Sequential measurement of IL-6 blood levels in patients with systemic inflammatory response syndrome (SIRS)/sepsis

This study was undertaken to investigate whether sequential measurement of blood interleukin (IL)-6 levels using chemiluminescent enzyme immunoassay (CLEIA) would be useful for the management of patients with systemic inflammatory response syndrome (SIRS)/sepsis. Forty consecutive patients with SIRS/sepsis admitted to ICU were involved in the study. Blood IL-6 level was measured everyday throughout their ICU stay at the clinical laboratory by CLEIA method. The platelet count and the sequential organ failure assessment (SOFA) score were measured consecutively. The blood IL-6 levels were elevated in SIRS/sepsis patients and were extremely high in patients with septic shock. There was no significant difference in the blood IL-6 level on admission between survivors (n=27) and non-survivors (n=13). However, the mean blood IL-6 level during ICU stay was significantly higher in the non-survivors (p<0.05). There were significant correlation between the peak IL-6 blood level and the lowest platelet count, and between the peak IL-6 blood level and the maximum SOFA score, respectively. The platelet count became lowest 2.0+/-2.0 days later on average, and the SOFA score became maximal 2.5+/-1.4 days later on average following the day when IL-6 reached its peak value. Sequential measurement of blood IL-6 levels by CLEIA is useful in evaluating the severity and in predicting the outcome of the patients with SIRS/sepsis.     

A variant protein from phi29 replication gene, gene 1, did not form homo-polymer due to a single amino acid substitution near the carboxyl terminus

For study of the self-association of the product of psi29 gene 1, one variant which has a substitution at the 71(st) amino acid was used. By glycerol gradient sedimentation, the product of wild-type gene 1 existed both as large aggregate and oligomer, whereas the variant was detected as a single peak of monomer size. According to experiments using His-tagged proteins and Ni-NTA magnetic beads, the variant made only a little self-associated complex. From these results, a site essential for self-association was suggested to exist close to the carboxyl terminus of the product of psi29 gene 1.     

Isolation of a series of single missense mutants of a dna gene of phage psi29, gene 1, utilizing their inhibitory effect on E. coli growth

Gene 1 product (gp1) of Bacillus subtilis phage psi29 is known to promote DNA replication of the phage. Although its role in the DNA replication is not clear, gp1 is reported to exhibit multiple characteristics, including RNA binding, cell membrane localization, and self-association. To investigate these characteristics, we undertook the isolation of a series of missense mutants of gene 1 bearing substitutions at various regions. During cloning of gene 1, we found that its expression severely inhibited the growth of its host Escherichia coli cells. In this study, we utilized this growth-inhibition phenomenon to screen a random library muta-genized by error-prone PCR, expecting that mutants which could not inhibit cell growth would be affected in the authentic functions of gp1. Using this approach, we obtained 31 different mutants bearing single amino acid substitutions at 26 positions along the entire length of gp1. As a preliminary analysis of these mutants, we compared the deduced amino acid sequences of gp1s from psi29 and its related phages PZA, B103 and M2. Alignment of these sequences revealed three conserved regions, i.e. a hydrophobic region near the carboxyl terminus (assumed to be involved in the membrane localization and self-association of gp1), coiled-coil motif (essential for self-association), and a region of unknown function near the amino terminus. Interestingly, many of the substitutions in the isolated mutants occurred at strongly conserved residues in these regions and affected characteristic features of the regions (e.g. hydrophobicity of the hydrophobic region). These substitutions are expected to affect authentic functions of gp1, and the mutants will be useful for studies of the structure and functions of gp1.     

Thermostable aldehyde dehydrogenase from psychrophile,Cytophaga sp. KUC-1: enzymological characteristics and functional properties

We found the occurrence of NAD(P)(+)-dependent aldehyde dehydrogenase (EC1.2.1.5) in the cells of a psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, and purified to homogeneity. About 50% of the enzyme activity remained even after heating at 50 degrees C for 65min and the highest activity was observed in the range of 55-60 degrees C. The enzyme was thermostable and thermophilic, although it was derived from a psychrophile. The circular dichroism at 222nm of the enzyme showed a peak at 32 degrees C. This temperature was closely similar to the transition temperature in the Arrhenius plots. The stereospecificity for the hydride transfer at C4-site of nicotinamide moiety of NADH was pro-R. The gene encoding the enzyme consisted of an open reading frame of 1506-bp encoding a protein of 501 amino acid residues. The significant sequence identity (61%) was found between the Cytophaga and the Pseudomonas aeruginosa enzymes, although their thermostabilities are completely different.     

Polysaccharide-polynucleotide complexes VIII. Cation-induced complex formation between polyuridylic acid and schizophyllan

Raman spectroscopy has recently been applied ex vivo and in vivo to address various biomedical issues such as the early detection of cancers, monitoring of the effect of various agents on the skin, determination of atherosclerotic plaque composition, and rapid identification of pathogenic microorganisms. This leap in the number of applications and the number of groups active in this field has been facilitated by several technological advancements in lasers, CCD detectors, and fiber-optic probes. However, most of the studies are still at the proof of concept stage. We present a discussion on the status of the field today, as well as the problems and issues that still need to be resolved to bring this technology to hospital settings (i.e., the medical laboratory, surgical suites, or clinics). Taken from the viewpoint of clinicians and medical analysts, the potential of Raman spectroscopic techniques as new tools for biomedical applications is discussed and a path is proposed for the clinical implementation of these techniques.     

