[Effect of the presence of student-assistants on the quality of care perception by ambulatory patients]. / Medición de la calidad del trato al usuario tras la introducción del asistente-alumno en centros de salud familiar en Chile.

BACKGROUND: The time dedicated to patients and how they are treated are crucial in the evaluation of health care quality. Medical students acting as medical assistants could improve the perception of a good quality of care among ambulatory patients. AIM: To evaluate if the presence of Student-Assistants improves the patients' perception of health care quality in ambulatory primary care. PATIENTS AND METHODS: Quasi-experimental exploratory study. In two health care centers, patients answered a questionnaire about their perception of how they were treated at baseline and after an intervention period. In one center, prior to the appointment of the patient with the doctor, the student interviewed patients focusing on chief complaints and registered their vital signs, orally presenting this information to the clinician. In the other center, there was no student intervention. Patients answered the questionnaire at the end of appointments. RESULTS: At baseline 103 patients answered the questionnaire (58 in the experimental and 45 in the control center). After the intervention, 121 patients answered it (56 in the experimental and 65 in the control center). Basal scores were 6,25 and 6,06 in experimental and control center, respectively (p = NS). After the intervention, the scores were 6,49 and 6,15, respectively (p = 0,01). CONCLUSIONS: These data support the hypothesis that the presence of a Student-Assistant could improve the perception of patients about how they are treated at primary health care centers.

Medición de la calidad del trato al usuario tras la introducción del asistente-alumno en centros de salud familiar en Chile / Effect of the presence of student-assistants on the quality of care perception by ambulatory patients

Background: The time dedicated to patients and how they are treated are crucial in the evaluation of health care quality. Medical students acting as medical assistants could improve the perception of a good quality of care among ambulatory patients. Aim: To evaluate if the presence of Student-Assistants improves the patients' perception of health care quality in ambulatory primary care. Patients and Methods: Quasi-experimental exploratory study. In two health care centers, patients answered a questionnaire about their perception of how they were treated at baseline and after an intervention period. In one center, prior to the appointment of the patient with the doctor, the student interviewed patients focusing on chief complaints and registered their vital signs, orally presenting this information to the clinician. In the other center, there was no student intervention. Patients answered the questionnaire at the end of appointments. Results: At baseline 103 patients answered the questionnaire (58 in the experimental and 45 in the control center). After the intervention, 121 patients answered it (56 in the experimental and 65 in the control center). Basal scores were 6,25 and 6,06 in experimental and control center, respectively (p = NS). After the intervention, the scores were 6,49 and 6,15, respectively (p = 0,01). Conclusions: These data support the hypothesis that the presence of a Student-Assistant could improve the perception of patients about how they are treated at primary health care centers.

Electroantennogram responses of the Triatoma dimidiata complex to volatiles produced by its exocrine glands.

Members of the Triatoma dimidiata complex are vectors of the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease. Morphological and genetic studies indicate that T. dimidiata complex has three principal haplogroups in Mexico. However, whether there are differences in the olfactory physiology among the haplogroups of this complex and a possible correlation with their antennal phenotype are not yet known. Antennal responses to 13 compounds released from the metasternal and Brindley´s glands, which are involved in the alarm and mating-related behaviours of T. dimidiata were investigated using electroantennography (EAG). Overall, of the 13 compounds tested, seven triggered EAG responses in both sexes of three Mexican haplogroups. The sensitivity of the EAG responses show some relationship with the total number of chemo-sensilla present on the antennae. Antennal sensitivity was different between sexes and haplogroups of the T. dimidiata complex. Discriminant analysis of EAG sensitivity was significant, separating the three haplogroups. Our finding is consistent with morphological and genetic evidence for haplogroups distinction within the complex.

Intracellular calcium movements of boar spermatozoa during 'in vitro' capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model.

This work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca(2+) signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca(2+) labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca(2+) signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca(2+) . The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca(2+) -induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

The Wnt1 ligand/Frizzled 3 receptor system plays a regulatory role in the achievement of the 'in vitro' capacitation and subsequent 'in vitro' acrosome exocytosis of porcine spermatozoa.

