Ischemia-induced angiogenesis is impaired in aminopeptidase A deficient mice via down-regulation of HIF-1α

Aminopeptidase A (APA; EC 3.4.11.7) is a transmembrane metalloprotease with several functions in tumor angiogenesis. To investigate the role of APA in the process of ischemia-induced angiogenesis, we evaluated the cellular angiogenic responses under hypoxic conditions and the process of perfusion recovery in the hindlimb ischemia model of APA-deficient (APA-KO; C57Bl6/J strain) mice.Western blotting of endothelial cells (ECs) isolated from the aorta of APA-KO mice revealed that the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein in response to hypoxic challenge was blunted. Regarding the proteasomal ubiquitination, a proteasome inhibitor MG-132 restored the reduced accumulation of HIF-1α in ECs from APA-KO mice similar to control mice under hypoxic conditions. These were associated with decreased growth factor secretion and capillary formation in APA-KO mice. In the hindlimb ischemia model, perfusion recovery in APA-KO mice was decreased in accordance with a significantly lower capillary density at 2 weeks. Regarding vasculogenesis, no differences were observed in cell populations and distribution patterns between wild type and APA-KO mice in relation to endothelial progenitor cells.Our results suggested that Ischemia-induced angiogenesis is impaired in APA-KO mice partly through decreased HIF-1α stability by proteasomal degradation and subsequent suppression of HIF-1α-driven target protein expression such as growth factors. APA is a functional target for ischemia-induced angiogenesis.     

Effects of α2-Adrenergic Agonists and Antagonists on Photoreceptor Membrane Currents

Type B photoreceptors of the nudibranch mollusc Hermissenda crassicornis receive excitatory synaptic potentials (EPSPs) whose frequency is controlled by potential changes of a neighboring cell known as the S optic ganglion cell which is thought to be electrically coupled to the presynaptic source of these EPSPs, the E optic ganglion cell. The frequency of the EPSPs increases when a conditioned stimulus (light) is paired with an unconditioned stimulus (rotation) during acquisition of a Pavlovian conditioned response. The results of the present study are consistent with an adrenergic origin for these EPSPs. Noradrenergic agonists (greater than 100 microM), norepinephrine and clonidine, only slightly depolarize the type B cell but clearly prolong its depolarizing response to light. Serotonin, by contrast, causes hyperpolarization of the type B cell's resting potential as well as after a light step. Clonidine reduces voltage-dependent outward K+ currents (IA, an early current, ICa2+-K+, a late Ca2+-dependent current) that control the type B cell's excitability (and thus its light response and membrane potential). These effects of clonidine are reduced or blocked by the alpha 2-receptor antagonist, yohimbine (0.5 microM), but not the alpha 1-blocker, prazosin. The same yohimbine concentration also blocked depolarizing synaptic excitation of the type B cell in response to depolarization of a simultaneously impaled S optic ganglion cell. Histochemical techniques (both the glyoxylic acid method of de la Torre and Surgeon and the formaldehyde-induced fluorescence or Falck-Hillarp method) demonstrated the presence of a biogenic amine(s) within a single neuron in each optic ganglion as well as three or four cells within the vicinity of previously identified visual interneurons. No serotonergic neurons were found within the optic ganglion or in proximity to visual interneurons. A clonidine-like synaptic effect on type B cells, therefore, could amplify conditioning-specific changes of membrane currents by increasing type B depolarization and possibly, as well, by elevating intracellular second messengers.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

How long is a piece of Strix? Methods in measuring and measuring the measurers

An experiment to quantify intra- and interobserver error in anatomical measurements found that interobserver measurements can vary by over 14% of mean specimen length; disparity in measurement increases logarithmically with the number of contributors; instructions did not reduce variation or measurement disparity; scale of the specimen influenced the precision of measurement (relative error increasing with specimen size); different methods of taking a measurement yielded different results, although they did not differ in terms of precision, and topographical complexity of the elements being considered may potentially influence error (error increasing with complexity). These results highlight concerns about introduction of noise and potential bias that should be taken into account when compiling composite datasets and meta-analyses.     

Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis

Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 μmol O₂ (mg Chl a)^− 1 h^− 1 in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl₂. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 °C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells.     

