I-ROAD could be efficient in predicting severity of community-acquired pneumonia or healthcare-associated pneumonia.

INTRODUCTION: The ability to predict the prognosis of patients with pneumonia is critical, especially when making decisions regarding treatment regimens and sites of care. However, prognostic guidelines for healthcare-associated pneumonia (HCAP) have yet to be established. I-ROAD is the prognostic guideline of the Japanese Respiratory Society for hospital-acquired pneumonia (HAP). This study compared available prognostic guidelines to determine the usefulness of I-ROAD as a prognostic tool for patients with HCAP. METHODS: We conducted a retrospective review of all patients with pneumonia admitted to Kameda Medical Center, Japan, from January 2006 to September 2009. Patients were categorised into two groups, namely those with community acquired pneumonia (CAP) and those with HCAP. We compared the baseline characteristics, laboratory findings, identified pathogens, antibiotic regimens, clinical outcomes, pneumonic severity and prognostic accuracy of each guideline between the two patient groups. The severity of each disease was assessed on admission using the A-DROP, CURB-65, PSI and I-ROAD guidelines. RESULTS: Of the 302 patients evaluated, 228 (75.5%) were diagnosed with CAP and 74 (24.5%) with HCAP. Patients with HCAP were older and had a higher performance status than patients with CAP. The mortality rate in the CAP group tended to rise with increasing severity scores of prognostic guidelines. Although the severity scores of all prognostic guidelines could predict 30-day mortality in patients with CAP, I-ROAD exhibited a higher discriminatory power for patients with HCAP based on analysis of receiver-operating characteristic curves. CONCLUSION: I-ROAD could be more accurate than other prognostic guidelines for evaluating the severity of HCAP.

Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30-40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.

Roles of induced expression of MAPK phosphatase-2 in tumor development in RET-MEN2A transgenic mice.

Germline mutations in the RET tyrosine kinase gene are responsible for the development of multiple endocrine neoplasia 2A and 2B (MEN2A and MEN2B). However, knowledge of the fundamental principles that determine the mutant RET-mediated signaling remains elusive. Here, we report increased expression of mitogen-activated protein kinase phosphatase-2 (MKP-2) in carcinomas developed in transgenic mice carrying RET with the MEN2A mutation (RET-MEN2A). The expression of MKP-2 was not only induced by RET-MEN2A or RET-MEN2B mutant proteins but also by the activation of endogenous RET by its ligand, glial cell line-derived neurotrophic factor (GDNF). MKP-2 expression was also evident in the MKK-f cell line, which was established from a mammary tumor developed in a RET-MEN2A transgenic mouse. Inhibition of MKP-2 attenuated the in vitro and in vivo proliferation of MKK-f cells, which was mediated by the suppression of cyclin B1 expression. Furthermore, we found that MKP-2 is highly expressed in medullary thyroid carcinomas derived from MEN2A patients. These findings suggest that the increased expression of MKP-2 may play a crucial role in oncogenic signaling downstream of mutant RET, leading to deregulation of cell cycle.

Sex pheromones of the hair crab Erimacrus isenbeckii. II. Synthesis of ceramides.

To confirm their structures and to assess the pheromonal activity, novel ceramides, possible sex pheromones of the hair crab Erimacrus isenbeckii, were synthesized from D-galactose. The synthetic ceramides were identical with the natural ceramides.

Functional analysis of RET with Hirschsprung mutations affecting its kinase domain.

BACKGROUND AND AIMS: Many missense mutations in the RET proto-oncogene were found in familial and sporadic cases of Hirschsprung disease (HSCR). The aim of this study was to make clear the mechanisms of RET dysfunction by HSCR mutations identified in its kinase domain. METHODS: Ten kinase domain HSCR mutations were introduced into wild-type RET and constitutively activated RET with a multiple endocrine neoplasia 2A mutation, and the resulting mutant complementary DNAs were transfected into SK-N-MC primitive neuroectodermal tumor cells or NIH 3T3 fibroblast cells. The levels of activation of mutant RET and representative signaling molecules were investigated in the transfectants. RESULTS: E762Q, S767R, R972G, and M980T mutations partially impaired the RET kinase activity and the representative signaling pathways. In particular, these mutations severely impaired the phospholipase C-gamma signaling pathway in SK-N-MC cells. S765P, R873Q, F893L, R897Q, and E921K mutations resulted in a complete loss of the RET kinase activity. The P973L mutation markedly decreased the expression of the RET protein with normal kinase activity. CONCLUSIONS: Hirschsprung disease can result from these distant functional classes of kinase domain mutation of RET.

Homo-oligomer formation by basigin, an immunoglobulin superfamily member, via its N-terminal immunoglobulin domain.

