MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4.

Toll-like receptor 4 (TLR4) is a mammalian homologue of Drosophila Toll, a leucine-rich repeat molecule that can trigger innate responses against pathogens. The TLR4 gene has recently been shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS). TLR4 may be a long-sought receptor for LPS. However, transfection of TLR4 does not confer LPS responsiveness on a recipient cell line, suggesting a requirement for an additional molecule. Here, we report that a novel molecule, MD-2, is requisite for LPS signaling of TLR4. MD-2 is physically associated with TLR4 on the cell surface and confers responsiveness to LPS. MD-2 is thus a link between TLR4 and LPS signaling. Identification of this new receptor complex has potential implications for understanding host defense, as well as pathophysiologic, mechanisms.

The toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells.

The susceptibility to infections induced by Gram-negative bacteria is largely determined by innate immune responses to bacteria cell wall lipopolysaccharide (LPS). The stimulation of B cells by LPS enhances their antigen-presenting capacity and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing antibodies. Similar to macrophages and neutrophils, the LPS-induced activation of B cells is dependent on Toll-like receptor (TLR)4. Here, we demonstrate that the responses of B cells to LPS are also regulated by another TLR protein, RP105, which is predominantly expressed on mature B cells in mice and humans. The analysis of mice homozygous for the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS. Using originally LPS-unresponsive Ba/F3 cells expressing exogenous TLR4 and RP105, we demonstrate the functional cooperation between TLR4 and RP105 in LPS-induced nuclear factor kappaB activation. These data suggest the existence of the TLR4-RP105 signaling module in the LPS-induced B cell activation.

Solvent accessibility of the thrombin-thrombomodulin interface.

The kinetics of solvent accessibility at the protein-protein interface between thrombin and a fragment of thrombomodulin, TMEGF45, have been monitored by amide hydrogen/deuterium (H/2H) exchange detected by MALDI-TOF mass spectrometry. The interaction is rapid and reversible, requiring development of theory and experimental methods to distinguish H/2H exchange due to solvent accessibility at the interface from H/2H exchange due to complex dissociation. Association and dissociation rate constants were measured by surface plasmon resonance and amide H/2H exchange rates were measured at different pH values and concentrations of TMEGF45. When essentially 100% of the thrombin was bound to TMEGF45, two segments of thrombin became completely solvent-inaccessible, as evidenced by the pH insensitivity of the amide H/2H exchange rates. These segments form part of anion-binding exosite I and contain the residues for which alanine substitution abolishes TM binding. Several other regions of thrombin showed slowing of amide exchange upon TMEGF45 binding, but the exchange remained pH-dependent, suggesting that these regions of thrombin were rendered only partially solvent-inaccessible by TMEGF45 binding. These partially inaccessible regions of thrombin form both surface and buried contacts into the active site of thrombin and contain residues implicated in allosteric changes in thrombin upon TM binding.

Role of EP4 prostaglandin E2 receptor in the ischemic liver.

Prostaglandin E(2) (PGE(2)) mediates a variety of both innate and adaptive immunity responses through 4 distinct receptors, EP1-4. Recent studies have suggested the physiological and pathological role of EP4 in various inflammatory diseases. In this study, we investigated the importance of the EP4 receptor, and the efficacy of a selective EP4 agonist to alter hepatic ischemia/reperfusion (I/R) injury, an important cause of damage in liver resection and transplantation. We used an established murine I/R injury model, 70% partial hepatic ischemia for 90 minutes in male C57BL/6 mice. The local expression of EP4 messenger RNA (mRNA) in the naive and the ischemic liver at 2 hours after reperfusion was examined using RT-PCR analysis. Some mice received the EP4 selective agonist during I/R. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as markers of hepatic injury. EP4 expression in the liver was significantly up-regulated at 2 hours after reperfusion. Furthermore, treatment with EP4 agonist significantly inhibited hepatic injury at 6 hours after reperfusion. Our data suggest an inhibitory role of EP4 PGE(2) receptor in hepatic I/R injury and the therapeutic efficacy of a selective EP4 agonist for liver protection.