Mossy fiber Zn2+ spillover modulates heterosynaptic N-methyl-D-aspartate receptor activity in hippocampal CA3 circuits

Although Zn2+ is contained in large amounts in the synaptic terminals of hippocampal mossy fibers (MFs), its physiological role in synaptic transmission is poorly understood. By using the newly developed high-sensitivity Zn2+ indicator ZnAF-2, the spatiotemporal dynamics of Zn2+ was monitored in rat hippocampal slices. When high-frequency stimulation was delivered to the MFs, the concentration of extracellular Zn2+ was immediately elevated in the stratum lucidum, followed by a mild increase in the stratum radiatum adjacent to the stratum lucidum, but not in the distal area of stratum radiatum. The Zn2+ increase was insensitive to a non-N-methyl-d-aspartate (NMDA) receptor antagonist but was efficiently attenuated by tetrodotoxin or Ca2+-free medium, suggesting that Zn2+ is released by MF synaptic terminals in an activity-dependent manner, and thereafter diffuses extracellularly into the neighboring stratum radiatum. Electrophysiological analyses revealed that NMDA receptor-mediated synaptic responses in CA3 proximal stratum radiatum were inhibited in the immediate aftermath of MF activation and that this inhibition was no longer observed in the presence of a Zn2+-chelating agent. Thus, Zn2+ serves as a spatiotemporal mediator in imprinting the history of MF activity in contiguous hippocampal networks. We predict herein a novel form of metaplasticity, i.e., an experience-dependent non-Hebbian modulation of synaptic plasticity.     

Src homology region 2 domain-containing phosphatase 1 positively regulates B cell receptor-induced apoptosis by modulating association of B cell linker protein with Nck and activation of c-Jun NH2-terminal kinase

Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a key mediator in lymphocyte differentiation, proliferation, and activation. We previously showed that B cell linker protein (BLNK) is a physiological substrate of SHP-1 and that B cell receptor (BCR)-induced activation of c-Jun NH(2)-terminal kinase (JNK) is significantly enhanced in cells expressing a form of SHP-1 lacking phosphatase activity (SHP-1-C/S). In this study, we confirmed that SHP-1 also exerts negative regulatory effects on JNK activation in splenic B cells. To further clarify the role of SHP-1 in B cells, we examined how dephosphorylation of BLNK by SHP-1 affects downstream signaling events. When a BLNK mutant (BLNK Delta N) lacking the NH(2)-terminal region, which contains four tyrosine residues, was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK activation was inhibited. Among candidate proteins likely to regulate JNK activation through BLNK, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of dominant-negative forms of Nck inhibited BCR-induced JNK activation. Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing cells and coexpression of Nck SH2 mutants or a dominant-negative form of SEK1 reversed this phenotype. Collectively, these results suggest that SHP-1 acts on BLNK, modulating its association with Nck, which in turn negatively regulates JNK activation but exerts a positive effect on apoptosis.     

Trans-interactions of nectins induce formation of filopodia and Lamellipodia through the respective activation of Cdc42 and Rac small G proteins

Nectins and afadin constitute a novel cell-cell adhesion system that plays a cooperative role with cadherins in the organization of adherens junctions (AJs). Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules, and afadin is a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton. Rac and Cdc42 small G proteins have been implicated in the organization of AJs, but their modes of action remain unknown. The trans-interaction of E-cadherin has recently been shown to induce the activation of Rac, but not that of Cdc42. We show here that the trans-interactions of nectins induce the formation of filopodia and lamellipodia through the respective activation of Cdc42 and Rac. The Cdc42 activation is necessary, but not sufficient, for the Rac-induced formation of lamellipodia, whereas the Rac activation is not necessary for the Cdc42-induced formation of filopodia. These effects of nectins require their cytoplasmic tail but not their association with afadin. We propose here the functional relationship between nectins and the small G proteins in the organization of AJs.     

Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: partial characterization and implication in metamorphosis

It has been shown that larval skin (LS) grafts are rejected by an inbred strain of adult Xenopus, which suggests a mechanism of metamorphosis by which larval cells are recognized and attacked by the newly differentiating immune system, including T lymphocytes. In an attempt to define the larval antigenic molecules that are targeted by the adult immune system, anti-LS antibodies (IgY) were produced by immunizing adult frogs with syngeneic LS grafts. The antigen molecules that reacted specifically with this anti-LS antiserum were localized only in the larval epidermal cells. Of 53 and 59-60 kDa acidic proteins that were reactive with anti-LS antibodies, a protein of 59 kDa and with an isoelectric point of 4.5 was selected for determination of a 19 amino acid sequence (larval peptide). The rat antiserum raised against this peptide was specifically reactive with the 59 kDa molecules of LS lysates. Immunofluorescence studies using these antisera revealed that the larval-specific molecules were localized in both the tail and trunk epidermis of premetamorphic larvae, but were reduced in the trunk regions during metamorphosis, and at the climax stage of metamorphosis were detected only in the regressing tail epidermis. Culture of splenocytes from LS-immunized adult frogs in the presence of larval peptide induced augmented proliferative responses. Cultures of larval tail pieces in T cell-enriched splenocytes from normal frogs or in natural killer (NK)-cell-enriched splenocytes from early thymectomized frogs both resulted in significant destruction of tail pieces. Tissue destruction in the latter was enhanced when anti-LS antiserum was added to the culture. These results indicate that degeneration of tail tissues during metamorphosis is induced by a mechanism such that the larval-specific antigen molecules expressed in the tail epidermis are recognized as foreign by the newly developing adult immune system, and destroyed by cytotoxic T lymphocytes and/or NK cells.     

Interleukin-1beta expression in human gastric carcinoma with Epstein-Barr virus infection

The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1beta (IL-1beta) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1beta mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1beta increased the cell growth of a GC cell line, TMK1. Thus, IL-1beta may act as an autocrine growth factor in EBVaGC.