Activities of the EM10 protein from Echinococcus multilocularis in cultured mammalian cells demonstrate functional relationships to ERM family members

The ezrin-radixin-moesin (ERM) homolog EM10 is expressed by the larval stage of the parasite E. multilocularis and shows 46.9% overall identity in the primary structure with human ezrin. To determine whether EM10 has similar activities to ERM proteins, we investigated properties of the protein expressed in mammalian cells. In particular, we transiently expressed haemagglutinin-tagged (HA-tagged) versions of the full-length EM10 as well as the amino- and the carboxy-terminal halves of EM10 in HtTA-1 cells. In addition we stably transfected NIH-3T3 cells with untagged full-length EM10. The data demonstrate that EM10 polypeptides behave like their corresponding portions of radixin when transiently expressed in mammalian cells. The full-length and amino-terminal EM10 polypeptides were localized to cortical structures. Cells expressing the carboxy-terminal polypeptide of EM10 showed long actin-filled protrusions. Cells expressing full-length EM10 showed a reduction in endogenous moesin-staining at cortical structures. In stably transfected NIH-3T3 cells EM10 was not crisply localized but rather was diffuse throughout the cytoplasm. These cells showed a conspicuous loss of stress-fibers, a phenotype that was not seen in analogous experiments with ERM proteins. The results demonstrate both similarities and differences between the functional properties of EM10 and ERM proteins expressed in vertebrate cells.     

Role of PGE2 on gallbladder muscle cytoprotection of guinea pigs

H2O2 and taurochenodeoxycholic acid (TCDC) impair the contraction induced by CCK-8, ACh, and KCl without affecting the actions of PGE2 and damage functions of membrane proteins except for PGE2 receptors. The aim of this study was to examine whether the preserved PGE2 actions contribute to cytoprotective mechanisms against reactive oxygen species. Muscle cells from guinea pig gallbladder were obtained by enzymatic digestion. Levels of lipid peroxidation and activities of SOD and catalase were determined by spectrophotometry. Pretreatment with PGE2 prevented the inhibition of H2O2 or TCDC on agonist (CCK-8, ACh, and KCl)-induced contraction and reduced the expected increase in lipid peroxidation and activities of catalase and SOD caused by H2O2 and TCDC. Incubation with CCK-8 for 60 min desensitized CCK-1 receptors up to 30 min, whereas no receptor desensitization was observed after PGE2 pretreatment. Cholesterol-rich liposome treatment enhanced the inhibition of H2O2 and TCDC on agonists-induced contraction, including that of PGE2. Pretreatment with PGE2 before H2O2 and TCDC did not completely block their inhibition on agonist-induced contraction. Cholesterol-rich liposome treatment impaired the expected increase in catalase activities in response to PGE2. We conclude that pretreatment with PGE2 prevents the muscle cell damage caused by H2O2 and TCDC due to the resistance of PGE2 receptors to agonist-induced desensitization. The preservation of PGE2 receptors may be designed to conserve these cytoprotective functions that are, however, impaired by the presence of excess cholesterol in the plasma membrane.     

Identification of nonsulfated cholecystokinin-58 in canine intestinal extracts and its biological properties

Nonsulfated CCK(58) [CCK(58)(ns)] has not been considered to be of biological importance because CCK(58)(ns) binds poorly to the CCK(A) receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the COOH-terminal region of gastrin and CCK (antibody 5135) was used to monitor the purification of CCK molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange, HPLC step. Characterization of this minor immunoreactive peak demonstrated that it was CCK(58)(ns). CCK(58)(ns) is 14% as immunoreactive as sulfated CCK(8) [CCK(8)(s)]. Amino acid analysis demonstrated that CCK(58)(ns) was present at 50% the amount of CCK(58)(s). In addition, we found that CCK(58)(ns) does not potently displace an (125)I-labeled CCK(10) analog from the CCK(A) receptor in mouse pancreatic membranes and does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK(58)(ns) does bind to CCK(B) receptors and stimulates gastric acid secretion via this receptor. The presence of CCK(58)(ns) and its ability to selectively stimulate the CCK(B) receptor without stimulation of the CCK(A) receptor suggest that CCK(58)(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent.     

D-Serine as a putative glial neurotransmitter

Abundant recent evidence favors a neurotransmitter/neuromodulator role for D-serine. D-serine is synthesized from L-serine by serine racemase in astrocytic glia that ensheath synapses, especially in regions of the brain that are enriched in NMDA-glutamate receptors. D-serine is more potent than glycine at activating the 'glycine' site of these receptors. Moreover, selective degradation of D-serine but not glycine by D-amino acid oxidase markedly reduces NMDA neurotransmission. D-serine appears to be released physiologically in response to activation by glutamate of AMPA-glutamate receptors on D-serine-containing glia. This causes glutamate-receptor-interacting protein, which binds serine racemase, to stimulate enzyme activity and D-serine release. Thus, glutamate triggers the release of D-serine so that the two amino acids can act together on postsynaptic NMDA receptors. D-serine also plays a role in neural development, being released from Bergmann glia to chemokinetically enhance the migration of granule cell cerebellar neurons from the external to the internal granular layer.     

