Full-term development of hamster embryos produced by injection of round spermatids into oocytes

The golden hamster is a mammal in which microinjection of round spermatids into oocytes (ROSI) was first attempted. However, no live ROSI offspring have ever been obtained in this species. This is the first report of live hamster offspring obtained by round spermatid injection. Over 90% of oocytes, injected with round spermatids, were activated without any additional stimulation. The proportion of the oocytes that were fertilized normally and that developed to morulae and blastocysts was higher when the plasma membranes of the spermatids were broken before injection, as compared with when the membranes were left intact. Five percent of 57 ROSI morulae/blastocysts developed into live offspring after transfer to foster mothers.     

Down-Regulation of the Noradrenaline Transporter by Interferon-α in Cultured Bovine Adrenal Medullary Cells

Abstract: The aim of this study was to investigate the effect of long-term treatment with interferon (IFN)-α on the noradrenaline transporter of bovine adrenal medullary cells. Treatment of cultured adrenal medullary cells with IFN-α caused a decrease in uptake of [³H]noradrenaline by the cells in time (4–48 h)- and concentration (300–1,000 U/ml)-dependent manners. IFN-β also inhibited [³H]noradrenaline uptake to a lesser extent than did IFN-α, whereas IFN-γ had little effect. An anti-IFN-α antibody reduced the effect of IFN-α on [³H]noradrenaline uptake. Saturation analysis of [³H]noradrenaline uptake showed that the inhibitory effect of IFN-α was due to a reduction in the maximal uptake velocity ( V _max) values without altering apparent Michaelis constant ( K _m) values. Incubation of cells with IFN-α caused a translocation of protein kinase C from the soluble to the particulate fraction in the cells. The effect of IFN-α on [³H]noradrenaline uptake was diminished in protein kinase C-down-regulated cells. Incubation of cells with IFN-α for 48 h significantly reduced the specific binding of [³H]desipramine to crude plasma membranes isolated from cells. Scatchard analysis of [³H]desipramine binding revealed that IFN-α decreased the maximal binding ( B _max) values without any change in the dissociation constant ( K _D) values. These findings suggest that IFN-α suppresses the function of noradrenaline transporter by reducing the density of the transporter in cell membranes through, at least in part, a protein kinase C pathway.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca²⁺ release and Ca²⁺ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca²⁺ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP₃), cyclic AMP, and the intracellular Ca²⁺ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP₃ and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP₃, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP₃ production, the Ca²⁺ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP₃ channels. The potencies of the peptides to cause Ca²⁺ release in the presence of cinnarizine were similar. The Ca²⁺ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca²⁺ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca²⁺ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP₃ production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca²⁺ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca²⁺ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP₃ and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca²⁺ in adrenal chromaffin cells.     

Glycolipids Isolated from Aplysia kurodaiCan Activate Cyclic Adenosine 3', 5'-Monophosphate-Dependent Protein Kinase from Rat Brain

Abstract: Cyclic AMP (cAMP)-dependent protein kinase (cAMP-kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonogly-cosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3-O-MeGalβ 1→3GalNAcα 1→3 [6'- O -(2-aminoethylphosphonyl) Galα1→2] (2-aminoethylphosphonyl→6) Glcβ 1→4GICβ1→1ceramide (SGL-II), was most potent, giving half-maximal activation at 32.2 μ M. Activation of cAMP-kinase was maximal with 250 μ M SGL-II using kemptide as substrate. The effect of SGL-II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL-II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP-kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP-kinase. This inhibition was reversed by addition of SGL-II, as observed for cAMP. SGL-II was capable of partially dissociating cAMP-kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL-II. These results demonstrate that the glycolipids can directly activate cAMP-kinase in a manner similar, but not identical, to that of cAMP.     

Oxygen uptake during swimming in a hypobaric hypoxic environment

The purpose of this study was to determine oxygen uptake (VO2) at various water flow rates and maximal oxygen uptake (VO2max) during swimming in a hypobaric hypoxic environment. Seven trained swimmers swam in normal [N; 751 mmHg (100.1 kPa)] and hypobaric hypoxic [H; 601 mmHg (80.27 kPa)] environments in a chamber where atmospheric pressure could be regulated. Water flow rate started at 0.80 m.s-1 and was increased by 0.05 m.s-1 every 2 min up to 1.00 m.s-1 and then by 0.05 m.s-1 every minute until exhaustion. At submaximal water flow rates, carbon dioxide production (VCO2), pulmonary ventilation (VE) and tidal volume (VT) were significantly greater in H than in N. There were no significant differences in the response of submaximal VO2, heart rate (fc) or respiratory frequency (fR) between N and H. Maximal VE, fR, VT, fc, blood lactate concentration and water flow rate were not significantly different between N and H. However, VO2max under H [3.65 (SD 0.11) l.min-1] was significantly lower by 12.0% (SD 3.4)% than that in N [4.15 (SD 0.18) l.min-1]. This decrease agrees well with previous investigations that have studied centrally limited exercise, such as running and cycling, under similar levels of hypoxia.     