The aim of this work was to determine the existence of a functional Wnt/ß-catenin signaling pathway in boar spermatozoa, which would be linked with the already well-known GSK-3 signaling pathway. This was first confirmed by detecting the presence of the specific Frizzled 3 receptor in these cells. Furthermore, this signaling pathway was activated in boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome exocytosis (IVAE) by incubating cells with separate concentrations of Wnt1 ligand, the Wnt/ß-catenin signaling pathway-specific effector. Incubation with the Wnt1 ligand increased the rhythm of the time-dependent reduction in sperm viability during the achievement of both IVC and IVAE. This finding was concomitant with an increase in the percentage of spermatozoa with altered membrane fluidity and permeability determined through both merocyanine-540 and YO-PRO-1 stains. While the Wnt1 ligand did not affect total sperm motility during the achievement of the IVC, it induced a fast and transient increase in the overall motility patterns in spermatozoa subjected to IVAE. This IVAE-linked action was related to a decrease in the percentage of cells with high mitochondrial membrane potential and an increase in the percentage of cells with high intracellular Fluo-3-marked Ca(2+) content. In conclusion, our results suggest that the Wnt1 ligand-modulated Wnt/ß-catenin signaling pathway plays a relevant role in the modulation of both IVC and subsequent, progesterone-induced IVAE. Furthermore, our data indicate that the transduction pathways by which the Wnt1 ligand acts on IVC and IVAE are different, and that the Wnt/ß-catenin pathway is independent from GSK-3 activity in the achievement of IVC.

Morphology and physiology of the olfactory system of blood-feeding insects.

Several blood-feeding (hematophagous) insects are vectors of a number of diseases including dengue, Chagas disease and leishmaniasis which persistently affect public health throughout Latin America. The vectors of those diseases include mosquitoes, triatomine bugs and sandflies. As vector control is an efficient way to prevent these illnesses it is important to understand the sensory biology of those harmful insects. We study the physiology of the olfactory system of those insects and apply that knowledge on the development of methods to manipulate their behavior. Here we review some of the latest information on insect olfaction with emphasis on hematophagous insects. The insect olfactory sensory neurons are housed inside hair-like organs called sensilla which are mainly distributed on the antenna and mouthparts. The identity of many of the odor compounds that those neurons detect are already known in hematophagous insects. They include several constituents of host (vertebrate) odor, sex, aggregation and alarm pheromones, and compounds related to egg-deposition behavior. Recent work has contributed significant knowledge on how odor information is processed in the insect first odor-processing center in the brain, the antennal lobe. The quality, quantity, and temporal features of the odor stimuli are encoded by the neural networks of the antennal lobe. Information regarding odor mixtures is also encoded. While natural mixtures evoke strong responses, synthetic mixtures that deviate from their natural counterparts in terms of key constituents or proportions of those constituents evoke weaker responses. The processing of olfactory information is largely unexplored in hematophagous insects. However, many aspects of their olfactory behavior are known. As in other insects, responses to relevant single odor compounds are weak while natural mixtures evoke strong responses. Future challenges include studying how information about odor mixtures is processed in their brain. This could help develop highly attractive synthetic odor blends to lure them into traps.

Volatile compounds emitted by Triatoma dimidiata, a vector of Chagas disease: chemical analysis and behavioural evaluation.

In this study, we evaluated the responses of Triatoma dimidiata Latreille (Hemiptera: Reduviidae) to volatiles emitted by conspecific females, males, mating pairs and metasternal gland (MG) extracts with a Y-tube olfactometer. The volatile compounds released by mating pairs and MGs of T. dimidiata were identified using solid-phase microextraction and coupled gas chromatography-mass spectrometry (GC-MS). Females were not attracted to volatiles emitted by males or MG extracts; however, they preferred clean air to their own volatiles or those from mating pairs. Males were attracted to volatiles emitted by males, females, mating pairs, pairs in which the male had the MG orifices occluded or MG extracts of both sexes. However, males were not attracted to volatiles emitted by pairs in which the female had the MG orifices occluded. The chemical analyses showed that 14 and 15 compounds were detected in the headspace of mating pairs and MG, respectively. Most of the compounds identified from MG except for isobutyric acid were also detected in the headspace of mating pairs. Both females and males were attracted to octanal and 6-methyl-5-hepten-2-one, and males were attracted to 3,5-dimethyl-2-hexanol. Males but not females were attracted to a seven-compound blend, formulated from compounds identified in attractive MG extracts.

