Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.     

Structure of the ovary and "nurse cells" in a freshwater ostracod, Cyprinotus uenoi Brehm (Podocopida: Cypridoidea)

The adult female of the freshwater ostracod Cyprinotus uenoi Brehm, 1936 (Podocopida: Cypridoidea) has a pair of long, sac-like ovaries separately lying in the posterior part of the left and the right carapace valves. Oogonia and very early previtellogenic oocytes are located in the terminal germarium of each ovary. In the germarium, the oogonia occur in the most terminal region, and the very early previtellogenic oocytes are located in the remainder, arranged in order of size, the larger ones nearer the ovarian lumen. Most of the growing oocytes, previtellogenic and vitellogenic, are found in the ovarian lumen, the larger ones farther from the germarium. In the germarium, a cytoplasmic bridge connects a pair of adjoining germ cells, resulting from an incomplete cytokinesis of oogonial division. Among the previtellogenic and early vitellogenic oocytes in the ovarian lumen, "nurse cells" are found as small, spherical cells in mostly the same number as these oocytes. A cytoplasmic bridge connects each "nurse cell" to an adjoining oocyte. Based on the manner of connection and some morphological features, we consider that each "nurse cell" originates from one of each pair of adjoining germ cells connected by a cytoplasmic bridge in the germarium, as in the true nurse cells of several branchiopod crustaceans and insects with meroistic ovarioles.     

Transient O2-dependent effects of CO2 on ventilation in the anesthetized mouse.

In this study we sought to determine the effects of background hyperoxia on the ventilatory response to hypercapnia. We addressed this issue by examining the temporal profile of the first minute transients of minute ventilation, and its frequency and tidal components, in response to 5% and 10% CO2 each co-applied with the natural (balanced with air) and hyperoxic (balanced with O2) levels of oxygen. The study was performed on the urethane-anesthetized, tracheostomized, spontaneously breathing mouse, placed in a flow-through body plethysmograph. We identified an early suppressant effect of CO2-in-O2 on breathing frequency. The frequency declined to 88.5 +/-1.4% and 87.8 +/-1.9% relative to the pre-test, baseline level for 5% and 10% CO2, respectively. There was a compensatory rise in tidal volume and no major change in the overall ventilation. In contrast, CO2-in-Air resulted in ventilatory stimulation caused in equal measure by frequency and tidal components. Thus, the inhibitory effect on breathing frequency of the CO2-in-O2 resulted from the O2 content in the mixture and had the temporal characteristics consistent with carotid body function. In conclusion, transient O2-dependent effects can bear on the nascent hypercapnic ventilatory response. The complexity of the O2-CO2 interaction regarding the breathing pattern components should be taken into account while designing the optimal conditions for a hypercapnic test.     

Glucose Metabolism and Its Complicated Relationship with Tumor Growth and Perfusion in Head and Neck Squamous Cell Carcinoma

ObjectiveTo determine the relationship between tumor glucose metabolism and tumor blood flow (TBF) in head and neck squamous cell carcinoma (HNSCC).MethodsWe retrospectively analyzed 57 HNSCC patients. Tumor glucose metabolism was assessed by maximum and mean standardized uptake values (SUVmax and SUVmean) obtained by ¹⁸F-fluorodeoxyglucose positron-emission tomography. TBF values were obtained by arterial spin labeling with 3-tesla MRI. The correlations between both SUVs and TBF were assessed in the total series and among patients divided by T-stage (T1–T3 and T4 groups) and tumor location (pharynx/oral cavity and sinonasal cavity groups). Pearson’s correlation coefficients were calculated for significant correlations.ResultsSignificant correlations were detected: a negative correlation in the advanced T-stage group (TBF and SUV max: r, −0.61, SUVmean: r, −0.62), a positive correlation in the non-advanced T-stage pharynx/oral cavity group (TBF and SUVmax: r, 0.70, SUVmean: r, 0.73), a negative correlation in the advanced T-stage pharynx/oral cavity group (TBF and SUVmax: r, −0.62, SUVmean: r, −0.65), and a negative correlation in the advanced T-stage sinonasal cavity group (TBF and SUVmax: r, −0.61, SUVmean: r, −0.65).ConclusionSignificant correlations between glucose uptake and TBF in HNSCC were revealed by the division of T-stage and tumor location.     

Cystathionine Is a Novel Substrate of Cystine/Glutamate Transporter: IMPLICATIONS FOR IMMUNE FUNCTION*

The cystine/glutamate transporter, designated as system x_c⁻, is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system x_c⁻, xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system x_c⁻ and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine β-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system x_c⁻ and that the accumulation of cystathionine in immune tissues is exclusively mediated by system x_c⁻.     

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.     

1-Aminocyclopropane-1-carboxylic acid and its analogs alleviate heat stress damage in the marine red alga Neopyropia yezoensis (Rhodophyta)

Journal of Applied Phycology - Heat stress disrupts algal growth, development, and physiological processes, such as photosynthesis, and eventually decreases seaweed productivity. Previous studies...     

Weight Loss by Ppc-1, a Novel Small Molecule Mitochondrial Uncoupler Derived from Slime Mold

Mitochondria play a key role in diverse processes including ATP synthesis and apoptosis. Mitochondrial function can be studied using inhibitors of respiration, and new agents are valuable for discovering novel mechanisms involved in mitochondrial regulation. Here, we screened small molecules derived from slime molds and other microorganisms for their effects on mitochondrial oxygen consumption. We identified Ppc-1 as a novel molecule which stimulates oxygen consumption without adverse effects on ATP production. The kinetic behavior of Ppc-1 suggests its function as a mitochondrial uncoupler. Serial administration of Ppc-1 into mice suppressed weight gain with no abnormal effects on liver or kidney tissues, and no evidence of tumor formation. Serum fatty acid levels were significantly elevated in mice treated with Ppc-1, while body fat content remained low. After a single administration, Ppc-1 distributes into various tissues of individual animals at low levels. Ppc-1 stimulates adipocytes in culture to release fatty acids, which might explain the elevated serum fatty acids in Ppc-1-treated mice. The results suggest that Ppc-1 is a unique mitochondrial regulator which will be a valuable tool for mitochondrial research as well as the development of new drugs to treat obesity.