Endogenous lactate transport in Xenopus laevis oocyte: dependence on cytoskeleton and regulation by protein kinases

Carbon flux in Xenopus laevis oocyte is glycogenic and an endogenous monocarboxylate transporter is responsible for intracellular lactate uptake. The aim of the present study was to determine if direct activation of protein kinases C and A modulates the activity of lactate transporter, as well as to investigate the possible role of cytoskeleton in these regulatory phenomena. The modulation was studied in isolated Xenopus oocytes of stage V–VI by measuring ¹⁴C-lactate uptake, both in the absence and in the presence of cytoskeletal-perturbing toxins. We found that the basal lactate transporter activity depends on the integrity of the cytoskeleton since it is partially inhibited by cytoskeleton disorganisation. Both PKA and PKC activation caused a significant decrease in transport activity and this decrease could be blocked by specific protein kinase inhibitors. The evidenced effects were not additive. Transport inhibition was annulled by agents that destabilize actin filaments or microtubules. We conclude that both protein kinases A and C, whose effects are mediated by cytoskeleton, negatively regulate the endogenous lactate transporter of Xenopus oocyte, suggesting that these kinases may have a role in the control of cytosolic pyruvate/lactate pool in the oocyte. All the experiments in this study comply with the current laws of Italy.     

GSK3 beta mediates acentromeric spindle stabilization by activated PKC zeta

Upon fertilization, the mammalian egg undergoes a precise series of signaling events that orchestrate its conversion into a zygote. Mouse eggs contain acentrosomal spindle poles when arrested at meiotic metaphase II. The meiotic spindle is thought to provide a scaffold that mediates spatial and temporal regulation of the signaling pathways orchestrating post-fertilization events. Many kinases have been found to be enriched at the MII meiotic spindle, such as Protein Kinase C (PKC), and are thought to have an important role in regulating signaling events initiated through fertilization. In this study phosphorylated PKCζ (p-PKCζ) and Glycogen Synthase Kinase 3β (GSK3β) were found to be enriched at both acentrosomal spindle poles and the kinetochore region. Phosphorylated PKCζ (p-PKCζ) was immunopurified from MII eggs and was found to co-localize with known microtubule stabilizing components found in somatic cells, including GSK3β and Partition deficit protein 6 (Par6). Both fluorescence resonance energy transfer (FRET) and immunofluorescence confirmed the existence and close association of these proteins with p-PKCζ at the meiotic spindle. When GSK3β is phosphorylated on ser9 its activity is inhibited and the spindle is stabilized. However, when GSK3β is dephosphorylated (on ser9) it becomes active and the spindle is destabilized. The mechanism by which p-PKCζ maintains spindle organization appears to be through GSK3β and suggests that p-PKCζ phosphorylates GSK3β on the ser9 position inactivating GSK3β and consequently maintaining spindle stability during meiotic metaphase arrest.     

WHY DO PARASITIC CUCKOOS HAVE SMALL BRAINS? INSIGHTS FROM EVOLUTIONARY SEQUENCE ANALYSES

Brain size is under many opposing selection pressures. Estimating their relative influence and reconstructing the brain's evolutionary history have, however, proved difficult. Here, we confirm the suggestion that the brain of brood parasitic cuckoos is smaller in relation to their body weight than that of nonparasitic cuckoo species. Two hypotheses explaining reductions in brain size are tested, using phylogenetically controlled correlations and evolutionary pathway analyses. In a novel approach, the pathway models are combined to build the most likely evolutionary sequence of trait changes correlating with changes in brain size. Brain size changed before brood parasitism, followed by a shift toward less-productive habitats and an increase in migration. This sequence shows that brain size was not reduced as a consequence of a loss of cognitive skills related to chick provisioning, and it offers no support for the hypothesis that an increase in energetic demands or a reduction in energy availability selected for a reduction of brain size. Instead, the sequence suggests that the reduction in energetic demands due to the smaller brain size and parasitic breeding strategy may have enabled parasitic cuckoos to colonize new niches.     

Nepmucin/CLM-9, an Ig domain-containing sialomucin in vascular endothelial cells, promotes lymphocyte transendothelial migration in vitro

Nepmucin/CLM-9 is an Ig domain-containing sialomucin expressed in vascular endothelial cells. Here we show that, like CD31, nepmucin was localized to interendothelial contacts and to vesicle-like structures along the cell border and underwent intracellular recycling. Functional analyses showed that nepmucin mediated homotypic and heterotypic cell adhesion via its Ig domain. Nepmucin-expressing endothelial cells showed enhanced lymphocyte transendothelial migration (TEM), which was abrogated by anti-nepmucin mAbs that block either homophilic or heterophilic binding. Notably, the mAbs that inhibited homophilic binding blocked TEM without affecting lymphocyte adhesion. These results suggest that endothelial nepmucin promotes lymphocyte TEM using multiple adhesion pathways.     

PKC and MEK pathways inhibit caspase-9/-3-mediated cytotoxicity in differentiated cells

Many studies have indicated that differentiated cells inhibit drug-induced cytotoxicity but undifferentiated cells do not, though the mechanisms are unclear. Currently, HL-60 cells are induced to differentiate into macrophage-like cells with Phorbol-12-myristate-13-acetate (TPA) treatment (TPA-differentiated cells). Our study shows that caspase-9/-3-mediated cytotoxicity can be induced in undifferentiated HL-60 cells but not in TPA-differentiated HL-60 cells. However, caspase-9/-3-mediated cytotoxicity can be induced in TPA-differentiated cells if they are pretreated with a protein kinase C (PKC) or a mitogen activated protein kinase (MEK) inhibitor. Taken together, this study demonstrates that TPA-differentiated HL-60 cells inhibit caspases-9/-3-mediated cytotoxicity through the PKC and MEK signaling pathways.     

