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ObjectiveAntimelanoma differentiation–associated gene 5 (anti-MDA5)–positive dermatomyositis with interstitial lung disease (DM-ILD) progresses rapidly and has a poor prognosis. Previously, we reported the efficacy of a combination therapy comprising high-dose glucocorticoids (GCs), calcineurin inhibitors (CNIs), and intravenous cyclophosphamide (IV CYC) in a multicenter clinical trial (UMIN000014344). In the present study, we evaluated the long-term outcomes and effects of induction therapy on the maintenance of remission.MethodsAll participants from our previous trial were followed up for > 5 years. Seventy-three other patients with anti-MDA5–positive DM-ILD from our institute were retrospectively integrated into the previous trial for further analysis. Sixty-eight patients achieved remission and survived for > 6 months. Based on the induction treatment, we classified the patients into 2 groups: (1) group T (n = 56), with triple combination therapy (GCs, CNIs, and IV CYC), and (2) group C (n = 12), with monotherapy/dual therapy. The recurrence-free and drug-withdrawal rates of immunosuppressive agents were compared.ResultsThe overall survival and recurrence-free survival rates at 5 years were 100% for the participants in the previous trial. The 5-year cumulative withdrawal rates for CNIs and GCs were 70% and 53%, respectively. In a comprehensive analysis, the recurrence-free rates in group T were higher than those in group C (90% vs 56%; P< 0.05). The drug-withdrawal rates of CNIs and GCs at 10 years in group T were also higher than those in group C (79% vs 0% and 43% vs 0%, respectively; P< 0.05).ConclusionTriple combination therapy in the induction phase can reduce the risk of recurrence and facilitate drug withdrawal in anti-MDA5–positive DM-ILD.

Background: Spondyloarthritis (SpA) is an autoimmune and autoinflammatory musculoskeletal disease characterised by systemic enthesitis. Recent research has focused on subclinical inflammatory bowel disease (IBD) in SpA pathogenesis. SKG mice, harbouring the Zap70 W163C mutation, increase autoreactive Th17 cells intrinsically, and in a conventional environment, they exhibit spontaneous arthritis with fungal factors. Under SPF conditions, they show SpA features, including enteritis, after peritoneal injection of β-1,3-glucan. This study aimed to clarify whether oral dextran sulfate sodium (DSS) administration, utilised in IBD model mice, can provoke SpA features in SKG mice under SPF conditions, focusing on the relationship between gut microorganisms and SpA pathogenesis. Methods: BALB/c and SKG mice were administered oral DSS, and their body weights, arthritis, and enthesitis scores were recorded. In another cohort, antibiotics (meropenem and vancomycin) or an anti-fungal agent (amphotericin B) was administered orally before DSS administration. The splenic Th1 and Th17 cell populations were examined before and after DSS administration using flow cytometry. Furthermore, the amount of circulating bacterial DNA in whole blood was measured by absolute quantitative polymerase chain reaction (qPCR), and the number and characteristics of bacterial species corresponding to these circulating DNA were analysed by next-generation sequencing (NGS). Results: Ankle enthesitis as a peripheral SpA feature was elicited in half of DSS-administered SKG mice, and none of the BALB/c mice. Pre-administration of antibiotics suppressed enthesitis, whilst an anti-fungal agent could not. Th1 and Th17 cell levels in the spleen increased after DSS administration, and this was suppressed by pre-administration of antibiotics. SKG mice have a larger amount of bacterial DNA in whole blood than BALB/c mice before and 1 day after the initiation of DSS administration. The number of bacterial species in whole blood increased after DSS administration in BALB/c and SKG mice. Some genera and species significantly specific to the DSS-treated SKG mouse group were also detected. Conclusion: Oral DSS administration alone elicited peripheral enthesitis in SKG mice with bacterial translocation accompanied by increased splenic Th1 and Th17 cell levels. Pre-administration of antibiotics ameliorated these DSS-induced SpA features. These findings suggest that intestinal bacterial leakage plays a pivotal role in SpA pathogenesis.

An epidermoid cyst (EC) is a benign soft tissue lesion that can form in any part of the body. However, the occurrence of EC in the maxilla is extremely rare. Herein, we report a rare case of an intraosseous EC located in the posterior region of the maxilla. A 13-year-old woman was referred to our department for evaluation of an intraosseous radiolucent lesion in the right maxilla. The asymptomatic lesion appeared well-defined and unilocular (15 × 20 mm) on the panoramic radiograph and computed tomography scan, and was surgically extracted under general anesthesia. Based on the results of the histopathological examination, a final diagnosis of EC was confirmed. EC should be included in the differential diagnosis of radiolucent lesions of the jaws and maxilla.