Influence of respiratory network drive on phrenic motor output evoked by activation of cat pre-Botzinger complex

We have previously demonstrated that microinjection of dl-homocysteic acid (DLH), a glutamate analog, into the pre-B?tzinger complex (pre-B?tC) can produce either phasic or tonic excitation of phrenic nerve discharge during hyperoxic normocapnia. Breathing, however, is influenced by input from both central and peripheral chemoreceptor activation. This influence of increased respiratory network drive on pre-B?tC-induced modulation of phrenic motor output is unclear. Therefore, these experiments were designed to examine the effects of chemical stimulation of neurons (DLH; 10 mM; 10-20 nl) in the pre-B?tC during hyperoxic modulation of CO2 (i.e., hypercapnia and hypocapnia) and during normocapnic hypoxia in chloralose-anesthetized, vagotomized, mechanically ventilated cats. For these experiments, sites were selected in which unilateral microinjection of DLH into the pre-B?tC during baseline conditions of hyperoxic normocapnia [arterial PCO2 (PaCO2) = 37-43 mmHg; n = 22] produced a tonic (nonphasic) excitation of phrenic nerve discharge. During hypercapnia (PaCO2 = 59.7 +/- 2.8 mmHg; n = 17), similar microinjection produced excitation in which phasic respiratory bursts were superimposed on varying levels of tonic discharge. These DLH-induced phasic respiratory bursts had an increased frequency compared with the preinjection baseline frequency (P < 0.01). In contrast, during hypocapnia (PaCO2 = 29.4 +/- 1.5 mmHg; n = 11), microinjection of DLH produced nonphasic tonic excitation of phrenic nerve discharge that was less robust than the initial (normocapnic) response (i.e., decreased amplitude). During normocapnic hypoxia (PaCO2 = 38.5 +/- 3.7; arterial Po2 = 38.4 +/- 4.4; n = 8) microinjection of DLH produced phrenic excitation similar to that seen during hypercapnia (i.e., increased frequency of phasic respiratory bursts superimposed on tonic discharge). These findings demonstrate that phrenic motor activity evoked by chemical stimulation of the pre-B?tC is influenced by and integrates with modulation of respiratory network drive mediated by input from central and peripheral chemoreceptors.     

Origin and evolution of Japanese encephalitis virus in southeast Asia

Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu-->Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the Indonesia-Malaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the Indonesia-Malaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens.     

Comparative analysis of diffusive and stress induced nutrient transport efficiency in the lacunar-canalicular system of osteons

Marker migration experiments suggest that cyclic mechanical loading of cortical bone in vivo increases marker penetration into bone. Is this a result of stress induced fluid flow or of stress stimulation of active transport processes? Active lacunar-canalicular transport of nutrients was suggested by Ham in 1979 on the basis of the presence of actin filaments in osteocyte processes and their suspected role in cell motility. In addition, Tanaka in 1984 observed active transport of microperoxidase in bone and Tanaka-Kamioka et al. in 1998 observed experimentally that osteocyte processes are able to actively change their form. In this study we performed parametric and comparative analyses of the transport efficiencies of diffusion and stress generated fluid flow of (glucose) nutrients in lacunar-canalicular systems in cortical bone. The result obtained is that neither diffusion nor stress induced fluid flow is capable of sustaining osteocyte viability. It is possible that cyclic stress stimulates an active nutrient transport mechanism to supplement stress flows.     

Upregulation of the gene encoding a cytoplasmic dynein intermediate chain in senescent human cells

Normal human somatic cells, unlike cancer cells, stop dividing after a limited number of cell divisions through the process termed cellular senescence or replicative senescence, which functions as a tumor-suppressive mechanism and may be related to organismal aging. By means of the cDNA subtractive hybridization, we identified eight genes upregulated during normal chromosome 3-induced cellular senescence in a human renal cell carcinoma cell line. Among them is the DNCI1 gene encoding an intermediate chain 1 of the cytoplasmic dynein, a microtubule motor that plays a role in chromosome movement and organelle transport. The DNCI1 mRNA was also upregulated during in vitro aging of primary human fibroblasts. In contrast, other components of cytoplasmic dynein showed no significant change in mRNA expression during cellular aging. Cell growth arrest by serum starvation, contact inhibition, or gamma-irradiation did not induce the DNCI1 mRNA, suggesting its specific role in cellular senescence. The DNCI1 gene is on the long arm of chromosome 7 where tumor suppressor genes and a senescence-inducing gene for a group of immortal cell lines (complementation group D) are mapped. This is the first report that links a component of molecular motor complex to cellular senescence, providing a new insight into molecular mechanisms of cellular senescence.     

Anti-semaphorin 3A antibodies rescue retinal ganglion cells from cell death following optic nerve axotomy

Damage to the optic nerve in mammals induces retrograde degeneration and apoptosis of the retinal ganglion cell (RGC) bodies. The mechanisms that mediate the response of the neuronal cells to the axonal injury are still unknown. We have previously shown that semaphorins, axon guidance molecules with repulsive cues, are capable of mediating apoptosis in cultured neuronal cells (Shirvan, A., Ziv, I., Fleminger, G., Shina, R., He, Z., Brudo, I., Melamed, E., and Brazilai, A. (1999) J. Neurochem. 73, 961-971). In this study, we examined the involvement of semaphorins in an in vivo experimental animal model of complete axotomy of the rat optic nerve. We demonstrate that a marked induction of type III semaphorin proteins takes place in ipsilateral retinas at early stages following axotomy, well before any morphological signs of RGC apoptosis can be detected. Time course analysis revealed that a peak of expression occurred after 2-3 days and then declined. A small conserved peptide derived from semaphorin 3A that was previously shown to induce neuronal death in culture was capable of inducing RGC loss upon its intravitreous injection into the rat eye. Moreover, we demonstrate a marked inhibition of RGC loss when axotomized eyes were co-treated by intravitreous injection of function-blocking antibodies against the semaphorin 3A-derived peptide. Marked neuronal protection from degeneration was also observed when the antibodies were applied 24 h post-injury. We therefore suggest that semaphorins are key proteins that modulate the cell fate of axotomized RGC. Neutralization of the semaphorin repulsive function may serve as a promising new approach for treatment of traumatic injury in the adult mammalian central nervous system or of ophthalmologic diseases such as glaucoma and ischemic optic neuropathy that induce apoptotic RGC death.