Evolution of the cephalopod head complex by assembly of multiple molluscan body parts: Evidence from Nautilus embryonic development

Cephalopod head parts are among the most complex occurring in all invertebrates. Hypotheses for the evolutionary process require a drastic body-plan transition in relation to the life-style changes from benthos to active nekton. Determining these transitions, however, has been elusive because of scarcity of fossil records of soft tissues and lack of some of the early developmental stages of the basal species. Here we report the first embryological evidence in the nautiloid cephalopod Nautilus pompilius for the morphological development of the head complex by a unique assembly of multiple archetypical molluscan body parts. Using a specialized aquarium system, we successfully obtained a series of developmental stages that enabled us to test previous controversial scenarios. Our results demonstrate that the embryonic organs exhibit body plans that are primarily bilateral and antero-posteriorly elongated at stereotyped positions. The distinct cephalic compartment, foot, brain cords, mantle, and shell resemble the body plans of monoplacophorans and basal gastropods. The numerous digital tentacles of Nautilus develop from simple serial and spatially-patterned bud-like anlagen along the anterior-posterior axis, indicating that origins of digital tentacles or arms of all other cephalopods develop not from the head but from the foot. In middle and late embryos, the primary body plans largely change to those of juveniles or adults, and finally form a "head" complex assembled by anlagen of the foot, cephalic hood, collar, hyponome (funnel), and the foot-derived epidermal covers. We suggest that extensions of the collar-funnel compartment and free epidermal folds derived from multiple topological foot regions may play an important role in forming the head complex, which is thought to be an important feature during the body plan transition.     

Physicochemical Properties and Oxidative Inactivation of Soluble Lectin from Water Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Brain

Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G_50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard analysis was 6.6 × 10³ M⁻¹ showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H₂O₂ was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells. The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike those purified till date.     

蛋白磷酸酯酶2A调节微管相关蛋白1b的磷酸化

微管相关蛋白MAP1b的生物学活性受其磷酸化修饰的调节,后者则受相应的蛋白激酶和蛋白磷酸酯酶(PP)调控.为研究蛋白磷酸酯酶在脑内对MAP1b磷酸化的调控作用,采用有代谢活性的大鼠脑片作为模型,分别应用冈田酸(okadaic acid)和cyclosporin A选择性地抑制PP2A 和PP2B活性,来研究其对脑内蛋白磷酸酯酶MAP1b磷酸化的调控.采用特异性的MAP1bⅠ型磷酸化依赖性抗体522和免疫印迹技术检测MAP1bⅠ型磷酸化.结果表明,当PP2A被okadaic acid选择性抑制后,MAP1bⅠ型磷酸化明显增加.而PP2B被选择性地抑制后,MAP1b磷酸化的变化不大.免疫组化染色显示,MAP1b广泛分布于鼠大脑神经元和突起中,与对照组相比,在PP2A抑制的脑片中抗体522的免疫活性在神经元中明显升高.上述结果表明,PP2A是脑中调控MAP1bⅠ型磷酸化的主要蛋白磷酸酯酶.     

Functional restoration of acoustic units and adult‐generated neurons after hypothalamic lesion

The hypothalamus of the adult ring dove contains acoustic units that respond to species‐specific coo vocalization. Loss of nest coo leads to unsuccessful breeding. However, the recovery of nest coo in some doves suggests that these units are capable of self‐renewal. We have previously shown that lesioning the hypothalamus generates the addition of new neurons at the lesioned area. In this study, we sought to determine whether lesion‐induced new neurons are involved in the recovery of coo‐responsive units. We systematically recorded electrical activity in the ventromedial nucleus (VMN) of the hypothalamus, before and after lesion, for varying periods up to 3 months. Recordings were made when the birds were at rest (spontaneous discharge) and when the birds were exposed to acoustic stimulations (evoked discharge). Concurrently, the lesioned area was monitored for changes in cell types by using bromodeoxyuridine (BrdU) to label newly divided cells and NeuN to identify mature neurons. For 1 month after lesion, there was no sign of electrical activity, and only BrdU‐labeled cells were present. When the first electrical activity occurred, it displayed abnormal spontaneous bursting patterns. The mature discharge patterns (both spontaneous and evoked) occurred after detection of BrdU⁺/NeuN⁺ double‐labeled cells 2–3 months postlesion and were similar to those found in intact and sham‐lesioned birds. Double‐labeled cells bore morphologic characteristics of a neuron and were confirmed with z‐stack analysis using confocal laser scanning microscopy. Moreover, double‐labeled cells were not stained for glial fibrillary acidic protein (GFAP), suggesting that they were neurons. The number of coo‐responsive units was significantly correlated with that of BrdU⁺/NeuN⁺ cells. Furthermore, the marker for recording sites revealed that coo‐responsive units were colocalized with BrdU⁺/NeuN⁺ cells. Taken together, the evidence strongly suggests that lesion‐induced addition of new neurons promotes the functional recovery of the adult hypothalamus. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 197–213, 2004