NG,NG-Dimethyl-L-arginine (asymmetric dimethylarginine: ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Plasma ADMA concentrations have been reported to increase in connection with diseases associated with an impaired endothelial L-arginine/NO pathway. In this study, we investigated the metabolism of ADMA in circulating blood cell populations to elucidate the regulatory mechanism of elevation of plasma ADMA, a novel risk factor for cardiovascular disease. We found by RT-PCR and Western blot analyses that protein arginine methyltransferase (PRMT)1 and dimethylarginine dimethylaminohydrolase (DDAH)-1, responsible for the biosynthesis and degradation of ADMA respectively, are expressed in erythrocytes (ECs), leukocytes, and platelets. We also identified a major ADMA-containing protein in ECs as catalase, confirmed by GST-pull down assay to bind to PRMT1 in vitro. This is the first report that the ADMA-metabolizing system, including the arginine methylation of proteins and the breakdown of free ADMA, occurs in circulating blood cell-populations, and that catalase in ECs might be a potential protein targeted by PRMT1.

We produced a monoclonal antibody (mAb) against NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM–100 nMADMA, and ADMA levels in human plasma and urine were found to be 0.78 μMand 51.3 μmol/g of creatinine respectively.

Tropomyosin had been identified as a major allergen in shrimp. The digestion and absorption of tropomyosin (Pen j 1) from kuruma prawn were investigated by ex vivo, in vitro, and in vivotechniques in order to elucidate the relationship between the allergenicity of the allergen and its gastrointestinal behavior. Pen j 1 transported the Caco-2 monolayer in a dose-dependent manner, and also enhanced the permeability of lucifer yellow, a marker of paracellular transportation, at high concentrations of the allergen. Studies with everted sacs revealed that Pen j 1 was rapidly degraded to small peptides (MW<3.5 kDa) and amino acids by intestinal proteases and absorbed from enterocytes. Furthermore, Pen j 1 orally administered to rats tended to remain in the stomach rather than in the small intestine, after which the allergen moved to the epithelial cells. These observations suggest that Pen j 1 may be absorbed viathe gastric mucosa prior to its digestion in the intestines.

Tri a Bd 27K, a major wheat allergen, is a glycoprotein. Tri a Bd 27K was found to occur in multiple forms by two-dimensional polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against the allergen. Furthermore, it was found that only Tri a Bd 27K components, which have N-linked glycan moieties with fucose residues, bound to IgE antibodies in the sera of wheat-sensitive patients.

We produced a monoclonal antibody (mAb) as a probe for detection of Tri a Bd 27K, a major wheat allergen. The mAb recognized the allergen purified from wheat flour, and the epitope on the allergen to the mAb was determined to be amino acid sequence 154VPWVVVDGKPL164of Tri a Bd 27K. Of the amino acid residues on the epitope, the amino acid residues responsible for the binding to the mAb were found to be W156, D160, G161, and P163.

8S-Lipoxygenase (8S-LOX) is known as a mouse homolog of human 15S-LOX-2. 15S-LOX-2 was down-regulated in malignant transformation of prostate epithelial cells, and its overexpression caused cell cycle arrest. To determine whether 8S-LOX would have a growth inhibitory effect on prostate carcinoma, we obtained human prostate carcinoma PC-3 cells expressing 8S-LOX or 15S-LOX-2. The growth rate of cells measured by colorimetric assay was reduced by expression of 8S-LOX and 15S-LOX-2. The addition to enzyme-expressing cells of arachidonic acid enhanced the growth suppressive effect, whereas the expression of catalytically inactive mutants did not affect cell growth, suggesting that the effect was product-dependent. DNA microarray and quantitative reverse transcription-PCR analyses revealed that the c-mycmRNA coding region determinant-binding protein/insulin-like growth factor II mRNA-binding protein 1 (CRD-BP/IMP-1), known as an oncofetal protein, was down-regulated in 8S-LOX- and 15S-LOX-2-expressing PC-3 cells. Targeted knockdown of CRD-BP/IMP-1 resulted in inhibition of the DNA synthesis rate of PC-3 cells as measured by bromodeoxyuridine incorporation. We propose that expression of 8S-LOX and 15S-LOX-2 suppresses CRD-BP/IMP-1 expression, resulting in inhibition of human prostate carcinoma PC-3 cell proliferation.