Characterization of the interface structure of enzyme-inhibitor complex by using hydrogen-deuterium exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

We investigated the interaction between a thiol protease inhibitor, cystatin, and its target enzyme, papain, by hydrogen-deuterium (H/D) exchange in conjunction with successive analysis by collision-induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain, examining the mass of each fragment produced by hexapole-CID. In the absence of papain, amide hydrogens in short amino-terminal fragments, such as b10(2+) and b12(2+), were highly deuterated within 1 min. Although fewer fragments were observed for the cystatin-papain complex in the hexapole-CID spectra, significant reductions in initial deuterium content were recognized throughout the sequence of cystatin. This suggests that complex formation restricted the flexibility of the whole cystatin molecule. Detailed analyses revealed that a marked reduction in deuterium content in the region of residues 1-10 persisted for hours, suggesting that the flexible N-terminal region was tightly fixed in the binding pocket with hydrogen bonds. Our results are consistent with those of previous studies on the structure and inhibition mechanism of cystatin. We demonstrated here that enzyme-inhibitor interactions can be characterized by H/D exchange in combination with CID in a hexapole ion guide using ESI-FTICR MS rapidly and using only a small amount of sample.

Insulin-like growth factor-I supports formation and activation of osteoclasts.

Although the action of insulin-like growth factor-I (IGF-I) on bone formation has been extensively investigated, the effect of the factor on bone resorption is little known. We first examined the effect of IGF-I on bone resorption by preexistent osteoclasts by using unfractionated bone cells cultured on dentin slices. IGF-I had a dose-related effect of stimulating bone resorption by preexistent osteoclasts, whereas IGF-II did not. When IGF-I was added to cultures of bone cells after preexistent osteoclasts had degenerated on the dentin slices, IGF-I increased the number of osteoclastic multinucleate cells (MNCs) with tartrate-resistant acid phosphatase activity. Moreover, IGF-I augmented the area of pits produced by newly formed osteoclasts. These results suggest that IGF-I directly or indirectly stimulates osteoclast recruitment and activation. Therefore, we next examined the direct effect of IGF-I on osteoclastic MNC formation by using hemopoietic blast cells. In the presence of 1,25-dihydroxyvitamin D3, IGF-I, like granulocyte-macrophage colony-stimulating factor (GM-CSF), dose-dependently increased the number of TRAP-positive MNCs. This stimulatory effect of IGF-I was additive with that of GM-CSF. Both IGF-I and GM-CSF supported the survival of the blast cells, indicating that IGF-I as well as GM-CSF are supporting factors for osteoclast differentiation. In addition, the blast cells possessed high affinity binding sites for IGF-I, with a Kd of 0.8 nM. These data, thus, indicate that IGF-I stimulates osteoclastic bone resorption through its direct or indirect action of supporting the generation and activation of osteoclasts.

Innate recognition of lipopolysaccharide by Toll-like receptor 4/MD-2 and RP105/MD-1.

The Toll family of receptors has been implicated in innate recognition and subsequent activation of defense programs against pathogens such as bacteria and fungi. TLR4, for example, signals the presence of lipopolysaccharide (LPS), a membrane constituent of Gram-negative bacteria. LPS signaling via TLR4 is greatly enhanced by a molecule referred to as MD-2, which is associated with the extracellular domain of TLR4. The TLR4/MD-2 complex, therefore, recognizes LPS. RP105, another member of the Toll family, has a striking similarity to TLR4 in that it is associated with an MD-2-like molecule MD-1. B-cells lacking RP105 are severely impaired in LPS-induced proliferation and antibody production. Studies employing transfectants showed that RP105/MD-1, like MD-2, enhances the LPS signaling via TLR4. RP105/MD-1 thus constitutes an LPS-signaling complex on B-cells. These results suggest that a variety of cell surface molecules regulate LPS recognition/signaling by TLR4.

Involvement of TLR4/MD-2 complex in species-specific lipopolysaccharide-mimetic signal transduction by Taxol.

Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice, but not in humans. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS-responsiveness on TLR4. Here we examined whether or not TLR4/MD-2 complex mediates a Taxol-induced signal by using transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion. Our results demonstrated that co-expression of mouse TLR4 and mouse MD-2 was required for Taxol responsiveness, and that the TLR4/MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice. We also found that mouse MD-2, but not human MD-2, is involved in Taxol signaling, suggesting that MD-2 is responsible for the species-specific responsiveness to Taxol.