Basigin (Bsg) is a highly glycosylated transmembrane protein with two immunoglobulin (Ig)-like domains. A number of studies, including gene targeting, have demonstrated that Bsg plays pivotal roles in spermatogenesis, implantation, neural network formation and tumor progression. In the present study, to understand the mechanism of action of Bsg, we determined its expression status on the plasma membrane. Cotransfection of Bsg expression vectors with two different tags clarified that Bsg forms homo-oligomers in a cis-dependent manner on the plasma membrane. If the disulfide bond of the more N-terminally located Ig-like domain was destroyed by mutations, Bsg could not form oligomers. In contrast, the mutations of the C-terminal Ig-like domain or N-glycosylation sites did not affect the association. The association of mouse and human Bsgs, which exhibit high homology in the transmembrane and intracellular domains but low homology in the extracellular domain, was very weak as compared with that within the same species, suggesting the importance of the extracellular domain in the association. If the extracellular domain of the human Ret protein was replaced with the N-terminal Ig-like domain of Bsg, the resulting chimera protein was associated with intact wild-type Bsg, but not if the C-terminal Ig-like domain, instead of the N-terminal one, of Bsg was used. No oligomer formation took place between the intact wild-type Ret and Bsg proteins. In conclusion, these data indicate that the N-terminal Ig-like domain is necessary and sufficient for oligomer formation by Bsg on the plasma membrane.

Role of malate synthesis mediated by phosphoenolpyruvate carboxylase in guard cells in the regulation of stomatal movement.

To clarify the pathway and role of malate synthesis in guard cells, epidermal strips isolated from Vicia faba L. leaflets were treated with 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP), a specific inhibitor of phosphoenolpyruvate carboxylase (PEPC). When dark-closed stomata were illuminated, malate accumulated in guard cells and stomata opened; these were inhibited by 60% and 30%, respectively, by 5 mM DCDP treatment. When light-opened stomata were treated with DCDP, both malate level in guard cells and stomatal aperture decreased. Treatment with 5 mM DCDP partially inhibited CO2 incorporation into malate in guard cells. Treatment with mannitol at 0.4 M (osmotic stress) in the light increased malate level in guard cells and closed stomata. DCDP treatment decreased both malate level and stomatal aperture under stressed condition. These results show that malate synthesis in the light under both non-stressed and stressed conditions is dependent on PEPC activity. The extent of the decrease in malate level by DCDP treatment was larger under stressed condition than under nonstressed condition, suggesting that osmotic stress may enhance the activity of this pathway of malate synthesis which is induced by light. Role of malate synthesis in guard cells is discussed.

Isolation and characterization of Escherichia coli O157 from retail beef and bovine feces in Thailand.

Antibody to Escherichia coli O157 lipopolysaccharide was detected in the sera of healthy individuals more frequently in Southern Thailand than in Japan. The result suggested possible exposure of Thai people to E. coli O157. E. coli O157:H7 or O157:H(-) was isolated from four of 95 retail beef and one of 55 bovine feces samples collected in Southern Thailand by enrichment culture followed by immunomagnetic bead separation. Four of the five strains carried the stx(2) gene alone or in combination with the stx(1) gene. The strains were shown to be genetically distinct by an arbitrarily primed PCR method.

Ultraviolet light induces redox reaction-mediated dimerization and superactivation of oncogenic Ret tyrosine kinases.

The c-RET proto-oncogene encodes a receptor-type tyrosine kinase, and its mutations in the germ line are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B). Ret kinases are constitutively activated as a result of MEN2A mutations (Ret-MEN2A) or MEN2B mutations (Ret-MEN2B). Here we demonstrate that UV light (UV) irradiation induces superactivation of the constitutively activated Ret-MEN2A and Ret-MEN2B as well as activation of c-Ret. Before UV irradiation, small percentages of c-Ret (3-4%) and Ret-MEN2B (1-2%) and large percentages of Ret-MEN2A (30-40%) were dimerized through disulfide bonds. These dimerized Ret proteins were preferentially autophosphorylated, suggesting a close relation between up-regulated kinase activity and disulfide bond-mediated dimerization of Ret proteins. We found that UV irradiation promotes the disulfide bond-mediated dimerization of the Ret proteins, in close association with activation and superactivation of Ret kinases. UV irradiation also induced dimerization and activation of the extracellular domain-deleted mutant Ret (Ret-PTC-1). Interestingly, the levels of basic kinase activity and dimerization of Ret-PTC-1-C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-PTC-1 was replaced by alanine, were low and were not increased by UV irradiation. These results suggest that Ret-PTC-1 cysteine 376 is one of possibly multiple critical target amino acids of UV for Ret kinase activation. Overexpression of Cu/Zn superoxide dismutase in cells as a result of gene transfection prevented both the UV-mediated promotion of dimerization and the superactivation of Ret-MEN2A kinase. These results suggest that the UV-induced free radicals in cells attack intracellular domains of Ret to dimerize the kinase proteins for superactivation.