Tropomyosins have been identified as a common allergen in crustaceans, but their allergenicity is not well understood. In the present study, we isolated an allergen, Pen j 1, a tropomyosin from kuruma prawn Penaeus japonicus, and determined its N-terminal amino acid sequence. The cDNA encoding the allergen was cloned by 5′- and 3′-rapid amplification of cDNA ends (RACE), and was found to code for a protein which consists of 284 amino acid residues. Sequencing analyses indicated for the first time that mature tropomyosin is formed by the elimination of a leader peptide of nine amino acid residues. To elucidate the binding sites of IgE antibodies in the sera of shrimp-sensitive patients, various recombinant peptides were expressed in Escherichia colias fusion proteins with glutathione S-transferase (GST), and the examined with regard to reactivity with IgE antibodies. The IgE-binding epitopes were found to locate over the whole sequence of the allergen, and the IgE antibodies in the sera were found to recognize strongly its C-terminal region.

We have reported that orally administrated acetate contributed to suppression of lipogenesis in the liver and to reduction of lipid accumulation in the adipose tissue of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. The aim of this study was to investigate the effect of acetate on skeletal muscle and adipose tissues. Treatment with acetate showed a higher rate of oxygen consumption and a smaller size of lipid droplets in white adipose and brown adipose tissues. An analysis by Northern blotting revealed that the transcripts of myoglobin and Glut4 genes in the abdominal muscle of the OLETF rats were increased by acetate treatment, while the transcripts of lipolytic genes increased in the white adipose and brown adipose tissues. It is possible that acetate has effects on lipid metabolism in the skeletal muscles and the adipose tissues, and has functions that work against obesity and obesity-linked type 2 diabetes.

Tri a Bd 27K is the predominant allergen in wheat. In the present study, this allergen was purified to homogeneity from wheat flour. The N-terminal amino acid sequences of the purified allergen and the peptides obtained by its digestion, with trypsin were determined, and the allergen was shown to be a glycoprotein with an Asn-linked sugar moiety containing fucose residues.A cDNA encoding the allergen was obtained by polymerase chain reaction (PCR). The cDNA codes for a protein of 203 amino acid residues, with a molecular mass of 22,803 Da, that has two tentative sites glycosylated at Asn residues. Homology analysis suggested that the allergen might belong to a family of γ-interferon-inducible thiol reductases. The cDNA was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. However, unlike the allergen purified from wheat, recombinant Tri a Bd 27K was not immunoblotted with IgE antibodies in the serum of a wheat-sensitive patient.

Background: Pulmonary artery involvement (PAI) in Takayasu arteritis (TAK) can lead to severe complications, but the relationship between the two has not been fully clarified. Methods: We retrospectively investigated 166 consecutive patients with TAK who attended Kyoto University Hospital from 1997 to 2018. The demographic data, clinical symptoms and signs, comorbidities, treatments, and imaging findings were compared between patients with and without PAI. TAK was diagnosed based on the American College of Rheumatology Classification Criteria (1990) or the Japanese Clinical Diagnostic Criteria (2008). PAI was identified using enhanced computed tomography, magnetic resonance imaging, or lung scintigraphy. Results: PAI was detected in 14.6% (n= 24) of total TAK patients. Dyspnea (25.0% vs. 8.6%; p= 0.043), pulmonary arterial hypertension (PAH) (16.7% vs. 0.0%; p&lt; 0.001), ischemic heart disease (IHD) (29% vs. 9.3%; p= 0.018), respiratory infection (25.0% vs. 6.0%; p= 0.009), and nontuberculous mycobacteria (NTM) infection (20.8% vs. 0.8%; p&lt; 0.001) were significantly more frequent, and renal artery stenosis (0% vs. 17%; p= 0.007) was significantly less frequent in TAK patients with PAI than in those without PAI. PAI and biologics were risk factors for NTM. Conclusions: TAK patients with PAI more frequently have dyspnea, PAH, IHD, and respiratory infection, including NTM, than TAK patients without PAI.

The IgE-binding proteins in beer were examined by immunoblotting analysis with sera of patients sensitive to beer. Several proteins were immunoblotted with the sera, and among these, 18-kDa proteins were identified as new IgE-binding proteins in beer. Perhaps they originated from barley as a raw material.