'In vitro' capacitation and further 'in vitro' progesterone-induced acrosome exocytosis are linked to specific changes in the expression and acrosome location of protein phosphorylation in serine residues of boar spermatozoa.

The aim of this study is to determine changes in the expression and location of protein serine phosphorylation (pSer) during 'in vitro' capacitation (IVC) and 'in vitro' acrosome exocytosis (IVAE) in boar spermatozoa. This was performed in both mono- and bi-dimensional analyses of protein expression through Western blot, as well as through immunocytochemistry. Furthermore, IVC was induced through incubation in an IVC medium, and afterwards, progesterone-induced IVAE was performed. The mono-dimensional Western blot analysis showed the presence of a predominant pSer band of approximately 70-75 kDa, which was accompanied by fainter bands, especially three with molecular weights of approximately 50, 35 and 32 kDa. Neither IVC nor IVAE significantly modified this pattern. Bi-dimensional analyses showed a more complex pattern, with at least five protein clusters. The attainment of IVC caused the disappearance of the proteins with the highest molecular weight concomitantly with the appearance of pSer proteins of 75-kDa/pI 9.5 and 80-kDa/pI 10. The induction of IVAE caused the appearance of new pSer proteins of a 75-kDa/pI 6.5-7.5 and 75-kDa/pI 10. Immunocytochemistry showed that the main pSer expression in boar expression before the attainment of IVC was located at the midpiece. The IVC induced the appearance of acrosomal pSer, which was greatly increased during IVAE. Our results indicate that the changes in serine protein phosphorylation associated with IVC and IVAE comprise not only the appearance of specific phosphorylated proteins, such as the pSer-75 kDa, but also changes in pI and displacements in the sperm location of phosphorylated proteins, like the specific acrosomal pSer signal induced during IVC.

'In vitro' capacitation and acrosome reaction are concomitant with specific changes in mitochondrial activity in boar sperm: evidence for a nucleated mitochondrial activation and for the existence of a capacitation-sensitive subpopulational structure.

The main scope of this manuscript is to analyse the dynamics of mitochondrial activity in boar sperm subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome reaction (IVAR). This was determined after analysis of the rhythm of O(2) consumption and concomitant changes in the mitochondria activity-specific JC-1 staining. Results showed that IVC, and especially IVAR, was concomitant with a peak in O(2) consumption (from 1.61 ± 0.08 nmol O(2)/min/10(7) viable sperm at 0 h of incubation to 2.62 ± 0.12 nmol O(2) /min/10(7) viable sperm after 5 min of IVAR induction). These results were accompanied by parallel changes in the mean intensity of JC-1 staining. Based on JC-1, mitochondrial activation followed a nucleated pattern, with specific, activation starting points at the midpiece from which mitochondrial activation was spread. Moreover, four separate sperm subpopulations were detected following the JC-1 orange-red/green ratio, and the observed changes in the mean JC-1 staining during IVC and IVAR were related to concomitant changes in both the orange-red/green JC-1 ratio and the percentage of sperm included in each subpopulation. All of these results indicate that IVC and the first minutes of IVAR are accompanied by a progressive increase in mitochondrial activity, which reached a peak coincidental with the achievement of IVAR. Moreover, results suggest the presence of separate sperm subpopulations, which show a different mitochondrial sensitivity to IVC and IVAR. Finally, mitochondrial activation, at least under JC-1 staining, seems to originate in concrete nucleation points at the midpiece, thus suggesting thus a well-coordinated pattern in boar-sperm mitochondrial activity modulation.

IL-3 controls tau modifications and protects cortical neurons from neurodegeneration.