Image quality improvement using helium gas in low voltage variable pressure scanning electron microscopy.

An effective combination of the low voltage and variable pressure (VP) scanning electron microscopy (SEM) are discussed. In low voltage VP-SEM, helium gas is utilized for reducing the amount of scatter of the primary electron beam. Most samples can receive various benefits obtained from the combination of low voltage and low vacuum observation. Compared to a back-scattered electron (BSE) image in air, signal-to-noise ratio (SNR) of a BSE image taken with helium gas is 5.4 times under a pressure of 50 Pa and an accelerating voltage of 1.5 kV.

Different nuclear/cytoplasmic distributions of RET finger protein in different cell types.

The RET finger protein (RFP), which belongs to the B box zinc finger protein family, has a tripartite motif consisting of a Ring finger, a B box finger and a coiled-coil domain. The RET finger protein becomes oncogenic when its tripartite motif is fused with the tyrosine kinase domain of the RET protein. This study examined the RFP expression in normal and tumor tissues by immunohistochemistry. RFP was detected in the nuclei of various cells, including peripheral and central neurones, hepatocytes, adrenal chromaffin cells and male germ cells. Among them, RFP was expressed at high levels in male germ cells such as primary spermatocytes and round spermatids, and formed a perinuclear cap structure in primary spermatocytes. On the other hand, high levels of cytoplasmic expression of RFP were observed in some plasma cells as well as solitary plasmacytoma and multiple myeloma. These results suggested that different nuclear/cytoplasmic distributions of RFP might play a role in the regulation of growth or differentiation of different cell types.

The role of amino acids surrounding tyrosine 1062 in ret in specific binding of the shc phosphotyrosine-binding domain.

We investigated the role of the I-E-N-K-L (amino acids 1057-1061) sequence amino-terminal to Tyr1062 in Ret for binding of the Shc phosphotyrosine-binding (PTB) domain. Substitution of Ser for Ile1057 (I1057S), Ala for Asn1059 (N1059A), or Pro for Leu1061 (L1061P) in this sequence significantly decreased the transforming activity of Ret with the multiple endocrine neoplasm type 2A (MEN2A) mutation as well as the binding affinity of Shc to it in vivo and in vitro, indicating that these three amino acids play a role in Shc binding. In addition, as the RET protooncogene is translated as three isoforms of 1114 amino acids (Ret 51), 1106 amino acids (Ret 43), and 1072 amino acids (Ret 9) that differ from one another in the sequence carboxyl-terminal to Tyr1062, we examined whether these sequence differences influence the binding affinity of Shc to Ret. As a result, we found that the transforming activity of Ret 43 isoform with the MEN2A mutation and the binding affinity of Shc to it were very low, although the consensus sequence for the binding of the Shc PTB domain is conserved in the Ret 43 isoform. This finding suggested that the sequence carboxyl-terminal to Tyr1062 in Ret could also influence the binding affinity to Shc.

Biological and biochemical properties of Ret with kinase domain mutations identified in multiple endocrine neoplasia type 2B and familial medullary thyroid carcinoma.

Several mutations were identified in the kinase domain of the RET proto-oncogene in patients with multiple endocrine neoplasia (MEN) 2B, familial medullary thyroid carcinoma (FMTC) or sporadic medullary thyroid carcinoma. We introduced seven mutations (glutamic acid 768-->aspartic acid (E768D), valine 804-->leucine (V804L), alanine 883-->phenylalanine (A883F), serine 891-->alanine (S891A), methionine 918-->threonine (M918T), alanine 919-->proline (A919P) and E768D/A919P) into the short and long isoforms of RET cDNA and transfected the mutant cDNAs into NIH3T3 cells. The transforming activity of the long isoform of Ret with each mutation was much higher that that of its short isoform. Based on the levels of the transforming activity, these mutant RET genes were classified into two groups; a group with high transforming activity (A883F, M918T and E768D/A919P) and a group with low transforming activity (E768D, V804L, S891A and A919P) (designated high group and low group). Interestingly, the level of transforming activity correlated with clinical phenotypes; high group Ret with the A883F or M918T mutation and low group Ret with the E768D, V804L or S891A mutation were associated with the development of MEN 2B and FMTC, respectively. In addition, we found that substitution of phenylalanine for tyrosine 905 present in the kinase domain abolished both transforming and autophosphorylation activities of low group Ret whereas it did not affect the activities of high group Ret.

Enhanced phosphatidylinositol 3-kinase activity and high phosphorylation state of its downstream signalling molecules mediated by ret with the MEN 2B mutation.

