Infrared spectroscopy of an endohedral water in fullerene.

An infrared absorption spectroscopy study of the endohedral water molecule in a solid mixture of H2O@C60 and C60 was carried out at liquid helium temperature. From the evolution of the spectra during the ortho-para conversion process, the spectral lines were identified as para-H2O and ortho-H2O transitions. Eight vibrational transitions with rotational side peaks were observed in the mid-infrared: ω1, ω2, ω3, 2ω1, 2ω2, ω1 + ω3, ω2 + ω3, and 2ω2 + ω3. The vibrational frequencies ω2 and 2ω2 are lower by 1.6% and the rest by 2.4%, as compared to those of free H2O. A model consisting of a rovibrational Hamiltonian with the dipole and quadrupole moments of H2O interacting with the crystal field was used to fit the infrared absorption spectra. The electric quadrupole interaction with the crystal field lifts the degeneracy of the rotational levels. The finite amplitudes of the pure v1 and v2 vibrational transitions are consistent with the interaction of the water molecule dipole moment with a lattice-induced electric field. The permanent dipole moment of encapsulated H2O is found to be 0.50 ± 0.05 D as determined from the far-infrared rotational line intensities. The translational mode of the quantized center-of-mass motion of H2O in the molecular cage of C60 was observed at 110 cm-1 (13.6 meV).

Evidence for the role of INSL3 on sperm production in boars by passive immunisation.

Insulin-like factor 3 (INSL3), previously called relaxin-like factor, is essential for foetal testis descent and has been implicated in sperm production in adult males. This study investigated the role of INSL3 in sperm production by examining the effect of neutralising INSL3 by passive immunisation on testicular function and sperm output in boars. Six male Duroc boars were randomly assigned to passive immunisation and control groups (n = 3 each). The immunisation group was intravenously injected with an IgG fraction of anti-INSL3 antibody developed against the B domain of INSL3 at 2-week intervals from 21-40 weeks of age. The control group was treated with normal IgG in the same manner. Antibody administration reduced testis weight and caused a fourfold increase in the frequency of apoptotic germ cells, which was associated with upregulation of the pro-apoptotic caspase 3 and BAX, and downregulation of the anti-apoptotic XIAP and BCL2, and a substantial marked reduction in sperm concentration. Neutralising INSL3 delivered by passive immunisation reduced testis weight and sperm concentration by inducing germ cell apoptosis, suggesting that INSL3 acts as a germ cell survival/anti-apoptotic factor in the maintenance of sperm production.

Surotomycin versus vancomycin in adults with Clostridium difficile infection: primary clinical outcomes from the second pivotal, randomized, double-blind, Phase 3 trial.

BACKGROUND: The available treatment options for Clostridium difficile infection (CDI) are limited by high recurrence rates. Surotomycin was a novel bactericidal cyclic lipopeptide in development to treat CDI that demonstrated non-inferiority to vancomycin in a Phase 2 trial. OBJECTIVES: To assess surotomycin safety and clinical response (non-inferiority versus vancomycin) at the end of treatment (EOT) of CDI. Additionally, to assess surotomycin response over time and sustained response at 30-40 days post-EOT (superiority versus vancomycin). PATIENTS AND METHODS: Patients with CDI were randomized (1:1) to receive twice-daily oral surotomycin 250 mg alternating with twice-daily placebo or four-times-daily oral vancomycin 125 mg for 10 days in this Phase 3, double-blind, multicentre, international trial. Clinical response over time and sustained clinical response were monitored until the end of the trial, through a follow-up period of 30-40 days. Clinical Trial Registration: NCT01598311. RESULTS: A total of 285 and 292 patients with confirmed CDI were randomized to receive surotomycin and vancomycin, respectively. Surotomycin-associated clinical response at EOT was non-inferior to vancomycin (surotomycin/vancomycin: 83.4%/82.1%; difference 1.4%, 95% CI - 4.9, 7.6). Following treatment with surotomycin, both clinical response over time (stratified log-rank test, P = 0.277) and sustained clinical response (63.3%/59.0%; difference 4.3%, 95% CI - 3.6, 12.2) did not demonstrate superiority versus vancomycin at end of trial. Both treatments were generally well tolerated. CONCLUSIONS: Surotomycin demonstrated non-inferiority to vancomycin for CDI clinical response at EOT. Surotomycin did not demonstrate superiority to vancomycin for clinical response over time or sustained clinical response rate.

