Caveolin-1 function at the plasma membrane and in intracellular compartments in cancer.

Caveolin-1 (CAV1) is commonly considered to function as a cell surface protein, for instance in the genesis of caveolae. Nonetheless, it is also present in many intracellular organelles and compartments. The contributions of these intracellular pools to CAV1 function are generally less well understood, and this is also the case in the context of cancer. This review will summarize literature available on the role of CAV1 in cancer, highlighting particularly our understanding of the canonical (CAV1 in the plasma membrane) and non-canonical pathways (CAV1 in organelles and exosomes) linked to the dual role of the protein as a tumor suppressor and promoter of metastasis. With this in mind, we will focus on recently emerging concepts linking CAV1 function to the regulation of intracellular organelle communication within the same cell where CAV1 is expressed. However, we now know that CAV1 can be released from cells in exosomes and generate systemic effects. Thus, we will also elaborate on how CAV1 participates in intracellular communication between organelles as well as signaling between cells (non-canonical pathways) in cancer.

Características antropométricas y somatotipo en seleccionados chilenos de remo / Anthropometric characteristics and somatotype in elite Chilean rowers

El objetivo del estudio fue describir las características antropométricas y somatotipo de los seleccionados chilenos de remo. Se evaluaron a 21 deportistas (6 mujeres y 15 hombres) con edades promedio de 18,66 ± 2,58 años para las mujeres y de 22,06 ± 4,71 años para los hombres. El estudio se realizó previo al clasificatorio Panamericano Lima 2019, al momento de las evaluaciones, los deportistas se encontraban concentrados en el centro de entrenamiento olímpico (CEO) ubicado en Curauma en la región de Valparaíso en Chile. Las evaluaciones antropométricas se realizaron en base al perfil restringido de 25 variables propuesto por Ross & Kerr (1991), que permitió fraccionar la masa corporal en 5 componentes (tejidos: piel, residual, óseo, adiposo y muscular). Se midieron también la envergadura, la altura ilioespinal y los pliegues del bíceps y supracrestídeo, todas las medidas se realizaron en base al protocolo de marcaje y evaluación propuesto por ISAK. Se uso el método de Carter y Heath para calcular el somatotipo, describiendo los componentes de endomorfía, mesomorfía y ectomorfía respectivamente. Los resultados mostraron un porcentaje de grasa en hombres de 19,34 ± 1,59 % y en mujeres de 27,08 ± 3,6 % (p=0,003), la masa muscular de hombres fue de 52,69 ± 1,78 % y en mujeres de 45,68 ± 4,19 % (p=0,003), se apreció un somatotipo mesomorfo balanceado tanto en hombres (1,7-5,3-2,2) como mujeres (2,5-3,7-2,7). Se concluye que los seleccionados chilenos de remo presentan un alto desarrollo de masa muscular y un bajo porcentaje de masa grasa, además de un somatotipo mesomorfo balanceado.

The aim of the study was to describe the anthropometric characteristics and somatotype of the Chilean selected rowers. Twenty one 21 athletes (6 women and 15 men) with an average age of 18.66 ± 2.58 years for women and 22.06 ± 4.71 years for men were evaluated. The study was conducted prior to the Pan American qualification in Lima 2019. At the time of the evaluations, the athletes were concentrated in the Olympic Training Center (CEO) located in Curauma in the Valparaíso region of Chile. The anthropometric evaluations were carried out based on the restricted profile of 25 variables proposed by Kerr and Ross that allowed the fractionation of the body mass into 5 components (tissue: skin, residual, bone, adipose and muscle). The wingspan, the iliospinal height and the folds of the biceps and supracrestide were also measured; all measurements were based on the protocol of marking and evaluation proposed by ISAK. Carter and Heath method was used to calculate the somatotype, describing the components of endomorphy, mesomorphy and ectomorphy respectively. The results showed a percentage of fat in men of 19.34 ± 1.59 % and in women of 27.08 ± 3.6 % (p = 0.003), the muscle mass of men was 52.69 ± 1.78 % and in women of 45.68 ± 4.19 % (p = 0.003), a balanced mesomorphic somatotype was observed in both men (1.7-5.3-2.2) and women (2.5-3, 7-2.7). It is concluded that the Chilean rowing selected have a high development of muscle mass and a low percentage of fat mass, in addition to a balanced mesomorphic somatotype.