Acetate has been found to have an inhibitory effect on the activity of carbohydrate-responsive element-binding protein (ChREBP) in cultured hepatocytes, this being a transcription factor that regulates several genes required for the conversion of glucose to fatty acids in the liver. The aim of this study was to investigate whether an oral administration of acetate would contribute to reducing lypogenic genes and protecting against obesity. We orally injected 5.2 mg/kg BW of acetate to obesity-linked type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. The treatment with acetate showed a marked reduction in lipid accumulation in the adipose tissue, protection against accumulation of fat in the liver, and improved glucose tolerance. An analysis by Northern blotting revealed that the transcripts of several lipogenic genes in the liver of OLETF rats were decreased by the acetate treatment. On the basis of those results, it was indicated that acetate was a potential compound to improve obesity and obesity-linked type 2 diabetes.

Di (2-ehtylhexyl) phthalate (DEHP) is a peroxisome proliferator and a drug having a hypolipidemic effect. The body-weight change of rats treated with DEHP was lower than that of rats in an untreated control group. Expressions of long-chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase, which are involved in fatty acid oxidation and acetate formation in mitochondria, showed an increase in the liver and testes of rats treated with DEHP. The expression of acetyl-CoA synthetase 1 was significantly decreased in the testes and relatively decreased in the liver, while the expression of acetyl-CoA synthetase 2 was significantly increased in the heart. Furthermore, the expressions of acetyl-CoA carboxylase in heart and testes showed a tendency to decrease. From these results, it is suggested that DEHP-treatment increased fatty acid oxidation and acetate formation in liver and testes, and that acetate utilization was increased in peripheral tissues such as the heart.

An NADPH-dependent α-keto amide reductase was purified from Saccharomyces cerevisiae. The molecular mass of the native enzyme was estimated to be 33 and 36 kDa by gel filtration chromatography and SDS–polyacrylamide gel electrophoresis, respectively. The purified enzyme showed a reducing activity not only for aromatic α-keto amides but also for aliphatic and aromatic α-keto esters. The internal sequence of the enzyme was identical with that of a hypothetical protein (ORF YDL 124w) coded by yeast chromosome IV.

The acylated plant pigments synthesized by lipase-catalyzed transesterification with aromatic acids were compared in respect of their light-resistance and radical-scavenging ability. With both the flavonols and anthocyanins, their acylated derivatives were more stable against illumination with fluorescent light than their non-acylated glucosides. Their radical-scavenging ability partially decreased or was retained by acylation to the glucoside molecules.

A 36-kDa allergen, Tri a Bd 36K, was purified from wheat albumin and characterized. The protein was similar to barley peroxidase BP-1 both in its amino acid sequence and peroxidase activity. The enzyme seemed to contain L-fucose and D-mannose and the glycan moiety reacted with IgE antibodies in a patient's serum.

Earthworm serine protease is more stable and is less affected by organic solvents and detergent than other proteases. However, it is inactivated, probably by autolysis, at 60°C or above under alkaline conditions. Further stabilization was managed by chemical modification of the enzyme with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and phenylglyoxal to protect the activity from the autolytic inactivation. Stabilization was possible also under acidic conditions, in which the stability of the enzyme was rather low, by immobilization with folded sheet mesoporous material. Thus, further stabilization of the enzyme has been achieved by chemical modification or immobilization.

The neurofibromatosis type 1 (NF1) tumor suppressor (neurofibromin) is thought to play crucial roles in cellular Ras‐ and cAMP‐dependent kinase (PKA)‐associated signals. In this study, we identified a cellular neurofibromin‐associating protein, NG,NG‐dimethylarginine dimethylaminohydrolase (DDAH) that is known as a cellular NO/NOS regulator. The interaction of DDAH was mainly directed to the C‐terminal domain (CTD) and to the cysteine/serine‐rich domain (CSRD) of neurofibromin, coinciding with the regions containing specific PKA phosphorylation sites. DDAH increased PKA phosphorylation of native neurofibromin in a dose‐dependent manner, especially affecting the phosphorylation of CSRD. These findings suggest that the PKA accessibility of neurofibromin was regulated via DDAH interaction, and this regulation may modulate the cellular function of neurofibromin that is implicated in NF1‐related pathogenesis.