Association of immunoglobulin G4 and free light chain with idiopathic pleural effusion.

The cause of pleural effusion remains uncertain in approximately 15% of patients despite exhaustive evaluation. As recently described immunoglobulin (Ig)G4-related disease is a fibroinflammatory disorder that can affect various organs, including the lungs, we investigate whether idiopathic pleural effusion includes IgG4-associated etiology. Between 2000 and 2012, we collected 830 pleural fluid samples and reviewed 35 patients with pleural effusions undiagnosed after pleural biopsy at Yamaguchi-Ube Medical Center. Importantly, IgG4 immunostaining revealed infiltration of IgG4-positive plasma cells in the pleura of 12 patients (34%, IgG4+ group). The median effusion IgG4 level was 41 mg/dl in the IgG4+ group and 27 mg/dl in the IgG4- group (P < 0·01). The light and heavy chains of effusion IgG4 antibodies of patients in the IgG4+ group were heterogeneous by two-dimensional electrophoresis, indicating the absence of clonality of the IgG4 antibodies. Interestingly, the κ light chains were more heterogeneous than the λ light chains. The measurement of the κ and λ free light chain (FLC) levels in the pleural fluids showed significantly different κ FLC levels (median: 28·0 versus 9·1 mg/dl, P < 0·01) and κ/λ ratios (median: 2·0 versus 1·2, P < 0·001) between the IgG4+ and IgG4- groups. Furthermore, the κ/λ ratios were correlated with the IgG4+ /IgG+ plasma cell ratios in the pleura of the IgG4+ group. Taken together, these results demonstrate the involvement of IgG4 in certain idiopathic pleural effusions and provide insights into the diagnosis, pathogenesis and therapeutic opportunities of IgG4-associated pleural effusion.

Efficacy and safety of ustekinumab in Japanese patients with severe atopic dermatitis: a randomized, double-blind, placebo-controlled, phase II study.

BACKGROUND: Ustekinumab, a fully human monoclonal antibody against interleukin-12/23, may potentially be effective for severe atopic dermatitis (AD) treatment. OBJECTIVES: To evaluate efficacy and safety of ustekinumab 45 mg and 90 mg in patients with severe AD. METHODS: In this randomized, placebo-controlled, phase II study, Japanese patients (aged 20-65 years) with severe or very severe AD entered a 12-week double-blind treatment period during which they received (1 : 1 : 1) ustekinumab 45 mg, 90 mg or placebo subcutaneous injections at weeks 0 and 4, with follow-up until week 24. The primary efficacy end point was percentage change from baseline in Eczema Area and Severity Index (EASI) score at week 12. Major secondary efficacy end points included the proportion of patients achieving EASI 50, EASI 75, Investigator's Global Assessment score 0-1, change from baseline Atopic Dermatitis Itch Scale and Dermatology Life Quality Index. RESULTS: A total of 79 patients were randomized [ustekinumab 45 mg (n = 24), 90 mg (n = 28), placebo (n = 27)]. Ustekinumab treatment showed nonsignificant improvement in least square mean change from baseline EASI score at week 12 [45 mg: -38·2%, 95% confidence interval (CI) -21·02-19·51; P < 0·94 and 90 mg: -39·8%, 95% CI -21·84-17·14; P < 0·81] vs. placebo (-37·5%). A nonsignificant improvement in major secondary efficacy end points was observed in both ustekinumab groups vs. placebo. The most common treatment-emergent adverse events were nasopharyngitis and worsened AD (higher in placebo vs. ustekinumab groups). CONCLUSIONS: Ustekinumab 45 mg and 90 mg did not demonstrate meaningful efficacy in Japanese patients with severe AD. The treatment was generally well tolerated.

Heterogeneous nucleation of catalyst-free InAs nanowires on silicon.

