Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca²⁺ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca ²⁺ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca ²⁺ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca ²⁺ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca ²⁺ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca ²⁺ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH ₄Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca ²⁺ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.     

Spontaneous meningoencephalitis by Staphylococcus aureus in an infant golden‐headed lion tamarin (Leontopithecus chrysomelas)

Non‐human primates are susceptible to many bacteria, some of which bear zoonotic potential. We report the pathologic features of spontaneous fulminating meningoencephalitis by Staphylococcus aureus in a captive infant golden‐headed lion tamarin (Leontopithecus chrysomelas) from Brazil.     

Evaluating the genetic structure of wild and commercial red cusk-eel (Genypterus chilensis) populations through the development of novel microsatellite markers from a reference transcriptome

Molecular Biology Reports - The red cusk-eel (Genypterus chilensis) is a native Chilean species with a high-value market, with the potential to diversify Chilean aquaculture. The objective of this...     

Neuronal ceroid lipofuscinosis related ER membrane protein CLN8 regulates PP2A activity and ceramide levels

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN8 deficiency causes a subtype of NCL, referred to as CLN8 disease. CLN8 is an ER resident protein with unknown function; however, a role in ceramide metabolism has been suggested. In this report, we identified PP2A and its biological inhibitor I2PP2A as interacting proteins of CLN8. PP2A is one of the major serine/threonine phosphatases in cells and governs a wide range of signaling pathways by dephosphorylating critical signaling molecules. We showed that the phosphorylation levels of several substrates of PP2A, namely Akt, S6 kinase, and GSK3β, were decreased in CLN8 disease patient fibroblasts. This reduction can be reversed by inhibiting PP2A phosphatase activity with cantharidin , suggesting a higher PP2A activity in CLN8-deficient cells. Since ceramides are known to bind and influence the activity of PP2A and I2PP2A, we further examined whether ceramide levels in the CLN8-deficient cells were changed. Interestingly, the ceramide levels were reduced by 60% in CLN8 disease patient cells compared to controls. Furthermore, we observed that the conversion of ER-localized NBD-C6-ceramide to glucosylceramide and sphingomyelin in the Golgi apparatus was not affected in CLN8-deficient cells, indicating transport of ceramides from ER to the Golgi apparatus was normal. A model of how CLN8 along with ceramides affects I2PP2A and PP2A binding and activities is proposed.     

Simultaneous direct determination of amiloride and triamterene in urine using isopotential fluorometry

A method for the simultaneous fluorometric determination of two diuretics in urine is proposed. The combination of matrix isopotential synchronous fluorescence (MISF) and first derivative techniques provides good analytical results. MISF spectra are obtained by calculating the isopotential trajectory in the three-dimensional fluorescence spectrum for a urine solution. In the spectral contour, the trajectory is taken to be the portion of the line that passes by the fluorescence maxima of both diuretics (lambda(ex) = 365 and lambda(em) = 413 nm for amiloride and lambda(ex) = 365 and lambda(em) = 437 nm for triamterene). Because contour lines connect points of identical intensity and the trajectory is part of a contour line, it is called "isopotential." Analyses was carried out in a 1/1 (v/v) ethanol/water mixture, using an apparent pH of 6.3 provided by 0.01 M sodium/citrate citric acid buffer. Urine samples are diluted 50 times and provide linear calibration plots at amiloride and triamterene concentrations up to 320 and 100 ng mL(-1), respectively. The goodness of the analytical signal was checked by using variance analysis. Signals recorded throughout the calibration range were subjected to three calibrations per each analyte, both in the absence and in the presence of variable amounts of the other analyte. Differences between individual calibrations and slopes were compared with those within individual calibrations. Based on the results, triamterene and amiloride can be accurately quantified in the presence of each other. The limit of detection calculated according to Clayton who uses error propagation throughout the calibration curve and a noncentralized security factor was 16.8 and 2.4 ng mL(-1) for amiloride and triamterene, respectively.     

Glucose-induced activation of rubidium transport and water flux in sunflower root systems

Excised 20-d-old sunflower roots (Helianthus annuus L. cv. Sun-Gro 393) were used to study the effect of different sugars on rubidium and water fluxes. The roots sensed and absorbed glucose from the external medium inducing the activation of rubidium accumulated in the root (Rb(+) root), the flux of exuded rubidium (J(Rb)) and, to a lesser degree, the exudation rate (J(v)). These effects were also triggered by fructose, but not by 6-deoxyglucose (6-dG), a glucose analogue which is not a substrate for hexokinase (HXK). The effect of 2-deoxyglucose (2-dG), an analogue that is phosphorylated but not further metabolized, was complex, suggesting an inhibitory effect on solute transport to the xylem. The amounts of glucose required to activate rubidium and water fluxes were similar to those previously reported to regulate different processes in other plants (0.5--10 mM). When sorbitol was used instead of glucose, neither rubidium uptake (Rb(+) root plus J(Rb)) nor J(v) was activated. It is proposed that glucose present in the root plays an important signalling role in the regulation of Rb(+) (K(+)) and water transport in plant roots.