Loss of mammalian Sprouty2 leads to enteric neuronal hyperplasia and esophageal achalasia

We report here that loss of the Sprouty2 gene (also known as Spry2) in mice resulted in enteric nerve hyperplasia, which led to esophageal achalasia and intestinal pseudo-obstruction. Glial cell line-derived neurotrophic factor (GDNF) induced hyperactivation of ERK and Akt in enteric nerve cells. Anti-GDNF antibody administration corrected nerve hyperplasia in Sprouty2-deficient mice. We show Sprouty2 to be a negative regulator of GDNF for the neonatal development or survival of enteric nerve cells.     

Contribution of redox-active iron and copper to oxidative damage in Alzheimer disease

Metal-catalyzed hydroxyl radicals are potent mediators of cellular injury, affecting every category of macromolecule, and are central to the oxidative injury hypothesis of Alzheimer disease (AD) pathogenesis. Studies on redox-competent copper and iron indicate that redox activity in AD resides exclusively within the neuronal cytosol and that chelation with deferoxamine, DTPA, or, more recently, iodochlorhydroxyquin, removes this activity. We have also found that while proteins that accumulate in AD possess metal-binding sites, metal-associated cellular redox activity is primarily dependent on metals associated with nucleic acid, specifically cytoplasmic RNA. These findings indicate aberrations in iron homeostasis that, we suspect, arise primarily from heme, since heme oxygenase-1, an enzyme that catalyzes the conversion of heme to iron and biliverdin, is increased in AD, and mitochondria, since mitochondria turnover, mitochondrial DNA, and cytochrome C oxidative activity are all increased in AD. These findings, as well as studies demonstrating a reduction in microtubule density in AD neurons, suggest that mitochondrial dysfunction, acting in concert with cytoskeletal pathology, serves to increase redox-active heavy metals and initiates a cascade of abnormal events culminating in AD pathology.     

Rhythmical contractions in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats

Rhythmical contractions accompanied by an increase in cytosolic Ca2+ concentrations were produced in ring preparations of endothelium-denuded pulmonary arteries from monocrotaline-treated rats, but not in those from vehicle-treated rats, 2-3 h after a resting tension of 15 mN (150-180% of the initial wall length of the artery) was applied. The rhythmical contractions were abolished by nicardipine and ryanodine. Cyclopiazonic acid reduced the relaxation phase of the rhythmical contractions, finally leading to a sustained contraction. Similarly, apamin caused a sustained contraction, whereas charybdotoxin increased the amplitude of the rhythmical contractions. Glibenclamide had no apparent effects on them. Indomethacin and the prostaglandin H2/thromboxane A2 receptor antagonist SQ29548 abolished the rhythmical contractions and reduced the tension, but the thromboxane synthase inhibitor ozagrel had no effect. These results suggest that optimal stretch induces rhythmical contractions in the pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats, to which both Ca2+ influx through voltage-operated Ca2+ channels and Ca2+ release from the endoplasmic reticulum seem to contribute. It is also suggested that small-conductance Ca(2+)-activated K+ channels participate in the relaxation phase of rhythmical contractions. Furthermore, prostaglandin H2 released from nonendothelial cells is likely to play a pivotal role in the induction of rhythmical contractions.     

Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations

Although various genetic factors have been implicated in human male infertility, the causative genes for the different types of idiopathic male infertility have not been elucidated. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense codon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2.     

Expression of synaptotagmin 1 in the taste buds of rat gustatory papillae

Synapses between taste receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the central nervous system. The synaptic vesicle cycle at the synapses involves vesicle docking, priming, fusion, endocytosis, and recycling. Many kinds of synaptic vesicle proteins participate in synaptic vesicle cycles. One of these, synaptotagmin 1, binds Ca(2+) phospholipids with high affinity and plays a role in Ca(2+) regulated neurotransmitter release in the central and peripheral nervous systems. However, the expression patterns of synaptotagmin 1 in rat taste tissues have not been determined. We therefore examined the expression patterns of synaptotagmin 1 and several cell specific markers of type II and III cells in rat taste buds. RT-PCR assay showed that synaptotagmin 1 mRNA was expressed in circumvallate papillae. In fungiform, foliate, and circumvallate papillae, the antibody against synaptotagmin 1 yielded the labeling of a subset of taste bud cells and intra- and subgemmal nerve processes. Double labeled experiments showed that synaptotagmin 1 positive cells co-expressed type III cell markers, PGP 9.5, and NCAM. Intragemmal nerve processes positive for synaptotagmin 1 co-expressed PGP 9.5. Conversely, all synaptotagmin 1 expressing cells did not co-expressed type II cell markers, PLCbeta2, or gustducin. These results show that synaptotagmin 1 may play some regulatory roles in vesicle membrane fusion events with the plasma membrane at the synapses of type III cells in rat taste buds.     

Immunofluorescence technique using HeLa cells expressing recombinant nucleoprotein for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.     