We report on the heterogeneous nucleation of catalyst-free InAs nanowires on Si(111) substrates by chemical beam epitaxy. We show that nanowire nucleation is enhanced by sputtering the silicon substrate with energetic particles. We argue that particle bombardment introduces lattice defects on the silicon surface that serve as preferential nucleation sites. The formation of these nucleation sites can be controlled by the sputtering parameters, allowing the control of nanowire density in a wide range. Nanowire nucleation is accompanied by unwanted parasitic islands, but careful choice of annealing and growth temperature allows us to strongly reduce the relative density of these islands and to realize samples with high nanowire yield.

Immunocytochemical staining for stratifin and OCIAD2 in bronchial washing specimens increases sensitivity for diagnosis of lung cancer.

OBJECTIVE: Brushing or washing cytology taken at bronchoscopy is a standard diagnostic procedure for lung cancer. The present study evaluated the sensitivity of immunocytochemical diagnosis of lung cancer using bronchial washing materials. METHODS: We collected bronchial washing samples taken at bronchoscopy between July 2012 and July 2013 at Tsukuba University Hospital and studied 106 cases that were finally diagnosed as lung cancer. We collected exfoliated cells using a thin-layer advanced cytology assay system (TACAS(™)) and performed cytological diagnosis using Papanicolaou staining. As controls, we randomly selected 30 tumour-negative cases from among samples collected during the same period. Using these materials, we also examined the expression of stratifin (14-3-3 sigma) (n = 92) and OCIAD2 ovarian immunoreactive antigen domain 2) (n = 106) by immunocytochemistry, as these are considered to be broad spectrum immune markers for lung adenocarcinoma including early invasive lung adenocarcinoma. RESULTS: Using Papanicolaou staining, 52 out of 106 lung cancers (49.1%) were diagnosed as positive. However, positivity was increased to 63.0% by immunocytochemistry using anti-stratifin or anti-OCIAD2 antibodies. Biopsies were taken in 103/106 cases and cancer was diagnosed in 60/103, (58.3%). The sensitivity of stratifin or OCIAD2 was significantly higher than that of Papanicolaou staining (P = 0.027), but immunocytochemistry detected false-positive cells in 3/30 cases (10%) for stratifin and 2/30 cases (7%) for OCIAD2. CONCLUSION: Immunocytochemical staining for stratifin and OCIAD2 improved diagnostic sensitivity for lung cancers but diagnostic specificity was lower than that for cytology alone. The immunostains carried up to a 10% risk of a false-positive result and therefore positive staining must be confirmed by morphological evidence of malignancy.

ITPKC and CASP3 polymorphisms and risks for IVIG unresponsiveness and coronary artery lesion formation in Kawasaki disease.

Functional single-nucleotide polymorphisms (SNPs) in inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) (rs28493229) and caspase-3 (CASP3) (rs113420705; formerly rs72689236) are associated with susceptibility to Kawasaki's disease (KD). To evaluate the involvement of these 2 SNPs in the risk for intravenous immunoglobulin (IVIG) unresponsiveness, we investigated 204 Japanese KD patients who received a single IVIG dose of 2 g kg(-1) (n=70) or 1 g kg(-1) daily for 2 days (n=134). The susceptibility allele of both SNPs showed a trend of overrepresentation in IVIG non-responders and, in combined analysis of these SNPs, patients with at least 1 susceptible allele at both loci had a higher risk for IVIG unresponsiveness (P=0.0014). In 335 prospectively collected KD patients who were treated with IVIG (2 g kg(-1)), this 2-locus model showed a more significant association with resistance to initial and additional IVIG (P=0.011) compared with individual SNPs. We observed a significant association when all KD patients with coronary artery lesions were analyzed with the 2-locus model (P=0.0031). Our findings strongly suggest the existence of genetic factors affecting patients' responses to treatment and the risk for cardiac complications, and provide clues toward understanding the pathophysiology of KD inflammation.

Efficacy and safety of switching from basal insulin to sitagliptin in Japanese type 2 diabetes patients.

