPa] vs. 4.50 kPa [IQR: 3.82-5.35 kPa]; P=0.009) liver stiffness were significantly higher in patients without liver lesions as compared to patients with liver lesions., Conclusion: Our study demonstrated a significant negative association between focal liver lesions and increased liver stiffness in patients following Fontan procedures.

Objectives: Identify predictors of residual carcinoma or carcinoma-in-situ (CIS) at hysterectomy following cervical conizations with CIS and positive margins or endocervical curettage (ECC) or microinvasive cervical cancer., Methods: Patients with cervical conization with CIS and positive margins, ECC or microinvasive carcinoma who underwent hysterectomy within 6 months of conization were identified. Conization and hysterectomy specimens were re-reviewed to assess volume of disease, ECC and margin status and residual carcinoma. Standard statistical tests were used., Results: 83 patients were included. 34 (41%) had residual carcinoma in the hysterectomy specimen: 23 CIS, 9 microinvasive and 2 invasive disease. In patients with squamous histology predictors of residual disease included a positive ECC (p=0.04), combined endocervical margin and ECC (69% if both positive, 38% either positive, 11% if both negative, p=0.01) and volume of disease ≥ 50% (p=0.01). In patients with glandular histology no factor predicted residual disease. Type of conization, >2 involved quadrants, and the presence of microinvasion in the conization specimen did not predict residual disease. No patient with squamous histology had >Stage IA1 disease at hysterectomy, whereas 2 (2.4%) with adenocarcinoma had >Stage IA1 disease at hysterectomy., Conclusions: Residual carcinoma or CIS is present in nearly half of hysterectomies after conization with CIS and positive ECC, margins or microinvasion. Patients with squamous histology may not require repeat conization prior to definitive therapy. No factors predict residual disease with adenocarcinoma. In women with AIS with negative margins and ECC and no microinvasion, it appears reasonable to proceed with simple hysterectomy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: To determine the impact of venous thromboembolism (VTE) during primary treatment of ovarian clear cell carcinoma (OCCC) on survival., Methods: After Institutional Review Board approval, 74 cases of OCCC were retrieved from our pathology files. Clinical and pathological data were obtained by medical record and pathology review. Standard statistical analyses were performed., Results: Among 74 patients with OCCC, VTE was diagnosed in 11 (15%) during primary treatment and 7 (9%) at time of cancer recurrence. 56 (76%) patients never developed VTE. Patients with VTE during OCCC primary treatment had shorter progression-free survival (PFS) and overall survival (OS) than OCCC patients without VTE (median PFS 11 vs. 76 months, p=0.01, median OS 19 vs. 90 months, p=0.001). Patients with VTE during OCCC primary treatment had a 3.9-fold increase in risk of recurrence (p=0.007) and a 6.3-fold increase in risk of death (p<0.001). After controlling for cancer stage, VTE during OCCC primary treatment remained an independent prognostic factor for death (HR=3.6, p=0.005). No patient died of VTE., Conclusions: VTE during OCCC primary treatment is associated with a significantly higher risk of cancer recurrence and death. This increased risk is not attributable to VTE-related mortality and raises the possibility that a paracrine circuit involving thrombosis might contribute to a more aggressive tumor biology., (© 2013.)