A scanning electron microscopy of the interstitial tissue of the boar testis

In this study, the architecture of the interstitial tissue of the boar testis was examined by using scanning and transmission electron microscopes. The boar testis was remarkable for the abundance of interstitial tissue, and Leydig cells having many microvilli in their surface were almost round in shape. Both bundles of collagen fibers and networks of reticular fibers were observed around the Leydig cells. The capillary in the interstitial tissue of the boar was a muscle type, and both pericytes and collagen fibers were observed around the capillaries. The lymphatic capillary was poorly developed in the interstitial tissues of the boar testis. Endothelial cells were the only component of the capillary wall, and anchoring filaments were often observed on the abluminal surface of the endothelium.     

Regulation of cardiac IKs potassium current by membrane phosphatidylinositol 4,5-bisphosphate

Regulation of the slowly activating component of delayed rectifier K+ current (IKs) by membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns-(4,5)P2) was examined in guinea pig atrial myocytes using the whole-cell patch clamp method. IKs was elicited by depolarizing voltage steps given from a holding potential of -50 mV, and the effect of various test reagents on IKs was assessed by measuring the amplitude of tail current elicited upon return to the holding potential following a 2-s depolarization to +30 mV. Intracellular application of 50 microM wortmannin through a recording pipette evoked a progressive increase in IKs over a 10-15-min period to 208.5 +/- 14.6% (n = 9) of initial magnitude obtained shortly after rupture of the patch membrane. Intracellular application of anti-PtdIns(4,5)P2 monoclonal antibody also increased the amplitude of IKs to 198.4 +/- 19.9% (n = 5). In contrast, intracellular loading with exogenous PtdIns(4,5)P2 at 10 and 100 mum produced a marked decrease in the amplitude of IKs to 54.3 +/- 3.8% (n = 5) and 44.8 +/- 8.2% (n = 5), respectively. Intracellular application of neomycin (50 microM) or aluminum (50 microM) evoked an increase in the amplitude of IKs to 161.0 +/- 13.5% (n = 4) and 150.0 +/- 8.2% (n = 4), respectively. These results strongly suggest that IKs channel is inhibited by endogenous membrane PtdIns(4,5)P2 through the electrostatic interaction with the negatively charged head group on PtdIns(4,5)P2. Potentiation of IKs by P2Y receptor stimulation with 50 microM ATP was almost totally abolished when PtdIns(4,5)P2 was included in the pipette solution, suggesting that depletion of membrane PtdIns(4,5)P2 is involved in the potentiation of IKs by P2Y receptor stimulation. Thus, membrane PtdIns(4,5)P2 may act as an important physiological regulator of IKs in guinea pig atrial myocytes.     

A region C-terminal to the proline-rich core of p47phox regulates activation of the phagocyte NADPH oxidase by interacting with the C-terminal SH3 domain of p67phox

Activation of the phagocyte NADPH oxidase requires the regulatory proteins p47(phox) and p67(phox), each harboring two SH3 domains. p67(phox) interacts with p47(phox) via simultaneous binding of the p67(phox) C-terminal SH3 domain to both the proline-rich region (PRR) of amino acid residues 360-369 and its C-terminally flanking region of p47(phox); the role of the interaction in oxidase regulation has not been fully understood. Here we show that the p47(phox)-p67(phox) interaction is disrupted not only by deletion of the PRR but also by substitution for basic residues in the extra-PRR (K383E/K385E). The substitution impaired oxidase activation partially in vitro and much more profoundly in vivo, indicating the significance of the p47(phox) extra-PRR. Replacement of Ser-379 in the extra-PRR, a residue known to undergo phosphorylation in stimulated cells, by aspartate attenuates the interaction and thus results in a defective superoxide production, suggesting that phosphorylation of Ser-379 is involved in oxidase regulation.