Interleukin-3 (IL-3) regulates the proliferation, survival and differentiation of haematopoietic cells via interaction with specific cell-surface receptors. IL-3 is expressed in several non-hematopoietic cell types. Studies have demonstrated the presence of IL-3 in the central nervous system, however, its physiological role in these cells is poorly understood. Previously we have been demonstrated that IL-3 prevents neuronal death induced by fibrillary ß amyloid in these cells, by PI 3-kinase and Jak/STAT pathway activation. In this study, we demonstrated that IL-3 significantly reduced Aß-promoted neurite degeneration and toxicity. Thus, this cytokine provides cellular protection against Aß neurotoxicity in primary cortical neuronal cells, by modulating microtubular dynamics and prevention of tau cleavage and hyperphosphorylation. We also demonstrates that IL-3 is expressed in the "in vivo" mouse model of AD, Tg2576, which also expresses human AßPP with the Swedish mutation. In summary, these results suggest that IL-3 could play a neuroprotective role in AD.

Rol de la analgesia y anestesia epidural en cirugía abdominal / The role of epidural anesthesia and analgesia in abdominal surgery

Desde 1980 que la anestesia peridural (AP) se ha propuesto para manejar el dolor postoperatorio, en especial en cirugías abdominales. A pesar de que ésta es percibida por varios autores, como la analgesia ideal para las cirugías abdominales mayores, hay algunos que prefieren la administración de antiinflamatorios no esteroidales o de opiodes por vía periférica, ya sea en bolos, infusión continua o controlados por el paciente (PCA). Si bien esta última provee mejor analgesia y satisfacción que la administración convencional, no ha demostrado mejorar la morbimortalidad quirúrgica, cosa que sí lo ha hecho la AP. Se realiza una revisión de la literatura con el objetivo de analizar los efectos benéficos y adversos de la anestesia peridural en los pacientes sometidos a una cirugía mayor abdominal. Se concluye que el uso de anestesia epidural intraoperatoria y postoperatoria está asociado a una disminución de la incidencia, severidad de las alteraciones fisiológicas perioperatorias y morbilidad postoperatoria.

Dynamics of motile-sperm subpopulation structure in boar ejaculates subjected to "in vitro" capacitation and further "in vitro" acrosome reaction.

Incubation of diluted boar sperm from fresh ejaculates in a previously established "in vitro" capacitation medium induced a significant, time-dependent increase in several mean parameters of sperm motility, such as curvilinear velocity (VCL), linear velocity (VSL), mean velocity (VAP), linearity coefficient (LIN), straightness coefficient (STR) and wobble coefficient (WOB). Furthermore, motile boar-sperm semen samples were structured in four definite subpopulations. Subpopulation 1 showed the lowest values of VCL, VSL and VAP and also low values of linearity. Subpopulation 2 showed the second lowest values of VCL and VAP and higher values of LIN and STR. Subpopulation 3 was characterized by high values of velocity and low values of linearity. Finally, Subpopulation 4 was characterized by high values of velocity and linearity. "In vitro" capacitation and further acrosome reaction induced changes in the motility characteristics of each subpopulation as well as in their percentage distribution, Subpopulations 3 and 4 being those that showed the most significant changes. However, despite these changes, the observed, overall four-subpopulation structure was firmly maintained during the entire "in vitro" capacitation and acrosome-reaction process. Our results suggest that capacitation-induced motility changes are related to specific changes in the percentage of each motile-sperm subpopulation in the ejaculate without losing the overall, specific four-subpopulation structure. In this way, the maintenance of a four-subpopulation structure seems to be important in the control of the whole ejaculate physiology.

Adenosine A(2B) receptor mediates an increase on VEGF-A production in rat kidney glomeruli.

Up-regulation of the glomerular expression and the activity of vascular endothelial growth factor-A (VEGF) have been identified as an early pathogenic event for the progression of diabetic nephropathy. Currently, however the mediators are not yet clearly recognized. In this study we identified all four adenosine receptor (AR) subtypes, i.e. A(1), A(2A), A(2B) and A(3) in isolated rat kidney glomeruli. We localized the expression of A(2B)AR in podocytes, the primary VEGF producing cells. The ex vivo treatment of kidney glomeruli with adenosine or a general AR agonist NECA, increases VEGF protein content. In addition, NECA treatment elicits VEGF release. These effects were blocked by the A(2B)AR selective antagonist MRS1754 supplementation. Furthermore, we showed that A(2B)AR activation was necessary to promote a higher expression of VEGF in kidney glomeruli upon exposure to high d-glucose concentration, a pathogenic condition like those observed in diabetic nephropathy.