Seven monoclonal antibodies (mAbs) against the wheat allergen, Tri a Bd 17 K, were prepared to obtain mAbs suitable for a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for determination of the allergen. Two of the mAbs strongly immunoblotted the allergen purified from wheat flour. However, only one (1G11) of them was found to be suitable for sandwich ELISA. Epitope mapping against mAb-1G11 on the allergen showed that the mAb recognized the peptide containing Lys-38 and Gln-39 of the allergen. We developed a sandwich ELISA method consisting of Aleuria aurantia lectin for fixing the allergen and 1G11 as the first antibody that enabled 4-4,000 ng/well of the allergen to be determined.

The neurofibromatosis type 1 (NF1) tumor suppressor (neurofibromin) is thought to play crucial roles in cellular Ras- and cAMP-dependent kinase (PKA)-associated signals. In this study, we identified a cellular neurofibromin-associating protein, NG, NG-dimethylarginine dimethylaminohydrolase (DDAH) that is known as a cellular NO/NOS regulator. The interaction of DDAH was mainly directed to the C-terminal domain (CTD) and to the cysteine/serine-rich domain (CSRD) of neurofibromin, coinciding with the regions containing specific PKA phosphorylation sites. DDAH increased PKA phosphorylation of native neurofibromin in a dose-dependent manner, especially affecting the phosphorylation of CSRD. These findings suggest that the PKA accessibility of neurofibromin was regulated via DDAH interaction, and this regulation may modulate the cellular function of neurofibromin that is implicated in NF1-related pathogenesis.

A micro-assay method for evaluating the allergenicity of soybean allergen was developed by using the mouse antiserum against Gly mBd 30K, a major soybean allergen, and RBL-2H3, a rat mucosal mast cell line. The antiserum against Gly mBd 30K was prepared by subcutaneously immunizing BALB/c mice with the allergen. The behavior by affinity-chromatography and the properties against heat treatment show that the reaginic activity of the antiserum resided in the IgE antibody specific for Gly mBd 30K. The developed assay method is shown to be useful for simulating IgE mediated type-I allergy and to be highly sensitive for detecting the allergen.

At least 15 allergenic proteins have been found in soybean using the sera of soybean-sensitive patients with atopic dermatitis [T. Ogawa et al., J. Nutr. Sci. Vitaminol., 35, 555–565 (1991)]. In the present study, a monoclonal antibody (mAb) against Gly m Bd 28K, one of the major allergens of soybean, was prepared, and Gly m Bd 28K was purified from defatted soybean flakes by five purification steps, including immunoaffinity chromatography with the mAb as a ligand. The purified allergen was found to be a glycoprotein with a molecular mass of 26 kDa. During the purification process the allergen was converted to more acidic proteins with the same molecular mass, suggesting that the allergen is unstable. The sugar composition and amino acid sequence of Gly m Bd 28K suggest that the allergen is a new glycoprotein with an Asn-linked sugar moiety. The distribution of the allergen in soybean products was examined by an immunoblotting technique with the mAb.

Yersinia pseudotuberculosiswhich were screened out depending on auto‐agglutination and Ca2+dependency, were examined for their production of hemagglutinin (HA), and its purification and characterization were performed. The HA with a broad reactivity with various mammalian erythrocytes was recovered from the culture supernatant of these strains grown at 37 C but not 25 C. HAs from two strains, R148R and T1040, were purified by salt precipitation, gel filtration and anion‐exchange chromatography by HPLC. Both purified HAs were cysteine‐deficient acidic protein with an apparent molecular weight in the range of 15,000 to 16,000. N‐terminal amino acid sequences of the first 25 residues were found to share 12% identity with that of afimbrial adhesin from enterotoxigenic Escherichia coli2230. Immunoelectron microscopy and immunodiffusion test with polyclonal antiserum raised against the purified R148RHA demonstrated that the HA was associated with the amorphous aggregates which were detached from bacteria. These results suggest that the HA of Y. pseudotuberculosisbelongs to a third type of HA produced by the yersinial species.

In the food, particularly the feed, industry, large quantities of soya bean protein products are used for the formulation of end-products destined for human or animal consumption. These basic ingredients and the final end products often have to fulfill specific requirements regarding the presence of antinutritional compounds (ANCs) and their allergenic potential. Therefore, a special need exists for the identification and characterization of ANCs and the causative factors behind allergy and hyper sensitivity. This paper describes the results of a comparative study of a previously identified unknown antigenic soya bean P22-25 protein with the first, recently described Gly m I (or P34) soya bean allergen. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting analysis were carried out using calf anti-soya protein sera and a murine anti-Gly m I (or P34) monoclonal antibody in order to detect the antigenic soya bean P22-25 protein and the allergen Gly m (or P34) respectively. The results showed that these proteins were different. In addition, a number of industrial soya bean products were analyzed for the presence of both apparent antigenicity (essentially ss-conglycinin and P22-25) using calf anti-soya protein sera and the allergen Gly m I (or P34), and both ss-conglycinin, P22-25 and P34 could be detected to a varying extent in the products analyzed.

