Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.     

Chicks from a high and low feather pecking line of laying hens differ in apomorphine sensitivity

Proactive rodents show a larger behavioral response to apomorphine (APO) than reactive copers, suggesting a more sensitive DA system in proactive individuals. Previously, chicks from a high feather pecking (HFP) and low feather pecking line (LFP) have been suggested to display a proactive and reactive coping strategy, respectively. Therefore, at approximately 4 weeks of age, the behavior of 48 LFP and 48 HFP chicks in response to an APO injection was studied using an open field. Another objective of the present study was to determine whether behavioral variation (in an open field) between HFP and LFP birds, after APO injection, is also reflected by variation of D(1) and D(2) receptor densities in the brain. Receptor binding capacities were assessed by measuring specific binding of tritiated D(1) and D(2) receptor ligands in different regions of the brain of control HFP and LFP chicks. In the present study, it is shown that indeed HFP chicks display a more enhanced behavioral response to acute APO treatment (0.5 mg/kg BW) than LFP birds in an open field. This difference was not reflected by variation of D(1) and D(2) receptor densities in the brain between both lines.     

Use of two parallel oxygenators in a very large patient (2.76 m2) for an acute "A" dissecting aortic aneurysm repair

The very large patient (weight 142 kg, height 197 cm, body surface 2.76 m2) was referred to acute operation with dissecting type A ascending aortic aneurysm. The calculated blood flow was 6.63 l/min. To anticipate potential difficulties with perfusion and oxygenation two oxygenators connected in parallel were incorporated into the circuit. Bentall procedure with ACB to the RCA was performed. The perfusion was uneventful. Bypass time was 259 minutes, cross clamp time 141 minutes, circulatory arrest 7 minutes. The highest oxygenators gas flow was 2.6 l/min with maximum FiO2 0.42. The use of two in parallel connected oxygenators is a very effective, easy and safe method in such extreme perfusions, offering to the perfusionist a great reserve of oxygenator output.     

An ELISA serum assay for autoantibodies to HSP70 in immune-mediated hearing loss

A Western blot to detect anti-HSP70 autoantibodies has been reported to be of diagnostic value for immune-mediated hearing loss patients. While setting up this Western blot in our lab, we detected two main problems. First, some patients were positive for antibodies to a 70-kDa protein when tested against a whole cell lysate, but negative if the antigen used was purified HSP70. Second, if high amounts of purified HSP70 were loaded on the gel, both patients and healthy controls were positive. We have developed and optimized an ELISA as an alternative to the Western blot. This assay is more appropriate to identify positive and negative individuals because it is semi-quantitative. The ELISA is also more sensitive, requiring very low concentrations of the antigen and thus minimizing false positives. Finally, we demonstrated that immune-mediated hearing loss patients recognize mainly the native form of HSP70, a fact that potentially leads to false negatives when a denaturing Western blot assay is used for diagnosis. To test the diagnostic value of the ELISA, we performed a blind test with 70 hearing loss patients, as well as 30 healthy controls. A sensitivity of 84% and a specificity of 93% were obtained, superior to what has been reported so far for the Western blot.     

New phenotypic, functional and electrophysiological characteristics of KG-1 cells

Myeloid dendritic cells (DC) are representatives of a rare and phenotypically diverse population of professional antigen presenting cells possessing high functional heterogeneity and flexibility. Here we studied the phenotypic, functional and electrophysiological characteristics of KG-1 cells, an erythroleukemia model cell line, which shares morphological and physiological similarities with immature and mature myeloid DC. We compared the expression of internalizing receptors and other cell surface molecules, antigen uptake and migration of unstimulated and activated KG-1 cells with the characteristics of immature and mature DC. Unstimulated KG-1 cells were less potent in capturing extracellular materials than immature DC. In contrast to monocyte-derived DC KG-1 cells stimulated by PMA and ionomycin ceased to migrate along the MIP-3beta chemokine gradient despite their high expression of CCR7 chemokine receptor and MDR, a transporter implicated in DC migration. Moreover, we determined the ion channel repertoire of KG-1 cells before and after treatment with PMA and ionomycin by using the patch-clamp technique. We found that both unstimulated and activated KG-1 cells expressed time- and voltage-independent, ChTx sensitive intracellular Ca(2+)-gated potassium conductance suggesting the presence of K(Ca) channels in their membranes. Based on our results we propose that KG-1 cells resemble myeloid DC but also possess unique phenotypic, functional and electrophysiological characteristics.     

Change in sympathetic activity,cardiovascular functions and plasma hormone concentrations due to cold water immersion in men

The purpose of this study was to determine whether or not repeated short-term cold water immersions can induce a change in the activity of the sympathetic nervous system and, consequently, in cardiovascular functions in healthy young athletes. Changes in some plasma hormone concentrations were also followed. A single cold water immersion (head-out, at 14°C, for 1 h) increased sympathetic nervous system activity, as evidenced by a four-fold increase (P < 0.05) in plasma noradrenaline concentration. Plasma adrenaline and dopamine concentrations were not increased significantly. Plasma renin-angiotensin activity was reduced by half (P < 0.05) during immersion but plasma aldosterone concentration was unchanged. Stimulation of the sympathetic nervous system during immersion did not induce significant changes in heart rate, but induced peripheral vasoconstriction (as judged from a decrease in skin temperature) and a small increase (by 10%) in systolic and diastolic blood pressures. No clear change in reactivity of the sympathetic nervous system was observed due to repeated cold water immersions (three times a week, for 6 weeks). Neither the plasma renin-angiotensin activity, aldosterone concentration nor cardiovascular parameters were significantly influenced by repeated cold water immersions. A lowered diastolic pressure and an increase in peripheral vasoconstriction were observed after cold acclimation, however. Evidently, the repeated cold stimuli were not sufficient to induce significant adaptational changes in sympathetic activity and hormone production.