Expression of the GM-CSF receptor in ovine spermatozoa: GM-CSF effect on sperm viability and motility of sperm subpopulations after the freezing-thawing process.

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine capable of stimulating proliferation, maturation and function of haematopoietic cells. Receptors for this cytokine are composed of two subunits, alpha and beta, and are expressed in myeloid progenitors and mature mononuclear phagocytes, monocytes, eosinophils and neutrophils, as well as in other non-haematopoietic cells. We have previously demonstrated that bull spermatozoa express functional GM-CSF receptors that signal for increased glucose and vitamin-C uptake and enhance several parameters of sperm motility in the presence of glucose or fructose substrates. In this study, we have analyzed the expression of GM-CSF receptors in ovine spermatozoa and studied the effect of GM-CSF on sperm viability and motility after the freezing-thawing process. Immunolocalization and immunoblotting analyses demonstrated that ovine spermatozoa (Xisqueta race) expressed GM-CSF receptors. In addition, GM-CSF partially counteracted the impairing action of freezing/thawing on the percentage of total motility, as well as on the specific motility patterns of each of the separate, motile sperm subpopulations of ram ejaculates subjected to this protocol. These results suggest that GM-CSF can play a role in the resistance of ram spermatozoa to environmental thermal stress.

Gluconeogenesis-linked glycogen metabolism is important in the achievement of in vitro capacitation of dog spermatozoa in a medium without glucose.

In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [(14)C]glycogen after the addition to l-CCM with [(14)C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

The protein kinase Cdk5. Structural aspects, roles in neurogenesis and involvement in Alzheimer's pathology.

A set of different protein kinases have been involved in tau phosphorylations, including glycogen synthase kinase 3beta (GSK3 beta), MARK kinase, MAP kinase, the cyclin-dependent kinase 5 (Cdk5) system and others. The latter system include the catalytic component Cdk5 and the regulatory proteins p35, p25 and p39. Cdk5 and its neuron-specific activator p35 are essential molecules for neuronal migration and for the laminar configuration of the cerebral cortex. Recent evidence that the Cdk5/p35 complex concentrates at the leading edge of axonal growth cones, together with the involvement of this system in the phosphorylation of neuronal microtubule-asociated proteins (MAPs), provide further support to the role of this protein kinase in regulating axonal extension in developing brain neurons. Although the aminoacid sequence of p35 has little similarity with those of normal cyclins, studies have shown that its activation domain may adopt a conformation of the cyclin-folded structure. The computed structure for Cdk5 is compatible with experimental data obtained from studies on the Cdk5/p35 complex, and has allowed predictions on the protein interacting domains. This enzyme exhibits a wide cell distribution, even though a regulated Cdk5 activity has been shown only in neuronal cells. Cdk5 has been characterized as a proline-directed Ser/Thr protein kinase, that contributes to phosphorylation of human tau on Ser202, Thr205, Ser235 and Ser404. Cdk5 is active in postmitiotic neurons, and it has been implicated in cytoskeleton assembly and its organization during axonal growth. In addition to tau and other MAPs, Cdk5 phosphorylates the high molecular weight neurofilament proteins at their C-terminal domain. Moreover, nestin, a protein that regulates cytoskeleton organization of neuronal and muscular cells during development of early embryos, and several other regulatory proteins appear to be substrates of Cdk5 and are phosphorylated by this kinase. Studies also suggest, that in addition to Cdk5 involvement in neuronal differentiation, its activity is induced during myogenesis, however, the mechanisms of how this activity is regulated during muscular differentiation has not yet been elucidated. Recent studies have shown that the beta-amyloid peptide (A beta) induces a deregulation of Cdk5 in cultured brain cells, and raises the question on the possible roles of this tau-phosphorylating protein kinase in the sequence of molecular events leading to neuronal death triggered by A beta. In this context, there are evidence that Cdk5 is involved in tau hyperphosphorylation promoted by A beta in its fibrillary form. Cdk5 inhibitors protect hippocampal neurons against both tau anomalous phosphorylations and neuronal death. The links between the studies on the Cdk5/p35 system in normal neurogenesis and its claimed participation in neurodegeneration, provide the framework to understand the regulatory relevance of this kinase system, and changes in its regulation that may be implicated in disturbances such as those occurring in Alzheimer disease.