Astrocyte-to-neuron communication through integrin-engaged Thy-1/CBP/Csk/Src complex triggers neurite retraction via the RhoA/ROCK pathway.

Two key proteins for cellular communication between astrocytes and neurons are αvß3 integrin and the receptor Thy-1. Binding of these molecules in the same (cis) or on adjacent (trans) cellular membranes induces Thy-1 clustering, triggering actin cytoskeleton remodeling. Molecular events that could explain how the Thy-1-αvß3 integrin interaction signals have only been studied separately in different cell types, and the detailed transcellular communication and signal transduction pathways involved in neuronal cytoskeleton remodeling remain unresolved. Using biochemical and genetic approaches, single-molecule tracking, and high-resolution nanoscopy, we provide evidence that upon binding to αvß3 integrin, Thy-1 mobility decreased while Thy-1 nanocluster size increased. This occurred concomitantly with inactivation and exclusion of the non-receptor tyrosine kinase Src from the Thy-1/C-terminal Src kinase (Csk)-binding protein (CBP)/Csk complex. The Src inactivation decreased the p190Rho GTPase activating protein phosphorylation, promoting RhoA activation, cofilin, and myosin light chain II phosphorylation and, consequently, neurite shortening. Finally, silencing the adaptor CBP demonstrated that this protein was a key transducer in the Thy-1 signaling cascade. In conclusion, these data support the hypothesis that the Thy-1-CBP-Csk-Src-RhoA-ROCK axis transmitted signals from astrocytic integrin-engaged Thy-1 (trans) to the neuronal actin cytoskeleton. Importantly, the ß3 integrin in neurons (cis) was not found to be crucial for neurite shortening. This is the first study to detail the signaling pathway triggered by αvß3, the endogenous Thy-1 ligand, highlighting the role of membrane-bound integrins as trans acting ligands in astrocyte-neuron communication.

Short- and long-term consequences of perinatal asphyxia: looking for neuroprotective strategies.

Perinatal asphyxia constitutes a prototype of obstetric complications occurring when pulmonary oxygenation is delayed or interrupted. A primary insult is first produced by the length of the time without oxygenation, leading to hypoxia/ischemia and death if oxygenation is not promptly established. A second insult is produced by re-oxygenation, eliciting a cascade of biochemical events for restoring function, implying, however, improper homeostasis. The effects observed long after perinatal asphyxia can be explained by over-expression of sentinel proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1), competing for oxidised nicotinamide adenine dinucleotide (NAD(+)) during re-oxygenation. Asphyxia also induces transcriptional activation of pro-inflammatory factors, including nuclear factor κB (NFκB) and its subunit p65, whose translocation to the nucleus is significantly increased in brain tissue from asphyxia-exposed animals, in tandem with PARP-1 overactivation, leading to the idea that sentinel protein inhibition constitutes a suitable therapeutic strategy. It is proposed that PARP-1 inhibition also down-regulates the expression of pro-inflammatory cytokines.Nicotinamide is a suitable PARP-1 inhibitor, whose effects have been studied in an experimental model of global perinatal asphyxia in rats, inducing the insult by immersing rat foetuses into a water bath for various periods of time. Following asphyxia, the pups are delivered, immediately treated, or given to surrogate dams for nursing, pending further experiments. Systemic administration of nicotinamide 1 h after the insult inhibited PARP-1 overactivity in peripheral and brain tissue, preventing several of the long-term consequences elicited by perinatal asphyxia, supporting the idea that it constitutes a lead for exploring compounds with similar or better pharmacological profiles.