Structure of melittin bound to phospholipid micelles studied using hydrogen-deuterium exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The structure of melittin bound to dodecylphosphocholine (DPC) micelles was investigated using hydrogen-deuterium (H/D) exchange in conjunction with collision induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of melittin with or without DPC micelles was analyzed at different time points examining the mass of each fragment ion produced by hexapole CID. When melittin existed alone in aqueous solution, more than 80% of amide hydrogens was exchanged within 10 s, and the deuterium content in each fragment ion showed high values throughout the experiments. When melittin was bound to DPC micelles, the percentage of deuterium incorporation into the fragment decreased remarkably at any time point. It increased little by little as the exchange period prolonged, indicating that some stable structure was formed by the interaction with DPC. The results obtained here were consistent with the previous studies on the helical structure of melittin carried out by NMR and CD analyses. The strategy using H/D exchange and MS analysis might be useful for studying structural changes of peptides and proteins caused by phospholipid micelles. It could also be applied to membrane-bound proteins to characterize their structure.

Characterization of mouse switch variant antibodies by matrix-assisted laser desorption ionization mass spectrometry and electrospray ionization mass spectrometry.

The amino acid sequences of mouse monoclonal antibodies have been characterized completely by mass spectrometry. Antibodies used in the present study were derived from mouse switch variant cell lines that produce four kinds of immunoglobulin Gs (IgGs). The amino acid sequences of these antibodies had not been estimated from the corresponding DNA sequence, so the sequences of IgGs derived from other strains were used as references in this study. Intra- and interchain disulfide bonds of the IgGs were reduced and carboxymethylated and the products were subjected to proteolytic digestion. The existence of N-linked oligosaccharides also was taken into account. The capabilities and limitations of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and capillary liquid chromatography-electrospray ionization mass spectrometry are discussed in the structural characterization of the antibodies. Based on our results, allotypes of the antibodies examined are discussed. This study shows that amino acid sequences of proteins, such as IgG, can be investigated without information about the corresponding DNA sequence if appropriate reference sequences derived from other strains can be used.

Adjuvant cyclophosphamide, methotrexate and 5-fluorouracil versus cyclophosphamide plus futraful for premenopausal patients with stage I-II and one- to three-node-positive breast cancer: results of a prospective randomized study.

A prospective randomized study was conducted to compare the adjuvant efficacy of 12 cycles of low-dose CMF (cyclophosphamide: CPA, methotrexate; MTX, 5-fluorouracil; 5-FU) with that of orally administered CPA plus FT (futraful) in premenopausal patients with stage I-II and one- to three-node-positive breast cancer. The 12-cycle CMF group (91 patients) received, 100 mg CPA orally on days 1 to 14 plus 20 mg MTX and 500 mg 5-FU intravenously (iv) on days 1 and 8 of each cycle. The CPA plus FT group (85 patients) received 100 mg CPA and 600 mg FT orally each day for one year. The background characteristics of the two groups were comparable. At 5 and 10 years, there were non-significant trends towards better disease-free and overall survival rates in the CMF group. Both treatments were well tolerated, but more patients in the CPA plus FT group refused to continue chemotherapy because of continuous gastrointestinal disturbances. No clear benefit of adding low-dose MTX to CPA and fluoropyrimidines was observed in this subgroup of Japanese patients. Further studies will be required to clarify the superiority of conventional-dose of CMF treatment to orally administered CPA plust FT treatment.

Structure of extracellular polysaccharides of Escherichia coli strains 36M, 72M, and 29M isolated from coligranuloma of chick intestine. I. Polysaccharide from E. coli 36M.

An extracellular polysaccharide was isolated from culture broth of Escherichia coli 36M, and fractionated on a column of Sephadex G-150 into two fractions; the high molecular weight portion (85% of the total polysaccharide) contained pyruvic acid, and showed a positive immune reaction with anti-Ps-I-serum obtained from a rabbit. The low molecular weight portion (15% of the total polysaccharide) showed a negative immune reaction. The methylation, Smith's degradation, partial acid hydrolysis and methanolysis of the higher molecular weight polysaccharide revealed a repeating structure as follows: (see article).