Flagellar Formation in C-Ring-Defective Mutants by Overproduction of FliI,the ATPase Specific for Flagellar Type III Secretion

The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the fliG, fliM, and fliN genes results in a Fla⁻ phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN- or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact.The flagellum is the ultrastructure for motility in many bacterial species (1). Flagellar assembly requires about 50 genes, among which about 20 gene products are incorporated in the complete flagellum (12). Most structural proteins and others necessary for assembly are exported through a flagellum-specific type III secretion apparatus housed within the basal body. The apparatus consists of at least six integral membrane proteins: FlhA, FlhB, FliP, FliQ, FliR, and FliO (for salmonellae and other species) (1, 12). Other proteins are also involved. FliI is the only known ATPase among flagellar proteins (2). FliI interacts with FliJ, which is of unknown function, and with a dimer of FliH, an inhibitor of FliI. The apparatus can be visualized by quick-freeze electron microscopy and has been termed the C (cytoplasmic) rod by virtue of its appearance and membrane-proximal location inside the C ring (7). The C ring is composed of three component proteins: FliG, FliM, and FliN (3). Mutations or deletions of any of these proteins cause a nonflagellate (Fla⁻) phenotype, strongly suggesting that the C ring is necessary for flagellar protein export (6, 22, 26). The trimer FliH₂-FliI specifically binds FliN (4, 15), suggesting that FliI docks at the periphery of the C ring through interactions with FliN-bound FliH, standing ready to escort export substrates to the secretion gate that is probably composed by FlhA, FlhB, and others (15).The C ring has long been studied with respect to motor function rather than export function. It has been proposed that FliG plays a major role in torque generation in concert with MotAB complexes, leaving the other two proteins, FliM and FliN, in minor and supporting roles (10, 11). However, as mentioned above, all three components are required for flagellar protein export (6, 22, 26). Together with the C ring, FliI pushes export substrates into the gate using the energy of ATP hydrolysis. Just recently, it was shown that FliI ATPase activity is not absolutely necessary for protein export and that increasing proton motive force (PMF) or reversion mutations in FlhA and FlhB can compensate for its absence (17, 21).In order to elucidate the roles that FliG, FliM, and FliN play in export, we employed C-ring-defective mutants. Here, we show that the overproduction of FliI allows flagellar formation in C-ring-defective mutants. We closely examined flagella formed in those mutants by electron microscopy, noting percentages of flagellation in each population, analyzing partially formed structures, and measuring hook length.     

Ganglioside GM1 mediates decapacitation effects of SVS2 on murine spermatozoa

Prior to fertilization, mammalian spermatozoa need to acquire fertilizing ability (capacitation) in the female reproductive tract. On the other hand, capacitated spermatozoa reversibly lose their capacitated state when treated with seminal plasma (decapacitation). Previously, we demonstrated that a mouse seminal plasma protein, SVS2, is a decapacitation factor and regulates sperm fertilizing ability in vivo. Here, we examined the mechanisms of regulation of fertilizing ability by SVS2. Capacitation appears to be mediated by dynamic changes in lipid rafts since release of the cholesterol components of lipid rafts in the sperm plasma membrane is indispensable for capacitation. When the ejaculated spermatozoa were stained with a cholera toxin subunit B (CTB) that preferably interacts with ganglioside GM1, another member of the lipid rafts, the staining pattern of the sperm was the same as the binding pattern of SVS2. Interestingly, SVS2 and CTB competitively bound to the sperm surface with each other, suggesting that the binding targets of both molecules are the same, that is, GM1. Molecular interaction studies by the overlay assay and the quartz crystal microbalance analysis revealed that SVS2 selectively interacts with GM1 rather than with other gangliosides. Furthermore, external addition of GM1 nullified SVS2-induced sperm decapacitation. Thus, ganglioside GM1 is a receptor of SVS2 and plays a crucial role in capacitation in vivo.     

TRPA1 agonists--allyl isothiocyanate and cinnamaldehyde--induce adrenaline secretion

Thermosensitive transient receptor potential (TRP) channels, especially TRPV1 and TRPA1, are activated by the pungent compounds present in spices. TRPV1 activation by the intake of capsaicin, the irritant in hot pepper, induces adrenaline secretion and increases energy consumption. TRPV1 is mainly expressed in the sensory neurons and coexpressed with TRPA1 at a high frequency. However, the mechanism underlying adrenaline secretion by TRPA1 agonists such as allyl isothiocyanate (AITC) and cinnamaldehyde (CNA), the pungent ingredients in mustard and cinnamon, is not known. We examined whether AITC and CNA could induce adrenaline secretion in anesthetized rats. An intravenous injection of AITC or CNA (10 mg/kg) increased adrenaline secretion. These responses disappeared completely in capsaicin-treated rats with an impaired sensory nerve function. Moreover, pretreatment with cholinergic blockers (hexamethonium and atropine) attenuated the AITC- or CNA-induced adrenaline secretion. These results suggest that TRPA1 agonists activate the sensory nerves and induce adrenaline secretion via the central nervous system.     

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.     

Polygalacturonase mRNA of Tomato: Size and Content in Ripe Fruits

In ripe tomato fruits, polygalacturonase (PG) mRNA comprisedabout 1% of the translatable RNAs in the poly(A)(+)RNA fraction.Sucrose density gradient centrifugation showed that this PGmRNA is similar in size to 18S rRNA, which suggests the presenceof a non-coding region. (Received June 19, 1984; Accepted October 23, 1984)