Objectives: Obesity impacts outcome in women with epithelial ovarian cancer (EOC), although its exact role and the molecular mechanisms remain poorly defined. Adipocytes secrete leptin and adiponectin, and the leptin to adiponectin (L:A) ratio is correlated with poor survival in other malignancies. We hypothesized that the L:A ratio is associated with survival in women with EOC., Methods: We queried the institutional tumor registry for patients with advanced stage EOC and identified a cohort of 161 women with banked fasting prediagnostic serum samples. Patients underwent cytoredutive surgery followed by platinum-based chemotherapy. Sera were assayed for leptin and adiponectin, and clinico-pathologic data were abstracted. Standard statistical tests were performed., Results: 161 patients met inclusion criteria. We identified a significant correlation between BMI and leptin and the L:A ratio, but not adiponectin, in this cohort (r=0.46, 0.46, and -0.13, respectively; p=0.001, 0.001, and 0.106). Women with low L:A ratios demonstrated statistically longer disease-specific survival (57 months) compared to those with median or high levels (49 and 37 months, respectively; p=0.02). On multivariate analysis, we determined that BMI and age, but not L:A ratio, retained significance as independent prognostic factors for survival (p=0.04, 0.004, and 0.895, respectively)., Conclusions: In this cohort, the L:A ratio correlated statistically with clinical outcome, but did not independently predict survival. Obesity remains a modifiable risk factor in women with EOC. Further studies are needed to determine if leptin and/or adiponectin may be potential therapeutic targets in obese women with EOC., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Stress may promote ovarian cancer progression through mechanisms including autonomic nervous system mediators such as norepinephrine and epinephrine. Beta blockers, used to treat hypertension, block production of these adrenergic hormones, and have been associated with prolonged survival in several malignancies. We sought to determine the association between beta blocker use and epithelial ovarian cancer (EOC) disease progression and survival., Methods: We performed an institutional retrospective review of patients with EOC treated between 1996 and 2006. Patients underwent cytoreductive surgery followed by platinum-based chemotherapy. Women were considered beta blocker users if these medications were documented on at least two records more than 6 months apart. Statistical tests included Fisher's exact, Kaplan-Meier, and Cox regression analyses., Results: 248 met inclusion criteria. 68 patients used antihypertensives, and 23 used beta blockers. Median progression-free survival for beta blocker users was 27 months, compared with 17 months for non-users (p=0.05). Similarly, overall disease-specific survival was longer for beta blocker users (56 months) compared with non-users (48 months, p=0.02, hazard ratio=0.56). Multivariate analysis identified beta blocker use as an independent positive prognostic factor, after controlling for age, stage, grade, and cytoreduction status (p=0.03). Overall survival remained longer for beta blocker users (56 months) when compared with hypertensive patients on other medications (34 months) and patients without hypertension (51 months) (p=0.007)., Conclusions: In this cohort of patients with EOC, beta blocker use was associated with a 54% reduced chance of death compared with that of non-users., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Fertilization is the process by which male and female haploid gametes (sperm and egg) unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding to the zona pellucida, acrosome exocytosis, sperm penetration through the zona and fusion of the sperm and egg plasma membranes. However, freshly ejaculated sperm are not capable of fertilizing an oocyte. They must first undergo a series of biochemical and physiological changes, collectively known as capacitation, before acquiring fertilizing capabilities. Several molecules are required for successful capacitation and in vitro fertilization; these include bicarbonate, serum albumin (normally bovine serum albumin, BSA) and Ca(2+). Bicarbonate activates the sperm protein soluble adenylyl cyclase (SACY), which results in increased levels of cAMP and cAMP-dependent protein kinase (PKA) activation. The response to bicarbonate is fast and cAMP levels increase within 60 s followed by an increase in PKA activity. Several studies with an anti-phospho-PKA substrate antibody have demonstrated a rapid increase in protein phosphorylation in human, mouse and boar sperm. The target proteins of PKA are not known and the precise role of BSA during capacitation is unclear. Most of the studies provide support for the idea that BSA acts by removing cholesterol from the sperm. The loss of cholesterol has been suggested to affect the bilayer of the sperm plasma membrane making it more fusogenic. The relationship between cholesterol loss and the activation of the cAMP/PKA pathway is also unclear. During early stages of capacitation, Ca(2+) might be involved in the stimulation of SACY, although definitive proof is lacking. Protein tyrosine phosphorylation is another landmark of capacitation but occurs during the late stages of capacitation on a different time-scale from cAMP/PKA activation. Additionally, the tyrosine kinases present in sperm are not well characterized. Although protein phosphorylation depends upon the balanced action of protein kinases and protein phosphatase, we have even less information regarding the role of protein phosphatases during sperm capacitation. Over the last few years, several reports have pointed out that the ubiquitin-proteasome system might play a role during sperm capacitation, acrosome reaction and/or sperm-egg fusion. In the present review, we summarize the information regarding the role of protein kinases, phosphatases and the proteasome during sperm capacitation. Where appropriate, we give examples of the way that these molecules interact and regulate each other's activities.

The aim of this work was to determine whether laminin (Ln), an extracellular matrix protein, induces the intracellular events that may be involved in producing the acrosome reaction in human sperm. To this end, we evaluated the effect of Ln on tyrosine phosphorylation, intracellular calcium concentration, proteasome activity, and phosphorylation in human sperm. Aliquots of highly motile sperm selected with a Percoll gradient, were incubated with different concentrations of Ln (0-20 μg/ml) for different periods (0-18 h). The percentage of viable acrosome-reacted sperm was evaluated using fluorescein isothiocyanate-labeled Pisum sativum agglutinin and Hoechst 33258 DNA dye. Tyrosine phosphorylation was evaluated by Western blot analysis. The chymotrypsin-like activity of the proteasome was evaluated with a fluorogenic peptide, and intracellular calcium concentration was measured with fura-2. The results indicate that Ln stimulated the acrosome reaction of human sperm in a dose-dependent manner. This increase was drastically inhibited in the presence of herbimycin A, SU6656, and epoxomicin. In addition, Ln increased proteasome activity and phosphorylation; both events were inhibited by herbimycin A and SU6656. Finally, Ln induced an increase in intracellular calcium concentration, which was inhibited by SU6656 and epoxomicin. These results suggest that Ln is able to induce the acrosome reaction. This effect may be mediated by Src kinase and the proteasome, with the consequent induction of a calcium influx.