A cDNA clone encoding 4-aminobenzoate hydroxylase (EC 1.14.13.27) has been isolated using a probe prepared by PCR on the basis of partially determined amino acid sequences of the enzyme. The cDNA contained 1380-base pair open reading frame encoding 460 amino acid residues (Mr 50 974), 14-base pair 5′-untranslated region and 123-base pair 3′-untranslated region including a poly(A) tail of 20 nucleotides. All of the partially determined amino acid sequences were shown to be included in the deduced amino acid sequence. Homology analyses showed that the two regions on the enzyme share other flavoproteins such as salicylate hydroxylase and p-hydroxybenzoate hydroxylase.

A nonfimbrial hemagglutinin (HA) of Yersinia pseudotuberculosiswas observed by immunoelectron microscopy using monospecific HA antiserum and protein A‐gold conjugate. The HA, an amorphous but morphologically identifiable entity, was located in a region distal to or detached from the outer edge of bacteria.

The thermolysis of N-t-alkyl-, and N-aryl-(2,2-dichlorocyclopropyl)methyleneamines yielded directly 1-t-alkyl, and 1-aryl-3-chloropyrroles as the major products along with a slight amount of the 2-chloro isomers. The transformation to the 3-chloropyrroles was enhanced in polar solvents. But basic additives directed the thermal rearrangement into the 1,2-bond cleavage of the cyclopropane ring, leading to the 2-chloropyrroles. On the other hand, (difluorocyclopropyl)methyleneamines were pyrolyzed exclusively to 3-fluoropyrroles under 1,3-bond cleavage. The ionic mechanism involving a heterolytic dissociation of chlorine atom under 1,3-bond scission of the cyclopropane ring is proposed for the rearrangement to 3-chloropyrroles, while a homolytic cleavage pathway is proposed for 3-fluoropyrroles. The present thermolysis supplies a unique preparative tool for 2-, and 3-halo-4-phenylpyrrole derivatives.

We found a novel protein in the postmitochondria supernatant fraction of rat liver, which is soluble in 5% perchloric acid and strongly inhibits protein synthesis in a rabbit reticulocyte lysate system. The protein extracted from the supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The protein was shown to consist of two identical subunits with a molecular mass of 14 kDa. By immunoscreening with the rabbit antisera against the protein, a cDNA encoding the protein was cloned and sequenced. The cDNA contained an open reading frame of 411 base pairs encoding a 136-amino acid protein with a molecular mass of 14,149 Da. The deduced amino acid sequence was completely identical with that constructed from all of the above peptides. Interestingly, the perchloric acid-soluble protein inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner from RNase A. The protein is likely to inhibit an initiation stage of cell-free protein synthesis. Among the rat tissues tested, the protein was located only in liver and kidney. These findings are the first report on a new inhibitor that may be involved in the regulation of protein synthesis in those tissues.

An enzyme catalyzing the hydrolysis of triacylglycerol was purified from an earthworm. The N-terminal amino acid sequence and the catalytic function of the purified enzyme were identical to those of Isozyme C, an isozyme of the earthworm-serine proteases. No other lipase proteins were found in the earthworm cells. The isozyme might act on the hydrolysis of triacylglycerol as well as the protein decomposition.

A wild-type soybean contains three major allergenic proteins, Gly mBd 30K, the α-subunit of conglycinin, and Gly mBd 28K. A genetically mutated soybean (Tohoku 124), which was originally developed as a cultivar lacking the α- and α′-subunits of conglycinin, was also found to lack Gly mBd 28K from immunoblot analysis using monoclonal antibodies specific to Gly mBd 28K. This finding indicates the possibility to prepare soy milk and soy proteins containing none of the three major allergenic soybean proteins from this cultivar. By applying the previous removal procedure [Samoto et al., Biosci. Biotech. Biochem., 60, 1911–1913 (1996)] to Tohoku 124, the substantially complete removal of the three major allergenic proteins from the soy milk was attained. The removal rates of Gly mBd 30K, α-subunit of conglycinin, and Gly mBd 28K were 99.8, 100, and 100%, respectively.

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