Structural studies of the Maillard reaction products of a protein using ion trap mass spectrometry.

The early stage products of the Maillard reaction of egg white lysozyme with D-glucose were studied. Incubation with D-glucose at 50 degrees C for 20 days caused reaction on the Lys and Arg residues of lysozyme as follows: all of the six Lys residues and 10 of the 11 Arg residues in lysozyme reacted with D-glucose; Arg 61 did not react with D-glucose. The Lys residues reacted with D-glucose with 1 mol of dehydration per mole of residue, and the Arg residues reacted with 2 mol of dehydration per mole of residue. The major constituent of the Amadori product with the epsilon-amino group of the Lys residue and the D-glucose was found to be the beta-pyranose form. The structure of the early stage product of the Maillard reaction of a protein with a sugar is the same as that of an amino acid with a sugar.

The Japanese National Registry data on pediatric CAPD patients: a ten-year experience. A report of the Study Group of Pediatric PD Conference.

OBJECTIVE: Over the past 10 years, we have collected data on pediatric patients less than 16 years of age from the National Registry of CAPD (continuous ambulatory peritoneal dialysis). We present our experience with this population. DESIGN: The database details the patient number, age, weight, height, outcome, cause of death, reason for terminating CAPD therapy, peritonitis, and catheter survival. PATIENTS: Of the 434 patients (239 males, 195 females), 37 patients (8.5%) were under 1 year of age and 164 patients (37.8%) were under 6 years of age. About half of the patients were less than 20 kg in weight, clearly indicating that CAPD was the treatment of choice in young children. The duration on CAPD for these patients was less than 2 years for 233 patients (54%), and was 5 years or more in 48 patients (11%). RESULTS: The outcome of the total patient population of 434 as of May, 1991, is as follows: 229 patients (52.8%) were being successfully treated with CAPD, 47 patients (10.8%) died, and 78 patients (18.0%) received a kidney transplantation. The patient survival rate was 85.6% at 3 years and 81.7% at 5 years. The technique survival rate was 74.9% at 3 years and 63.5% at 5 years. The rate of peritonitis was one episode over 28.6 patient-months. The mean catheter duration was 1.68 years. Peritonitis rate, catheter survival rate, and the rate of tunnel infection were worse in children less than 6 years of age than in older children. CONCLUSION: The excellent patient and technique survival rates indicate that CAPD is an effective treatment for children with end-stage renal disease in Japan. The high infection rates in younger children indicate that extra careful management is needed for this young age group.

The three dimensional distribution of chromium and nickel alloy welding fumes.

In the present study, the fumes generated from manual metal arc (MMA) and submerged metal arc (SMA) welding of low temperature service steel, and the chromium and nickel percentages in these fumes, were measured at various horizontal distances and vertical heights from the arc in order to obtain a three dimensional distribution. The MMA welding fume concentrations were significantly higher than the SMA welding fume concentrations. The highest fume concentration on the horizontal was shown in the fumes collected directly above the arc. The fume concentration vertically was highest at 50 cm height and reduced by half at 150 cm height. The fume concentration at 250 cm height was scarcely different from that at 150 cm height. The distribution of the chromium concentration vertically was analogous to the fume concentration, and a statistically significant difference in the chromium percentages was not found at the different heights. The nickel concentrations were not statistically significant within the welding processes, but the nickel percentages in the SMA welding fumes were statistically higher than in the MMA welding fumes. The highest nickel concentration on the horizontal was found in the fumes collected directly above the arc. The highest nickel concentration vertically showed in the fume samples collected at 50 cm height, but the greater the height the larger the nickel percentage in the fumes.

Cephalometric comparisons of craniofacial and upper airway structures in young children with obstructive sleep apnea syndrome.

We studied 15 children of preschool age who had obstructive sleep apnea syndrome to evaluate their dentofacial morphology in relation to the pharyngeal airway space. We found that (1) sleep apnea was often associated with mandibular retrognathia, (2) the lower incisors tended to exhibit a retrocline, (3) there were no significant differences in angular and linear measurements in the cranial base between patients with sleep apnea and a control group of 30 nonapneic children, and (4) the apneic children had a narrower epipharyngeal airway space than did the controls. These findings suggest that obstructive sleep apnea is probably caused by both adenoidal hypertrophy and abnormal development of the facial skeleton. We highly recommend cephalometric analysis as a valuable tool for conducting the presurgical evaluation of sleep apnea in children of preschool age.