Human semen is composed of a heterogeneous population of spermatozoa with varying degrees of structural and functional differentiation and normality, which result in subpopulations of different quality. Using a discontinuous Percoll gradient, we separated three subsets of spermatozoa (65/45%, 90/65% and 90% fractions) from normozoospermic semen samples from healthy donors and proceeded to characterise their morphology, viability, motility and proteasome activity. In addition, the presence of proteasome subunit transcripts was investigated using reverse transcription-polymerase chain reaction (RT-PCR). The results obtained showed significant differences in sperm motility, viability and morphology between the cells collected from each of the fractions. In particular, normal sperm morphology was 4.5 times higher in the 90% pellet in comparison with the 65/45% interface. In addition, there were significant differences in proteasomal activity between spermatozoa recovered from the 90% pellet and spermatozoa recovered from the 65/45% interface. Finally, there was a positive correlation between sperm proteasomal enzymatic activity and sperm motility and normal morphology after separation by a discontinuous Percoll gradient. The results of the RT-PCR revealed the presence of transcripts for the proteasome subunits β1, β2 and β5 in the human spermatozoa analysed. In conclusion, poor quality spermatozoa isolated from a Percoll gradient display an intrinsic proteasome activity deficiency, which may be associated with their low fertilising potential., (© 2010 Blackwell Verlag GmbH.)

The proteasome is a multicatalytic cellular complex present in human sperm that plays a significant role during several steps of mammalian fertilization. Here, we present evidence that the proteasome is involved in human sperm capacitation. Aliquots of highly motile sperm were incubated with proteasome inhibitors MG132 or epoxomicin. The percentage of capacitated sperm, the chymotrypsin-like activity of the proteasome, cAMP content, and the pattern of protein phosphorylation were assayed by using the chlortetracycline hydrochloride assay, a fluorogenic substrate, the cAMP enzyme immunoassay kit, and Western blot analysis, respectively. Our results indicate that treatment of sperm with proteasome inhibitors blocks the capacitation process, does not alter cAMP concentration, and changes the pattern of protein phosphorylation. To elucidate how proteasome activity is regulated during capacitation, sperm were incubated with: 1) tyrosine kinase (TK) inhibitors (genistein or herbimycin A); 2) protein kinase (PK) A inhibitors or activators (H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKC inhibitors (tamoxifen or staurosporin) at different capacitation times. The chymotrypsin-like activity and degree of phosphorylation of the proteasome were then assayed. The results indicate that sperm treatment with TK and PKA inhibitors significantly decreases the chymotrypsin-like activity of the proteasome during capacitation. Immunoprecipitation and Western blot results suggest that the proteasome is phosphorylated during capacitation in a TK- and PKA-dependent pathway. In conclusion, we suggest that the sperm proteasome participates in the capacitation process, and that its activity is modulated by PKs.

Background: Previously we showed that the human sperm proteasome plays significant roles during mammalian fertilization. Here we studied the effect of fibronectin (Fn), an extracellular matrix protein present in the cumulus oophorus of the oocyte, on proteasome activity, acrosome reaction, intracellular calcium concentration ([Ca(2+)](i)) and protein tyrosine phosphorylation of human sperm., Methods: Aliquots of motile sperm were incubated for 15 min (T0), 5 h (T5) and 18 h (T18), at 37 degrees C, 5% CO(2) and 95% air with Fn (0-100 microg/ml). The chymotrypsin- and trypsin-like activity of the proteasome was measured using the fluorogenic substrates, Suc-Leu-Leu-Val-Tyr-AMC and Boc-Gln-Ala-Arg-AMC, respectively. At T18, sperm aliquots were incubated for 15 min with Fn and/or progesterone in the presence or absence of epoxomicin (a proteasome inhibitor). The percentage of viable acrosome reacted sperm was evaluated using the Fluorescein isothiocyanate (FITC)-labeled Pisum sativum agglutinin. Tyrosine phosphorylation was evaluated by western blot and [Ca(2+)](i) using fura 2., Results: Fn stimulated both enzymatic activities of the proteasome and the acrosome reaction of human sperm. Progesterone enhanced and epoxomicin drastically inhibited the effect of Fn. Fn treatment also increased the [Ca(2+)](i). Western blot analysis revealed that Fn increased tyrosine protein phosphorylation and that some proteasome subunits became tyrosine phosphorylated upon Fn treatment., Conclusions: These results suggest that Fn activates the proteasome and induces the acrosome reaction in human sperm. This effect may involve binding with specific receptors (integrins) on the sperm surface and the activation of tyrosine kinases.