Basal-supported oral therapy (BOT) is often used to treat poorly controlled type 2 diabetes. However, patients sometimes experience nocturnal and early morning hypoglycemia. Thus, maintaining targeted glycemic control by BOT is limited in some patients. We assessed the efficacy and safety of replacing basal insulin by sitagliptin therapy in Japanese type 2 diabetes patients on BOT. Forty-nine subjects were sequentially recruited for the 52-week, prospective, single arm study. Patients on BOT therapy were switched from basal insulin to sitagliptin. The primary endpoint was change in HbA1c in 52 weeks. The secondary endpoints were dropout rate, changes in body weight, frequency of hypoglycemia, and relationship between change in HbA1c and insulin secretion capacity evaluated by glucagon loading test. The average dose of basal insulin was 15.0±8.4 units. Sixteen subjects (31.3%) were dropped because replacement by sitagliptin was less effective for glycemic control. In these subjects, diabetes duration was longer, FPG and HbA1c at baseline were higher, and insulin secretion capacity was lower. Change in HbA1c in 52 weeks was - 4 mmol/mol (95% CI - 5 to - 4 mmol/mol) (p<0.05). Change in body weight was - 0.71 kg (95% CI - 1.42 to - 0.004 kg) (p<0.05). Frequency of hypoglycemia was decreased from 1.21±1.05 to 0.06±0.24 times/month. HbA1c level was improved if C-peptide index (CPI) was over 1.19. In conclusion, basal insulin in BOT can be replaced by sitagliptin with a decrease in HbA1c level and frequency of hypoglycemia in cases where insulin secretion capacity was sufficiently preserved.

Non-conventional yeast strains: Unexploited resources for effective commercialization of second generation bioethanol.

The conventional yeast (Saccharomyces cerevisiae) is the most studied yeast and has been used in many important industrial productions, especially in bioethanol production from first generation feedstock (sugar and starchy biomass). However, for reduced cost and to avoid competition with food, second generation bioethanol, which is produced from lignocellulosic feedstock, is now being investigated. Production of second generation bioethanol involves pre-treatment and hydrolysis of lignocellulosic biomass to sugar monomers containing, amongst others, d-glucose and D-xylose. Intrinsically, S. cerevisiae strains lack the ability to ferment pentose sugars and genetic engineering of S. cerevisiae to inculcate the ability to ferment pentose sugars is ongoing to develop recombinant strains with the required stability and robustness for commercial second generation bioethanol production. Furthermore, pre-treatment of these lignocellulosic wastes leads to the release of inhibitory compounds which adversely affect the growth and fermentation by S. cerevisae. S. cerevisiae also lacks the ability to grow at high temperatures which favour Simultaneous Saccharification and Fermentation of substrates to bioethanol. There is, therefore, a need for robust yeast species which can co-ferment hexose and pentose sugars and can tolerate high temperatures and the inhibitory substances produced during pre-treatment and hydrolysis of lignocellulosic materials. Non-conventional yeast strains are potential solutions to these problems due to their abilities to ferment both hexose and pentose sugars, and tolerate high temperature and stress conditions encountered during ethanol production from lignocellulosic hydrolysate. This review highlights the limitations of the conventional yeast species and the potentials of non-conventional yeast strains in commercialization of second generation bioethanol.

Serum undercarboxylated osteocalcin level increases with 48 weeks of teriparatide treatment in pre-treated elderly rheumatoid arthritis patients who use anti-resorptive drugs.

AIM: The serum undercarboxylated osteocalcin (ucOC) level, a biochemical bone marker of vitamin K insufficiency, is often affected by anti-osteoporosis drugs. There have been no reports regarding the relationship between ucOC and teriparatide. SUBJECTS AND METHODS: We conducted a prospective observational study of 26 female rheumatoid arthritis (RA) patients. The patients were divided into 3 groups: those who underwent a direct switch from anti-resorptive drugs to teriparatide (12 cases), those who started teriparatide without pre-treatment (5 cases), and the control patients (9 cases). The median age (interquartile range) of the patients in each group was 75 (67-77), 82 (78-84), and 69 (62-80) yr, respectively. All patients, except controls, received 48-week treatments of teriparatide. We analyzed the median 48-week changes from baseline of the serum ucOC levels with the Steel-Dwass method. RESULTS: The median change from baseline in the direct switch group was higher than that in other groups (p<0.05). CONCLUSIONS: The serum ucOC levels increased with treatment of teriparatide in elderly RA patients, especially when the patients received pre-treatment.