[Radioimmunoassay of arginine vasopressin in unextracted random urine: clinical application for discrimination of diabetes insipidus].

The concentration of arginine vasopressin in unextracted random urine was directly measured by radioimmunoassay. The urinary AVP value (Vm) measured by RIA, their AVP value (Vc) revised by urinary creatinine and their AVP value (Vc/op) revised by urinary creatinine and urinary osmotic pressure were applied for discriminate diagnosis of diabetes insipidus (DI). In children with central DI, Vm was significantly lower then that of normal subjects and the distribution of Vm was different from that of normal subjects. In children with renal DI, the distribution of Vc/op was different from that of normal subjects. In conclusion the children with central DI and renal DI can be discriminated from the normal children in comparison with Vm and Vc/op.

[Prophylactic use of concentrated antithrombin III preparation in children with nephrotic syndrome].

Decreased plasma level of antithrombin III was assumed to be one of the major factors underlying hypercoagulable state in nephrotic syndrome. Concentrated antithrombin III preparation was given to 8 children with nephrotic syndrome with a plasma antithrombin III activity of less than 70%, to evaluate the effect on hypercoagulable state. Plasma antithrombin III activity was elevated to more than 70% in 7 of 8 children after treatment, while plasma levels of plasmin-alpha 2 plasmin inhibitor complex and FDP-D dimer were not significantly decreased. One patient developed brain infarction after the treatment, suggesting that prophylactic administration of concentrated antithrombin III preparation is not fully protective against thrombotic complications in nephrotic syndrome.

[A coagulation of fibrinolytic study in children with nephrotic syndrome: evaluation of hypercoagulability by measuring with plasmin- alpha 2 plasmin inhibitor complex and FDP D-dimer].

Increased plasma plasmin-alpha 2 plasmin inhibitor complex (PIC) and FDP D-dimer (D-dimer) reflect in vivo activation of the coagulation and fibrinolytic systems. We measured plasma PIC, D-dimer and other coagulation and fibrinolytic parameters in 42 children with nephrotic syndrome. Higher levels of both plasma PIC (0.67 +/- 0.48 micrograms/ml, P < 0.01) and D-dimer (154 +/- 105 ng/ml, P < 0.001) were observed at the active stage of nephrotic syndrome, compared to the remission stage (0.43 +/- 0.18 micrograms/ml, 72 +/- 48 ng/ml, respectively). In addition, plasma D-dimer correlated significantly with serum albumin, urinary protein, plasma fibrinogen, aPTT, and plasma antithrombin III activity. Although no thromboembolic complication was diagnosed clinically in the study period, increased plasma PIC or D-dimer may reflect a hypercoagulable state or subclinical thrombosis in nephrotic syndrome.

[Severe Legionella micdadei pneumonia effectively treated with hemofiltration therapy].

A 42-year-old man was admitted because of fever, productive cough, and progressive dyspnea. Chest x-ray films and computed tomographic scans disclosed dense consolidation in the left and right lung fields. No pathogenic agent was found despite extensive bacteriological examinations. Based on serological findings, the patient was given a diagnosis of acute pneumonia caused by Legionella micdadei. It has been reported that Legionnaire's disease is easily complicated by fatal systemic illnesses such as disseminated intravascular coagulation (DIC) and multiple organ failure. In fact, the patient suffered from severe hypotension and DIC on admission. Treatments against systemic complications were started together with intravenous administration of antibiotics including erythromycin. Continuous intravenous cathecolamin, however, failed to alleviate the patient's shock. We therefore applied endotoxin eliminating therapy using a polymyxin-B-column (PMX) and continuous hemofiltration (CHF). The patient recovered from critical shock immediately after the start of PMX, which together with CHF, alleviated his systemic complications. Although the factors responsible for fatal systemic complications in Legionnare's disease are not well-documented, our findings suggested that some substances removable by PMX and CHF play an important role in pathogenesis.