Peripheral blood mononuclear cells stimulate progesterone production by luteal cells derived from pregnant and non-pregnant women: possible involvement of interleukin-4 and interleukin-10 in corpus luteum function and differentiation

Human luteal cells have been reported to express human leukocyteantigen-DR and lymphocyte functional antigen-3 on the cell surface,suggesting physiological interaction between luteal cells andT-lymphocytes through the menstrual cycle into early pregnancy.To elucidate the role of peripheral lymphocytes on corpus luteumdifferentiation, the effect of peripheral blood mononuclearcells (PBMC) on steroidogenesis by luteal cells was investigated.The production of Th-2 cytokines such as interleukin (IL)-4and IL-10 by the co-cultured cells was also examined, and theeffects of these cytokines on progesterone production by lutealcells were investigated. Corpora lutea were obtained from eightnon-pregnant women in the luteal phase and five women in earlypregnancy for luteal cell culture. PBMC were isolated from unrelatedwomen in the follicular phase, secretory phase, and early pregnancy.After co-culture with allogenic PBMC for 48 h, progesteroneproduction was significantly enhanced by PBMC from the secretoryphase and early pregnancy in the non-pregnant luteal cell culture.In the pregnant luteal cell culture, a significant increasein progesterone production was also observed by the co-culturewith PBMC from women in early pregnancy, showing that PBMC havea luteotrophic effect. The stimulatory effects of PBMC werealso observed in co-culture conditions which prevented directcell-to-cell interaction with luteal cells, showing the minorinfluence of mixed lymphocyte reaction. By co-culture with PBMC,the production of IL-10, but not IL-4, was significantly augmentedin luteal cell culture derived from non-pregnant women, whereasthe production of both IL-4 and IL-10 was significantly enhancedin the luteal cell culture derived from pregnant women. Moreover,IL-4 and IL-10 promoted progesterone production by culturedluteal cells, especially in the luteal cell culture derivedfrom corpora lutea of early pregnancy. These findings indicatethat PBMC stimulate progesterone production by luteal cellsand suggest the involvement of PBMC in corpus luteum functionand differentiation probably via the Th-2-type lymphocytes.     

Increased matrix metalloproteinase-9 activity in human ovarian cancer cells cultured with conditioned medium from human peritoneal tissue

Ovarian cancer cells disseminate by attachment to the peritoneal mesothelial cell surface of the abdominal cavity. We therefore investigated the influence of conditioned medium (CM) from human peritoneal tissues and mesothelial cells on the secretion of matrix metalloproteinases (MMPs) by ovarian cancer cells. The molecular weights of MMPs stimulating factors derived from human peritoneal tissues and mesothelial cells were estimated using microconcentrators with various cut-off membranes. Human peritoneal tissues were obtained from 12 surgical patients, and mesothelial cells were isolated from three peritoneal specimens. Exposure to CM from peritoneal tissue caused a concentration-dependent increase of the MMP-2 and MMP-9 bands in CM from NOM1 ovarian cancer cells, as shown by zymography. There was a significant difference in the increase of MMP-2 and MMP-9 (2.46-fold and 7.14-fold, respectively, at 0.4mg/ml protein; P < 0.005). CM from mesothelial cells also significantly increased the secretion of MMP-9 by NOM1 cells. The molecular size of possible MMP-9-stimulating factors secreted by peritoneal tissues and mesothelial cells was above M 100000. Further, CM of peritoneal tissues and mesothelial cells also induced the invasiveness of NOM1 cells. These findings suggest that mesothelial cells may secrete some factors which predominantly induce the MMP-9 production and increase invading cell numbers.     

Intercellular adhesion molecule-1 in patients with idiopathic interstitial pneumonia

This study focuses on a possible role of intercellular adhesion molecule-1 (ICAM-1) in interstitial pulmonary diseases. We determined a soluble form of ICAM-1 in serum and bronchoalveolar lavage fluid (BALF) using ELISA in patients with usual interstitial pneumonia (UIP), bronchiolitis obliterance organizing pneumonia (BOOP), or nonspecific interstitial pneumonia (NSIP). In addition, we investigated the expression of ICAM-1 in the lung tissues of these patients by means of immunohistochemical staining. Serum levels of soluble ICAM-1 were significantly higher in patients with UIP or NSIP than in healthy subjects, and were also high in patients with BOOP. The soluble ICAM-1 in BALF tended to be higher in patients with UIP, BOOP, or NSIP than in normal subjects. A significant correlation was seen between soluble levels of ICAM-1 in serum and BALF. In the immunostaining of ICAM-1 of the lung tissues, ICAM-1 expression was more pronounced in patients with UIP than in those with BOOP or NSIP. The increased expression of ICAM-1 was seen in type II alveolar epithelium and vascular endothelium in patients with interstitial pneumonia. A positive correlation was observed between the degree of ICAM-1 expression in the lung tissues and the BALF levels of soluble ICAM-1. The expression of ICAM-1 in type II alveolar epithelium suggests that ICAM-1 plays a specific role in the fibrotic process of the lung, and that the measurement of soluble ICAM-1 in sera and BALF could be a useful marker for evaluating the progression of fibrosis.