Sitagliptin add-on to low dosage sulphonylureas: efficacy and safety of combination therapy on glycaemic control and insulin secretion capacity in type 2 diabetes.

AIMS: To assess the efficacy and safety of combination therapy with sitagliptin and low dosage sulphonylureas on glycaemic control and insulin secretion capacity in Japanese type 2 diabetes. METHODS: Eighty-two subjects were sequentially recruited for the 52-week, prospective, single arm study. Sitagliptin was added on to sulphonylureas (glimepride or gliclazide) with or without metformin. The primary endpoint was a change in A1C. The secondary endpoints were changes in BMI, insulin secretion capacity, blood pressure and urinary albumin excretion, unresponsive rate, and hypoglycaemia. Insulin secretion capacity was evaluated by glucagon loading test. RESULTS: Change in A1C was -0.80% (95% CI -0.90 to -0.68) (p < 0.001). Change in BMI, systemic and diastolic blood pressure, and urinary albumin excretion were -0.38 kg/m(2) (95% CI -0.72 to -0.04) (p < 0.05), -6.7/-3.6 mmHg (95% CI -10.0 to -3.4/-4.8 to -2.4) (p < 0.001), and -43.2 mg/gCr (95% CI -65.7 to -20.8) (p < 0.001) respectively. Mild hypoglycaemia was observed in three cases. The unresponsive rate was 6.1%. Glucagon loading test showed that 0-min and 6-min CPR at baseline and 52-week were not significantly changed: 0-min CPR, 1.58 ± 0.58-1.71 ± 0.73 ng/ml; 6-min CPR, 3.48 ± 1.47-3.58 ± 1.21 ng/ml. Insulin secretion capacity, CPI and SUIT index at baseline did not predict the efficacy of the combination therapy. The final dosages of glimepiride and gliclazide were 1.44 ± 0.90 mg and 34.5 ± 15.3 mg respectively. The dosage of sitagliptin was increased from 50 mg to 69.0 ± 24.5 mg in 52-week. CONCLUSIONS: The combination therapy with sitagliptin and low dosage sulphonylureas was safe and effective for glycaemic control. Glucagon loading test indicated that 1 year administration of sitagliptin and sulphonylureas preserved insulin secretion capacity.

Evaluation of the efficacy and safety of the combination of gemcitabine and nedaplatin for elderly patients with advanced non-small-cell lung cancer.

OBJECTIVE: The aim of the present study was to retrospectively assess the safety and efficacy of the combination of gemcitabine and nedaplatin in elderly patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Patients ≥75 years with previously untreated NSCLC who underwent chemotherapy consisting of gemcitabine (800 mg/m(2) on days 1 and 8) and nedaplatin (80 mg/m(2) on day 1) every 3 weeks were retrospectively analyzed. RESULTS: Of the 35 patients, 28 were men and 7 were women, with a mean age of 78 years (range 75-87); 10 patients had stage IIIB disease and 25 patients had stage IV disease. The overall response rate was 45.7% (95% confidence interval 28.8-63.4). The median survival time was 14 months (range 3-44). Grade 3-4 toxicities included neutropenia in 74.3%, thrombocytopenia in 48.6%, anemia in 34.3%, hepatic dysfunction in 11.4%, and infection in 2.9%. There were no treatment-related deaths. There were no differences in response rate and survival between patients aged 75-79 years and patients ≥80 years, although grade 3-4 thrombocytopenia and anemia were significantly more frequent in patients ≥80 years. CONCLUSION: Our results suggest that the combination of gemcitabine and nedaplatin is effective and well tolerated for selected elderly patients with advanced NSCLC.

Interaction potential and infrared absorption of endohedral H2 in C60.