Magnitudes y unidades para dosimetría a pacientes en radiodiagnóstico e intervencionismo / Quantities and units for patient dosimetry in diagnostic and interventional radiology

Because diagnostic and interventional radiology procedures represent one of the main sources of irradiation by ionizing radiation in the population, it has become a priority to become familiar with the quantities and units that account for patient dosimetry. There are countless documents and international recommendations on names, concepts, definitions and areas of application for various quantities and units used in patient dosimetry, in interventional and diagnostic radiology procedures. However, national legislation is not updated in this regard and does not provide, in any of its documents, an updated glossary that enables finding this type of information quickly and precisely. Therefore, this review paper presents in a didactic way and in plain language, the main quantities and units to be used in the dosimetry of patients undergoing diagnostic and interventional radiology procedures.

Debido a que los procedimientos de radiodiagnóstico e intervencionismo representan una de las principales fuentes de irradiación a la población por radiaciones ionizantes, se vuelve prioritario conocer las magnitudes y unidades que dan cuenta de la dosimetría a los pacientes. Existen innumerables documentos y recomendaciones internacionales sobre nombres, conceptos, definiciones y campos de aplicación para diversas magnitudes y unidades utilizadas en la dosimetría de pacientes en procedimientos de radiodiagnóstico e intervencionismo. Sin embargo, la legislación nacional no se encuentra actualizada en este sentido y no contempla en ninguno de sus documentos, un glosario actualizado que permita encontrar en forma rápida y precisa este tipo de información. Por lo anterior, este trabajo de revisión presenta de manera didáctica y en un lenguaje sencillo, las principales magnitudes y unidades que se deben utilizar en la dosimetría de pacientes sometidos a procedimientos de radiodiagnóstico e intervencionismo.

Signaling pathways involved in neuron-astrocyte adhesion and migration.

Astrocytes in the normal brain possess a stellate shape reflecting their non-migratory properties. Alternatively, in neurodegenerative diseases or after injury, astrocytes become "reactive" in a process known as astrocytosis or reactive gliosis, retract their processes, become polarized and acquire front-to-rear asymmetry typical of migratory cells. On the other hand, neuronal migration is a common process during embryonic development, but only few types of neurons can migrate and differentiate during adult life in the central nervous system. Those that do migrate follow tracks made by glial cells and mainly give rise to interneurons. In vitro, molecular mechanisms involved in adhesion of cells to and migration on extracellular matrix proteins have been widely studied; however, signal transduction pathways explaining how particularly neurons and astrocytes, mutually modulate adhesion and migration are less well known. In this review, we describe and discuss how ligand/receptor interactions in astrocytes and neurons trigger signaling events leading to actin and microtubule reorganization, changes in cell morphology, as well as cell adhesion and migration. The biological significance these cell-cell interactions and signaling events might have in the brain are discussed.

The caveolin-1 connection to cell death and survival.

Caveolins are a family of membrane proteins required for the formation of small plasma membrane invaginations called caveolae that are implicated in cellular trafficking processes. In addition to this structural role, these scaffolding proteins modulate numerous intracellular signaling pathways; often via direct interaction with specific binding partners. Caveolin-1 is particularly well-studied in this respect and has been attributed a large variety of functions. Thus, Caveolin-1 also represents the best-characterized isoform of this family with respect to its participation in cancer. Rather strikingly, available evidence indicates that Caveolin-1 belongs to a select group of proteins that function, depending on the cellular settings, both as tumor suppressor and promoter of cellular traits commonly associated with enhanced malignant behavior, such as metastasis and multi-drug resistance. The mechanisms underlying such ambiguity in Caveolin-1 function constitute an area of great interest. Here, we will focus on discussing how Caveolin-1 modulates cell death and survival pathways and how this may contribute to a better understanding of the ambiguous role this protein plays in cancer.

Severe alterations in expression and localisation of {alpha}6{beta}4 integrin in salivary gland acini from patients with Sjogren syndrome.