We compared the intracellular signalling pathways through Ret tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF), multiple endocrine neoplasia (MEN) 2A, or MEN 2B mutation. Tyrosine phosphorylation of Grb2-associated binder-1 (Gab1) and activation of phosphatidylinositol 3-kinase (PI 3-kinase) were induced at higher levels by GDNF stimulation or the MEN 2B mutation than by the MEN 2A mutation. Tyrosine-phosphorylated Gab1 was a major component that interacted with the active PI 3-kinase in vivo. In addition, we found that p62Dok and PKB/Akt were phosphorylated in a PI 3-kinase-dependent manner and the levels of their phosphorylation were significantly higher in the MEN 2B transfectant than in the MEN 2A transfectant. Tyrosine phosphorylation of p62Dok resulted in its complex formation with the Ras GTPase-activating protein (RasGAP) and the Nck adaptor protein. These findings thus suggested that high levels of activation of PI 3-kinase and of phosphorylation of its downstream signalling molecules may be associated with the clinical phenotype of MEN 2B.

Characterization of the promoter region of the human RFP gene.

The RFP gene encodes a Ring finger protein that has a tripartite motif consisting of a Ring finger, a B-box finger and a coiled-coil domain. In the present study, we cloned and characterized the promoter region of the human RFP gene. The nucleotide sequence of the promoter was GC-rich and had no typical TATA and CAAT boxes. Instead, it contained one AP2 and two Sp1 binding sites within 100 base pairs upstream of the transcription initiation site. Analysis by the luciferase assay revealed that the activity of this promoter region is very strong in both human and mouse cell lines, although the activity in human cells was approximately 10-15 fold higher than that in mouse cells. In addition, the AP2 and Sp1 binding sites appeared to synergistically function for the promoter activity. Thus, the promoter of the RFP gene could be useful for high levels of expression of various genes in culture cells.

Implication of expression of GDNF/Ret signalling components in differentiation of bone marrow haemopoietic cells.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) mediate their actions through a unique multicomponent receptor system composed of Ret receptor tyrosine kinase and glycosyl-phosphatidylinositol-linked cell surface proteins (designated GFRalpha-1 and GFRalpha-2). In the present study, expression of these signalling components in the process of differentiation of haemopoietic cells was investigated. Ret was expressed at variable levels in normal and malignant cells of the myelomonocyte lineage. Immunohistochemical analysis of human and mouse tissues revealed that Ret expression was increased in intermediate mature myeloid cells such as promyelocytes and myelocytes and decreased in mature granulocytes and monocytes. Consistent with this observation, when THP-1 monocytic and HL-60 promyelocytic leukaemia cells expressing Ret were differentiated toward macrophages or granulocytes by treatment of 12-O-tetradecanoylphorbol-13-acetate (TPA) or all-trans retinoic acid (RA), Ret expression strikingly decreased during differentiation. Expression of GDNF, NTN, GFRalpha-1 and GFRalpha-2 was undetectable in THP-1 and HL-60 cells as well as in bone marrow haemopoietic cells. In contrast, bone marrow stromal cells appeared to express GDNF, GFRalpha-1 and GFRalpha-2 but not Ret. These findings suggested that the interaction between stromal cells and Ret-expressing haemopoietic cells in the bone marrow microenvironment may play a role in the differentiation of myelomonocyte-lineage cells through activation of the GDNF/Ret signalling pathway.

Rho-dependent and -independent tyrosine phosphorylation of focal adhesion kinase, paxillin and p130Cas mediated by Ret kinase.

Glial cell line-derived neurotrophic factor (GDNF) signals through a unique receptor system that includes Ret receptor tyrosine kinase and a glycosyl-phosphatidylinositol-linked cell surface protein. In the present study, we have identified several proteins in neuroblastoma cells that are phosphorylated on tyrosine in response to GDNF. The phosphorylated proteins include focal adhesion kinase (FAK), paxillin and Crk-associated substrate, p130Cas, all of which are known to be associated with focal adhesions. Of these, paxillin and p130Cas interacted with Crk proteins in GDNF-treated neuroblastoma cells. GDNF also induced reorganization of the actin cytoskelton. Tyrosine phosphorylation of FAK, paxillin and p130Cas was inhibited by cytochalasin D or two specific inhibitors of phosphatidylinositol-3' kinase (PI-3' kinase), wortmannin and LY294002, indicating that their tyrosine phosphorylation depends on the formation of actin stress fiber and activation of PI-3' kinase. In addition, phosphorylation of FAK but not of paxillin and p130Cas was markedly impaired by the Clostridium botulinum C3 exoenzyme that specifically ADP-ribosylates and inactivates Rho. These results suggested the presence of Rho-dependent and -independent signaling pathways downstream of PI-3' kinase that mediate tyrosine phosphorylation of FAK, paxillin and p130Cas through Ret kinase.