We have measured the temperature dependence of the infrared spectra of a hydrogen molecule trapped inside a C(60) cage, H(2)@C(60), in the temperature range from 6 to 300 K and analyzed the excitation spectrum by using a five-dimensional model of a vibrating rotor in a spherical potential. The electric dipole moment is induced by the translational motion of endohedral H(2) and gives rise to an infrared absorption process where one translational quantum is created or annihilated, ΔN = ±1. Some fundamental transitions, ΔN = 0, are observed as well. The rotation of endohedral H(2) is unhindered but coupled to the translational motion. The isotropic and translation-rotation coupling part of the potential are anharmonic and different in the ground and excited vibrational states of H(2). The vibrational frequency and the rotational constant of endohedral H(2) are smaller than those of H(2) in the gas phase. The assignment of lines to ortho- and para-H(2) is confirmed by measuring spectra of a para enriched sample of H(2)@C(60) and is consistent with the earlier interpretation of the low temperature infrared spectra [Mamone et al., J. Chem. Phys. 130, 081103 (2009)].

Infrared spectroscopy of endohedral HD and D2 in C60.

We report on the dynamics of two hydrogen isotopomers, D(2) and HD, trapped in the molecular cages of a fullerene C(60) molecule. We measured the infrared spectra and analyzed them using a spherical potential for a vibrating rotor. The potential, vibration-rotation Hamiltonian, and dipole moment parameters are compared with previously studied H(2)@C(60) parameters [M. Ge, U. Nagel, D. Hüvonen, T. Rõõm, S. Mamone, M. H. Levitt, M. Carravetta, Y. Murata, K. Komatsu, J. Y.-C. Chen, and N. J. Turro, J. Chem. Phys. 134, 054507 (2011)]. The isotropic part of the potential is similar for all three isotopomers. In HD@C(60), we observe mixing of the rotational states and an interference effect of the dipole moment terms due to the displacement of the HD rotation center from the fullerene cage center.

The persistence of drug-induced fever by teicoplanin--a case report.

OBJECTIVE: It was reported that the drug-induced fever of teicoplanin tended to persist after cessation of treatment. It is considered that the long half-life of teicoplanin causes the phenomenon. However there was no detailed report regarding plasma concentration of teicoplanin during onset of drug induced-fever. Therefore we investigated the relation between persistence of drug-induced fever and plasma concentration of teicoplanin. CASE: A 38-year-old male patient on the Left Ventricular Assist System (LVAS) was treated with teicoplanin for methicillin-resistant Staphylococcus aureus (MRSA) and he experienced drug-induced fever. Plasma concentrations of teicoplanin were measured not only during the treatment with the drug but also after it was discontinued. As such, plasma concentration was measured even when the fever had subsided. RESULTS: On Day 9 of treatment, the dose was increased from 400 to 600 mg, but the patient had a fever of about 38 - 39 °C. When the treatment was discontinued, it took 9 days for the fever to subside to a temperature of about 37 °C. The half-life of elimination of teicoplanin in the elimination phase is about 108 h, which is long. The fever persisted until the plasma concentration decreased to below 10 µg/ml, which is the effective trough concentration, and subsided when the estimated blood concentration was 7.5 µg/ml. CONCLUSIONS: We suggest that there is the possibility that the drug-induced fever due to teicoplanin persisted until the plasma concentration had decreased adequately. Close monitoring of plasma concentration is necessary, particularly when teicoplanin clearance is decreased such as in patients with renal dysfunction.

Comparative effects of ascobin and glutathione on copper homeostasis and oxidative stress metabolism in mitigation of copper toxicity in rice.

Copper (Cu) pollution of agricultural land is a major threat to crop production. Exogenous chemical treatment is an easily accessible and rapid approach to remediate metal toxicity, including Cu toxicity in plants. We compared the effects of ascobin (ASC; ascorbic acid:citric acid at 2:1) and glutathione (GSH) in mitigation of Cu toxicity in rice. Plants subjected to Cu stress displayed growth inhibition and biomass reduction, which were connected to reduced levels of chlorophylls, RWC, total phenolic compounds, carotenoids and Mg2+ . Increased accumulation of ROS and malondialdehyde indicated oxidative stress in Cu-stressed plants. However, application of ASC or GSH minimized the inhibitory effects of Cu stress on rice plants by restricting Cu2+ uptake and improving mineral balance, chlorophyll content and RWC. Both ASC and GSH pretreatments reduced levels of ROS and malondialdehyde and improved activities of antioxidant enzymes, suggesting their roles in alleviating oxidative damage. A comparison on the effects of ASC and GSH under Cu stress revealed that ASC was more effective in restricting Cu2+ accumulation (69.5% by ASC and 57.1% by GSH), Ca2+ and Mg2+ homeostasis, protection of photosynthetic pigments and activation of antioxidant defence mechanisms [catalase (110.4%), ascorbate peroxidase (76.5%) and guaiacol peroxidase (39.0%) by ASC, and catalase (58.9%) and ascorbate peroxidase (59.9%) by GSH] in rice than GSH, eventually resulting in better protection of ASC-pretreated plants against Cu stress. In conclusion, although ASC and GSH differed in induction of stress protective mechanisms, both were effective in improving rice performance in response to Cu phytotoxicity.