OBJECTIVES: In salivary glands from patients with Sjögren syndrome, overexpression of laminins 1 and 5 and disorganisation of the acinar basal lamina have been reported. Laminin 5 mediates association of the basal lamina with epithelial cells by forming adhesion complexes upon interaction with alpha6beta4 integrin. In the present work, mRNA and protein levels of alpha6beta4 integrin were determined and its localisation in salivary glands evaluated in patients with Sjögren syndrome. METHODS: Salivary glands of 12 patients with Sjögren syndrome and 8 controls were studied. The mRNA and protein levels of alpha6beta4 were determined by semiquantitative reverse transcriptase (RT)-PCR and western blot analysis, respectively. The subcellular localisation of alpha6beta4 and laminin were evaluated by confocal microscopy. RESULTS: In patients, no significant differences in alpha6 and beta4 mRNA levels were detected. However, beta4 integrin protein levels were significantly lower, whereas, changes in alpha6, were highly variable. In controls, alpha6beta4 was detected in the basolateral and basal surface of serous and mucous acini, respectively. In patients, alterations in alpha6beta4 distribution were particularly dramatic for acini with strong basal lamina disorganisation. alpha6beta4 was also detected in the cytoplasm and lateral plasma membrane in serous and mucous acini. CONCLUSION: Mild alterations in the basal lamina correlated with lateral redistribution of alpha6beta4 integrin and the formation of new cell-cell adhesions that help maintain acinar organisation and promote cell survival. Conversely, in cases with severe basal lamina alterations, lateral alpha6beta4 redistribution was no longer sufficient to maintain acinar cell survival. Thus, maintenance of equilibrium between cell-cell and cell-basal lamina attachment is required to sustain gland cell survival.

Reduced sulfation of muc5b is linked to xerostomia in patients with Sjögren syndrome.

OBJECTIVES: MUC5B contains sulfated and sialylated oligosaccharides that sequester water required for moisturising the oral mucosa. Xerostomia, in patients with Sjögren syndrome, is generally associated with reduced quantities, rather than altered properties, of saliva. Here, we determined the amount of MUC5B (mRNA and protein) as well as sulfation levels in salivary glands of patients with normal or altered unstimulated salivary flow. Localisation of MUC5B and sulfated MUC5B, as well as total levels sulfated groups were determined and compared with acini basal lamina disorganisation. PATIENTS AND METHODS: In all, 18 patients with normal or altered unstimulated salivary flow and 16 controls were studied. MUC5B mRNA and protein were evaluated in salivary glands by semiquantitative RT-PCR and Western blot analysis. MUC5B sulfation was determined by Western blotting. MUC5B and sulfo-Lewis(a) antigen localisation were assessed by immunohistochemistry. The total amount of sulfated oligosaccharides was determined microdensitometrically. RESULTS: No significant differences were detected in MUC5B mRNA and protein levels between controls and patients, while sulfo-Lewis(a) antigen levels were lower in patients. The number of sulfo-Lewis(a) positive mucous acini was reduced in patients but no correlation was observed between lower levels of sulfation and unstimulated salivary flow. Microdensitometric data confirmed the presence of reduced sulfated oligosaccharides levels in mucous acini from patients with highly disorganised basal lamina. CONCLUSION: Disorganisation of the basal lamina observed in patients with Sjögren syndrome may lead to dedifferentiation of acinar mucous cells and, as a consequence, alter sulfation of MUC5B. These changes are suggested to represent a novel mechanism that may explain xerostomia in these patients.

Aplicación de un protocolo de levantamiento radiométrico en instalaciones de radiodiagnóstico / Application of a radiometric inventory protocol in radiodiagnostics facilities

A radiometric study was carried out in two Radiodiagnostic Units, with seven rooms being assessed. Methodology of the Radiodiagnostics Quality Control Protocol ARCAL XLIX (Cooperation Agreement for the Promotion of Nuclear Science and Technology in Latin America and the Caribbean) of the International Atomic Energy Agency (IAEA) was used. From radiation protection viewpoint, effective dose rates on surface for different relevant positions were calculated.The eigthy-five percent (85 percent) of assessed positions showed effective dose rates within limit values established in the ARCAL XLIX Protocol. Nevertheless, occupationally-exposed personnel (OEP) placed at position A (controlled area) -one meter distant from the phantom- was found to be exceeding by 7 the ARCAL XLIX advised values.