Expression of GM-CSF receptors in male germ cells and their role in signaling for increased glucose and vitamin C transport.

We studied the expression and function of the granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in male germ cells. RT-PCR showed expression of mRNAs encoding the alpha- and beta-subunits of the GM-CSF receptor in human testis, and the presence of the alpha- and beta-proteins was confirmed by immunoblotting with anti-alpha and anti-beta-antibodies. Immunolocalization studies showed the level of expression of GM-CSF alpha- and beta-subunits in the germ line in the testis and in ejaculated spermatozoa. Receptor binding studies using radiolabeled GM-CSF revealed that bull spermatozoa have about 105 high-affinity sites with a K(d) of 222 pM and approximately 1100 low-affinity sites with a K(d) of 10 nM. GM-CSF signaled, in a time- and dose-dependent manner, for an increased uptake of glucose and vitamin C.

C-erbB-2 expression and its relationship with ploidy, p53 abnormalities and epidermal growth factor receptor content in human non-small cell lung cancer.

This study attempts to clarify the role of c-erbB-2 overexpression in human non-small cell lung cancer (NSCLC) and relate it with the p53 alterations, DNA index (D.I.) and epidermal growth factor receptor (EGFR) content in sixty four patients with NSCLC. c-erbB-2 and EGFR quantification were carried out from tissue homogenates using quantitative ELISA procedures. p53 alterations were determined by immunohistochemical (IHC) detection with the monoclonal antibody DO-7 and analysis for p53 mutations on exons 4 to 8 by single strand conformation polymorphism (SSCP). The D.I. was performed by flow cytometry. c-erbB-2 hyperexpression was found in 13 of 58 LC (22%), and it was closely associated with hyperdiploid tumors (D.I. >1.3; P = 0.00). The p53 abnormalities detected by SSCP were statistically more frequent in hyperdiploid tumors (16/25; P = 0.015) than in diploid ones (8/30). No relationship between the results of IHC p53 and SSCP was found. The patients with c-erbB-2 hyperexpressing tumors were prone to have frequent relapses (P = 0.03), although the patients with hyperdiploid NSCLC are the ones with the highest relapse rate (P = 0.008). From the results obtained in this study the following conclusions can be drawn: (a) c-erbB-2 hyperexpressing NSCLC are associated with abnormalities in other biological markers and with a greater rate of relapses; (b) SSCP seemed to be more specific that IHC to detect p53 molecular abnormalities; and (c) the D.I. is the parameter more tightly related with relapse.

Hexose transporter expression and function in mammalian spermatozoa: cellular localization and transport of hexoses and vitamin C.

We analyzed the expression of hexose transporters in human testis and in human, rat, and bull spermatozoa and studied the uptake of hexoses and vitamin C in bull spermatozoa. Immunocytochemical and reverse transcription-polymerase chain reaction analyses demonstrated that adult human testis expressed the hexose transporters GLUT1, GLUT2, GLUT3, GLUT4, and GLUT5. Immunoblotting experiments demonstrated the presence of proteins of about 50-70 kD reactive with anti-GLUT1, GLUT2, GLUT3, and GLUT5 in membranes prepared from human spermatozoa, but no proteins reactive with GLUT4 antibodies were detected. Immunolocalization experiments confirmed the presence of GLUT1, GLUT2, GLUT3, GLUT5, and low levels of GLUT4 in human, rat, and bull spermatozoa. Each transporter isoform showed a typical subcellular localization in the head and the sperm tail. In the tail, GLUT3 and GLUT5 were present at the level of the middle piece in the three species examined, GLUT1 was present in the principal piece, and the localization of GLUT2 differed according of the species examined. Bull spermatozoa transported deoxyglucose, fructose, and the oxidized form of vitamin C, dehydroascorbic acid. Transport of deoxyglucose and dehydroascorbic acid was inhibited by cytochalasin B, indicating the direct participation of facilitative hexose transporters in the transport of both substrates by bull spermatozoa. Transport of fructose was not affected by cytochalasin B, which is consistent for an important role for GLUT5 in the transport of fructose in these cells. The data show that human, rat, and bull spermatozoa express several hexose transporter isoforms that allow for the efficient uptake of glucose, fructose, and dehydroascorbic acid by these cells.