Molecular cloning and expression of the human interleukin 5 receptor.

Human interleukin 5 (IL-5) plays an important role in proliferation and differentiation of human eosinophils. We report the isolation of cDNA clones from cDNA libraries of human eosinophils by using murine IL-5 receptor alpha chain cDNA as a probe. Analysis of the predicted amino acid sequence indicated that the human IL-5 receptor has approximately 70% amino acid sequence homology with the murine IL-5 receptor and retains features common to the cytokine receptor superfamily. One cDNA clone encodes a glycoprotein of 420 amino acids (Mr 47,670) with an NH2-terminal hydrophobic region (20 amino acids), a glycosylated extracellular domain (324 amino acids), a transmembrane domain (21 amino acids), and a cytoplasmic domain (55 amino acids). Another cDNA encodes only the extracellular domain of this receptor molecule. Other cDNA clones encode molecules having diversified cytoplasmic domains. COS7 cells transfected with the cDNA expressed a approximately 60-kD protein and bound IL-5 with a single class of affinity (Kd = 250-590 pM). The Kd values were similar to that observed in normal human eosinophils. In contrast to the murine 60-kD alpha chain, which binds IL-5 with low affinity (Kd = approximately 10 nM), the human alpha chain homologue can bind IL-5 with much higher affinity by itself. RNA blot analysis of human cells demonstrated two transcripts (approximately 5.3 and 1.4 kb). Both of them were expressed in normal human eosinophils and in erythroleukemic cell line TF-1, which responds to IL-5. The human IL-5 receptor characterized in this paper is essential for signal transduction, because expression of this molecule in murine IL-3-dependent cell line FDC-P1 allowed these cells to proliferate in response to IL-5.

Reconstitution of the functional receptors for murine and human interleukin 5.

The murine interleukin 5 receptor (mIL-5R) is composed of two distinct subunits, alpha and beta. The alpha subunit (mIL-5R alpha) specifically binds IL-5 with low affinity. The beta subunit (mIL-5R beta) does not bind IL-5 by itself, but forms the high-affinity receptor with mIL-5R alpha. mIL-5R beta has been revealed to be the mIL-3R-like protein, AIC2B which is shared with receptors for IL-3 and granulocyte/macrophage colony-stimulating factor. We demonstrated here the reconstitution of the functional receptors for murine and human IL-5 on the mouse IL-2-dependent cell line, CTLL-2. CTLL-2 was transfected with the cDNAs for mIL-5R alpha and/or AIC2B. Only CTLL-2 transfectant expressing both mIL-5R alpha and AIC2B expressed the high-affinity receptor and proliferated in response to murine IL-5. Then CTLL-2 was transfected with the cDNAs for hIL-5R alpha and/or KH97 (beta c), the human homologue of AIC2B. Though beta c did not contribute much to binding affinity of hIL-5R, only CTLL-2 transfectant expressing both hIL-5R alpha and beta c proliferated in response to human IL-5. These results showed that the beta subunit is indispensable in IL-5 signal transduction. We further investigated the function of IL-5-specific alpha subunit in transmitting IL-5 signals. Mutant mIL-5R alpha, which lacks its whole cytoplasmic domain, was transfected into mouse IL-3-dependent cell line, FDC-P1 expressing AIC2B intrinsically. The resulting transfectant did not respond to IL-5, though the transfectant expressed the high-affinity IL-5R, indicating that the cytoplasmic portion of the alpha subunit also has some important role in IL-5-mediated signal transduction.