Se realizó un Levantamiento Radiométrico en dos servicios de imaginología con un total de 7 salas de radiodiagnóstico evaluadas. Se utilizó metodología del Protocolo de Control de Calidad en Radiodiagnóstico ARCAL (Acuerdo de Cooperación Regional para la promoción de la ciencia nuclear y tecnología en América Latina y el Caribe) XLIX del Organismo Internacional de Energía Atómica (OIEA). Se calcularon las tasas de dosis efectiva en superficie en diferentes posiciones de interés desde el punto de vista de la protección radiológica. El 85 por ciento de las posiciones evaluadas presentan tasas de dosis efectivas que cumplen con los valores límites establecidos en el protocolo ARCAL XLIX. No obstante el personal ocupacionalmente expuesto (POE) ubicado a un metro del simulador (área controlada) supera hasta en un factor 7 el límite propuesto en ARCAL XLIX.

Garantía de calidad y protección radiológica en las exposiciones médicas en Europa: un ejemplo a seguir / Quality assurance and radiation protection in medical exposures in europe: a role model

Medical exposures constitute the major source of ionizing radiation to which the world population is exposed to. Due to this fact, the European Community has implemented a number of agreements aimed at regulating these activities. For its part, Spain - through a series of Royal Ordinances - has developed a legal framework to broadly address not only Radiation Protection but medical exposure quality criteria as well. These regulations may be used as reference criteria to addressing same issues in our country.

Las exposiciones médicas constituyen la principal fuente de exposición a radiaciones ionizantes a la población mundial. Por esta razón, la Comunidad Europea ha desarrollado una serie de acuerdos, que tienen como objetivo regularizar estas actividades. En el mismo sentido España, a través de una serie de Decretos Reales estructuró un marco legal que considera aspectos amplios de Protección Radiológica, como así también criterios de calidad de las exposiciones médicas, las cuales pueden ser usadas como referencia para desarrollar estos temas en nuestro país.

Casein kinase 2 (CK2) increases survivin expression via enhanced beta-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription.

Increased expression of casein kinase 2 (CK2) is associated with hyperproliferation and suppression of apoptosis in cancer. Mutations in the tumor suppressor APC (adenomatous polyposis coli) are frequent in colon cancer and often augment beta-catenin-T cell factor (Tcf)/lymphoid enhancer binding factor (Lef)-dependent transcription of genes such as c-myc and cyclin-D1. CK2 has also been implicated recently in the regulation of beta-catenin stability. To identify mechanisms by which CK2 promotes survival, effects of the specific CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole were assessed. TBB and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole significantly decreased proliferation and increased apoptosis of HT29(US) colon cancer cells. RT-PCR and immunoblot analysis revealed that both inhibitors decreased survivin mRNA and protein levels in HT29(US) cells. Similar effects were observed with TBB in human DLD-1 and SW-480 colorectal cells as well as ZR-75 breast cancer cells and HEK-293T embryonic kidney cells. Expression of GFP-CK2alpha in HEK-293T cells resulted in beta-catenin-Tcf/Lef-dependent up-regulation of survivin and increased resistance to anticancer drugs. Augmented beta-catenin-Tcf/Lef-dependent transcription and resistance to apoptosis observed upon GFP-CK2alpha expression were abolished by TBB. Alternatively, HEK-293T cells expressing GFP-survivin were resistant to TBB-induced apoptosis. Finally, siRNA-mediated down-regulation of CK2alpha in HEK-293T cells coincided with reduced beta-catenin and survivin levels. Taken together, these results suggest that CK2 kinase activity promotes survival by increasing survivin expression via beta-catenin-Tcf/Lef-mediated transcription. Hence, selective CK2 inhibition or down-regulation in tumors may provide an attractive opportunity for the development of novel cancer therapies.

Basal lamina disorganisation of the acini and ducts of labial salivary glands from patients with Sjogren's syndrome: association with mononuclear cell infiltration.