Caveolin-1, the first member of caveolin family reported, is recognized as the structural component of caveola, a plasma membrane invagination or vesicles that are a subcompartment distinct from clathrin-coated pits. This protein is also known to be involved in cholesterol trafficking. The aim of this study was to determine the expression of caveolin-1 in adult rat Leydig cells. Testis sections incubated with an antibody to caveolin-1 showed, by immunohistochemistry, a moderate number of Leydig cells with different degrees of immunoreaction and a strong reaction in endothelial cells and in the lamina propia of seminiferous tubules. Caveolin- 1 was detected in the cell cytoplasm with a granular pattern and on the cell surface of Leydig cells cultured 24 h on uncoated, laminin-1 or type IV collagen coated coverslips. We also observed a milder reaction in 3 h cultures. Immunoreaction was also detected in Leydig cells with an antibody to tyrosine-phosphorylated caveolin-1. By double immunofluorescent technique, we observed co-localization of caveolin- I and 313-hydroxysteroid dehydrogenase. Western blot analysis revealed a band of about 22 kDa molecular weight that was recognized with both caveolin-1 and tyrosine-phosphocaveolin-1 antibodies. Caveolin-l is one of a few proteins with a demonstrated ability to bind cholesterol in vivo. In this context, the presence of caveolin- in Leydig cells may be related to cholesterol traffic--a rate limiting step in steroid biosynthesis.

We have previously shown that type IV collagen (alpha1 (IV) and alpha2 (IV) collagen chains) (Col-IV) inhibits testosterone (T) production by Leydig cells (LC). The aim of this study was to analyze mechanism/s by which Col-IV exerts this effect. No significant differences in the specific binding of hCG to LH/hCG receptors in LC cultured on uncoated or Col-IV coated plates were observed. An inhibition of cAMP production in hCG-stimulated LC cultured on Col-IV was detected. The inhibition exerted by Col-IV on T production in response to hCG was also observed when cells were stimulated with 8Bromo-cAMP. In addition, conversion of steroid precursors to T in LC cultured on uncoated and Col-IV coated plates was similar. On the other hand, we detected an increase of ERK1/2 phosphorylation in hCG-stimulated LC cultured on Col-IV. Genistein added to LC cultures reduced the ability of Col-IV to increase ERK1/2 phosphorylation and reverted the inhibitory effect of Col-IV on T production. An inhibitor of MEK, PD98059 added to LC cultures also reverted the inhibitory effect of Col-IV on T production. A decrease of steroidogenic acute regulatory protein (StAR) expression in hCG-stimulated LC cultured on Col-IV coated plates that could be reverted by addition of PD98059 to the cultures was also demonstrated. All together these results suggest that Col-IV inhibits T production in LC by binding to integrins, activating ERK1/2, decreasing cAMP production and decreasing StAR expression., ((c) 2005 Wiley-Liss, Inc.)

We previously demonstrated that laminin, a component of basement membranes, modulates pituitary hormone secretion. In the present study, we evaluated the effect of type IV collagen, another component of this membrane, on the release of prolactin (PRL) by anterior pituitary gland from adult male rats. Hemipituitaries were incubated for 3 h with type IV collagen or antibodies against it and PRL release was studied. Rabbit IgG to type IV collagen at concentrations of 10(-7) - 10(-5) M had a significant stimulatory effect on PRL release, in comparison to normal rabbit serum IgG or medium alone used as controls. Type IV collagen induced a significant inhibitory effect on basal release of PRL at a concentration of 30 microg/mL. A slight decrease in PRL release was detected in thyrotropin-releasing hormone-stimulated hemipituitaries incubated with type IV collagen at all concentrations used. These results suggest that type IV collagen, similar to laminin-1, modulates PRL released from hemipituitaries, in vitro.

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.