OBJECTIVE: To study the expression of laminin and type IV collagen as biomarkers of the organisation of the basal lamina of acini and ducts in labial salivary glands from patients with Sjögren's syndrome, and to relate this organisation to inflammatory cell invasion of acini and ducts. METHODS: Immunohistochemistry for laminin and type IV collagen was undertaken on sections of labial salivary glands from 30 patients with Sjögren's syndrome, 10 control subjects, and 24 controls with chronic sialoadenitis. Immunohistochemistry reaction, alterations to cell morphology, and the presence of inflammatory cells in acini and ducts were evaluated and scored using a semiquantitative method. RESULTS: Changes in the expression of laminin and type IV collagen in the basal lamina of acini and ducts of labial salivary glands from patients with Sjögren's syndrome were more pronounced than in labial salivary glands from control groups. A remarkable characteristic was the disorganisation of the basal lamina in the labial salivary glands in Sjögren's syndrome. The pattern of immunoreactivity of the basal lamina of other structures (for example, blood vessels) did not change. In Sjögren's syndrome, invasion of cytotoxic T lymphocytes was only observed in acini and ducts which had a disorganised basal lamina. CONCLUSIONS: The high state of disorganisation of the basal lamina of acini and ducts could allow invasion of cytotoxic T lymphocytes in Sjögren's syndrome, contributing to cell death and ductal hyperplasia.

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites.

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.

Radiación ionizante secundaria generada en equipos de cineangiografía coronaria digital y analógica: influencia de los sistemas externos de protección radiológica / Secondary ionizing radiation generated by analog and digital coronary cineangiographic equipment: influence of external protection devices

Background: Exposure to ionizing radiation is a known hazard of radiological procedures. Aim: To compare the emission of secondary ionizing radiation from two coronary angiographic equipments, one with digital and the other with analog image generation. To evaluate the effectiveness of external radiological protection devices. Material and methods: Environmental and fluoroscopy generated radiation in the cephalic region of the patient was measured during diagnostic coronary angiographies. Ionizing radiation generated in anterior left oblique projection (ALO) and in anterior right oblique projection (ARO) were measured with and without leaded protections. In 19 patients (group 1), a digital equipment was used and in 21 (group 2), an analog equipment. Results: Header radiation for groups 1 and 2 was 1194 ñ 337 and 364 ñ 222 µGray/h respectively (p<0.001). During fluoroscopy and with leaded protection generated radiation for groups 1 and 2 was 612 ñ 947 and 70 ñ 61 µGray/h respectively (p<0.001). For ALO projection, generated radiation for groups 1 and 2 was 105 ñ 47 and 71 ñ 192 µGray/h respectively (p<0.001). During filming the radiation for ALO projection for groups 1 and 2 was 7252 ñ 9569 and 1671 ñ 2038 µGray/h respectively (p = 0.03). Out of the protection zone, registered radiation during fluoroscopy for groups 1 and 2 was 2800 ñ 1741 and 1318 ñ 954 µGray/h respectively (p < 0.001); during filming, the figures were 15500 ñ 5840 and 18961 ñ 10599 µGray/h respectively (NS). Conclusions: Digital radiological equipment has a lower level of ionizing radiation emission than the analog equipment

Thy-1/CD3 coengagement promotes TCR signaling and enhances particularly tyrosine phosphorylation of the raft molecule LAT.

Clustering of the glycosyl-phosphatidylinositol (GPI)-anchored protein Thy-1 on the cell surface leads to T cell activation. However, despite the similarity to TCR-mediated events, cell signaling triggered by Thy-1 crosslinking, reportedly occurs in a manner independent of the TCR/CD3 complex. To investigate the relationship between responses resulting from Thy-1 or TCR engagement, a biochemically well defined system employing only affinity purified antibodies was used to crosslink these surface molecules and activation was assessed by monitoring tyrosine phosphorylation, intracellular calcium influx and IL-2 production. By these criteria, anti-CD3 mAbs moderately activated EL-4 thymoma or 2B4 hybridoma cell lines, while costimulation with anti-Thy-1-mAb strongly enhanced TCR signaling. Furthermore, a Thy-1 loss mutant cell line, did not respond to stimulation through CD3 despite expressing all essential signaling molecules. Together these results emphasized the existence of a poorly appreciated mutual interdependence between Thy-1 and CD3 for efficient cellular signaling. Thy-1/CD3-mediated activation enhanced mostly tyrosine phosphorylation of a 40 kDa protein which was identified as a transmembrane protein lacking N-linked oligosaccharides. These biochemical properties are identical to those described for a recently cloned adaptor protein called 'Linker for Activation of T cells' (LAT). Indeed, polyclonal Abs raised against a LAT-peptide (amino acids 103-131) specifically recognized the 40 kDa protein. LAT is present in microdomains of the plasma membrane enriched in sphingolipids, cholesterol, GPI-anchored proteins and a variety of signaling molecules. By contrast, the TCR/CD3 complex is excluded from these domains at least until stimulation takes place. Hence, we propose that Thy-1 promotes TCR/CD3 dependent signaling by facilitating LAT phosphorylation on tyrosine and the subsequent recruitment of downstream effector molecules.

LL95 monoclonal antibody mimics functional effects of ZP3 on mouse sperm: evidence that the antigen recognized is not hexokinase.

In the mouse, a 95 kD sperm protein has been identified as a putative receptor for the zona pellucida glycoprotein ZP3. The 95 kD sperm protein is a tyrosine kinase substrate, with phosphorylation on tyrosine stimulated upon zona pellucida binding. The latter finding is observed not only in live cells but also in isolated sperm membranes and in an electroeluted 95 kD protein. Stimulation of 95 kD protein tyrosine phosphorylation by zona pellucida is completely abolished by tyrosine kinase inhibitors, which effectively inhibit the sperm acrosome reaction. Since receptor oligomerization by ZP3 is essential for acrosome reaction triggering, we hypothesized that application of an external crosslinking agent will lead to the acrosome reaction, even in the absence of natural ligand ZP3. Here, we report the generation of a mouse monoclonal antibody (mAb) raised against the 95 kD protein. This antibody, termed LL95, mimics the bioactivities of ZP3 in inhibiting sperm-zona binding and inducing the acrosome reaction. The latter depends on receptor oligomerization. Immunolocalization revealed that the LL95 antigen is restricted to the head surface in the acrosomal region of live sperm. Thus, LL95 fulfills several criteria predicted for an antibody that recognizes a sperm receptor for the zona pellucida. Recently, it was reported that the amino acid sequence of the 95 kD protein we described corresponds to a mouse hepatoma hexokinase (Kalab et al., 1994: J Biol Chem 269:3810-3817). Although both hexokinase and LL95 antigen migrate at 95 kD in nonreducing gels, we show here that LL95 does not recognize hexokinase. Identification of different proteins is clear where hexokinase is a 116 kD protein and LL95 recognizes sperm proteins of 110 and 130 kD. Moreover, mAb anti-phosphotyrosine immunoprecipitates LL95 antigen under conditions where hexokinase is absent. Use of anti-hexokinase antibodies in gamete interaction assays failed to demonstrate any effect on either sperm-zona binding or acrosome reaction triggering. Finally, antihexokinase antibodies bind to a sperm tail antigen, thus direct involvement of hexokinase in gamete interaction seems improbable.

Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space.

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization--by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin--of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the antiacrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa.

Regulation of mouse gamete interaction by a sperm tyrosine kinase.

A 95-kDa mouse sperm protein has been previously identified as a putative receptor involved in the sperm-egg interactions that lead to fertilization. The ligand for this receptor is the zona pellucida glycoprotein ZP3. This constituent of the oocyte-specific extracellular matrix mediates not only sperm binding to the zona but also triggers acrosomal exocytosis. The latter, also termed the acrosome reaction, is a key regulatory event upon which fertilization is absolutely dependent. Previously, we showed that the 95-kDa protein that binds ZP3 is a substrate for tyrosine kinase, and its phosphotyrosine content increases after sperm-zona pellucida binding. Here, we show the presence of protein tyrosine kinase activity in sperm plasma membranes and in electroeluted 95-kDa protein. The tyrosine kinase activity of the isolated protein is stimulated by solubilized zona pellucida and inhibited by tyrphostin RG-50864, a membrane-permeable tyrosine kinase inhibitor. Furthermore, tyrphostin inhibits zona-triggered acrosomal exocytosis in a dose-dependent manner. These findings indicate that the 95-kDa protein participates in a critical regulatory event of gamete interaction; moreover, our experiments suggest that sperm protein tyrosine kinase may be an excellent target for the control of fertility.