Evaluation of changes in periodontal bacteria in healthy dogs over 6 months using quantitative real-time PCR.

Porphyromonas gulae, Tannerella forsythia and Campylobacter rectus are considered dominant periodontal pathogens in dogs. Recently, quantitative real-time PCR (qRT-PCR) methods have been used for absolute quantitative determination of oral bacterial counts. The purpose of the present study was to establish a standardized qRT-PCR procedure to quantify bacterial counts of the three target periodontal bacteria (P. gulae, T. forsythia and C. rectus). Copy numbers of the three target periodontal bacteria were evaluated in 26 healthy dogs. Then, changes in bacterial counts of the three target periodontal bacteria were evaluated for 24 weeks in 7 healthy dogs after periodontal scaling. Analytical evaluation of each self-designed primer indicated acceptable analytical imprecision. All 26 healthy dogs were found to be positive for P. gulae, T. forsythia and C. rectus. Median total bacterial counts (copies/ng) of each target genes were 385.612 for P. gulae, 25.109 for T. forsythia and 5.771 for C. rectus. Significant differences were observed between the copy numbers of the three target periodontal bacteria. Periodontal scaling reduced median copy numbers of the three target periodontal bacteria in 7 healthy dogs. However, after periodontal scaling, copy numbers of all three periodontal bacteria significantly increased over time (p<0.05, Kruskal-Wallis test) (24 weeks). In conclusion, our results demonstrated that qRT-PCR can accurately measure periodontal bacteria in dogs. Furthermore, the present study has revealed that qRT-PCR method can be considered as a new objective evaluation system for canine periodontal disease.

Measurement of specific IgE antibodies to Ses i 1 improves the diagnosis of sesame allergy.

BACKGROUND: The number of reported cases of allergic reactions to sesame seeds (Sesamum indicum) has increased significantly. The specific IgE tests and skin prick tests presently available for diagnosis of sesame allergy are all based on crude sesame extract and are limited by their low clinical specificity. Thus, oral food challenge (OFC) is still the gold standard in the diagnosis. OBJECTIVE: The aim was to identify the allergen components useful to diagnose sesame-allergic children with the goal to reduce the number of OFCs needed. METHODS: Ninety-two sesame-sensitized children were consecutively enrolled and diagnosed based on OFC or convincing history. Specific IgE to purified native 11S globulin (nSes i 11S), 7S globulin (nSes i 7S), 2S albumin (nSes i 2S), and two recombinant 2S albumins (rSes i 1 and rSes i 2) was measured by ELISA and/or ImmunoCAP (rSes i 1/streptavidin application). RESULTS: Based on area under curve (AUC) values from receiver operating characteristic (ROC) analysis, rSes i 1 was shown to have the best diagnostic performance of the allergen components in ELISA. The experimental rSes i 1 ImmunoCAP test had larger AUC (0.891; 95% CI, 0.826-0.955) compared to the commercially available sesame ImmunoCAP (0.697; 95% CI, 0.589-0.805). The clinical sensitivity and specificity for the rSes i 1 ImmunoCAP test at optimal cut-off (3.96 kUA /L) were 86.1% and 85.7%, respectively. CONCLUSION AND CLINICAL RELEVANCE: Sensitization to Ses i 1 is strongly associated with clinical sesame allergy. Measurement of specific IgE to rSes i 1 could reduce the numbers of OFCs needed.

Construction of novel immune-related signature for prediction of pathological complete response to neoadjuvant chemotherapy in human breast cancer.

BACKGROUND: The aim of this study was to construct a novel prediction model for the pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) using immune-related gene expression data. PATIENTS AND METHODS: DNA microarray data were used to perform a gene expression analysis of tumor samples obtained before NAC from 117 primary breast cancer patients. The samples were randomly divided into the training (n = 58) and the internal validation (n = 59) sets that were used to construct the prediction model for pCR. The model was further validated using an external validation set consisting of 901 patients treated with NAC from six public datasets. RESULTS: The training set was used to construct an immune-related 23-gene signature for NAC (IRSN-23) that is capable of classifying the patients as either genomically predicted responders (Gp-R) or non-responders (Gp-NR). IRSN-23 was first validated using an internal validation set, and the results showed that the pCR rate for Gp-R was significantly higher than that obtained for Gp-NR (38 versus 0%, P = 1.04E-04). The model was then tested using an external validation set, and this analysis showed that the pCR rate for Gp-R was also significantly higher (40 versus 11%, P = 4.98E-23). IRSN-23 predicted pCR regardless of the intrinsic subtypes (PAM50) and chemotherapeutic regimens, and a multivariate analysis showed that IRSN-23 was the most important predictor of pCR (odds ratio = 4.6; 95% confidence interval = 2.7-7.7; P = 8.25E-09). CONCLUSION: The novel prediction model (IRSN-23) constructed with immune-related genes can predict pCR independently of the intrinsic subtypes and chemotherapeutic regimens.

Susceptibility quantitative trait loci for pathogenic leucocytosis in SCG/Kj mice, a spontaneously occurring crescentic glomerulonephritis and vasculitis model.

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse, a model of human crescentic glomerulonephritis (CrGN) and systemic vasculitis, is characterized by the production of myeloperoxidase-specific anti-neutrophil cytoplasmic autoantibody (MPO-ANCA) and marked leucocytosis. This study was performed to identify the specific populations of leucocytes associated with CrGN and susceptibility loci for pathogenic leucocytosis. Four hundred and twenty female (C57BL/6 × SCG/Kj) F2 intercross mice were subjected to serial flow cytometry examination of the peripheral blood (PB). Kidney granulocytes and monocytes were examined histopathologically. Linkage analyses were performed with 109 polymorphic microsatellite markers. Correlation studies revealed that increase of the granulocytes, F4/80(+) cells, CD3(+) CD4(-) CD8(-) T cells and dendritic cells (DCs) in peripheral blood (PB) were associated significantly with glomerulonephritis, crescent formation and vasculitis. In kidney sections, F4/80(low) cells were observed in crescent, while F4/80(high) cells were around the Bowman's capsules and in the interstitium. Numbers of F4/80(+) cells in crescents correlated significantly with F4/80(+) cell numbers in PB, but not with numbers of F4/80(+) cells in the interstitium. Genome-wide quantitative trait locus (QTL) mapping revealed three SCG/Kj-derived non-Fas QTLs for leucocytosis, two on chromosome 1 and one on chromosome 17. QTLs on chromosome 1 affected DCs, granulocytes and F4/80(+) cells, but QTL on chromosome 17 affected DCs and granulocytes. We found CrGN-associated leucocytes and susceptibility QTLs with their positional candidate genes. F4/80(+) cells in crescents are considered as recruited inflammatory macrophages. The results provide information for leucocytes to be targeted and genetic elements in CrGN and vasculitis.

Efficacy and safety of canagliflozin in Japanese patients with type 2 diabetes: a randomized, double-blind, placebo-controlled, 12-week study.

AIMS: We examined the efficacy, safety and tolerability of canagliflozin, a sodium glucose co-transporter 2 inhibitor, in Japanese patients with type 2 diabetes (T2DM) undergoing diet and exercise therapy. METHODS: Patients aged 20-80 years with T2DM diagnosed ≥3 months previously, and HbA1c of 6.9-9.9% were randomized to 50, 100, 200 or 300 mg canagliflozin or placebo once daily for 12 weeks. The primary and secondary endpoints were changes in HbA1c, fasting plasma glucose (FPG), urinary glucose/creatinine and postprandial glycaemic parameters following a meal test. The safety assessments included adverse events (AEs) and clinical laboratory tests. RESULTS: Overall, 383 patients were randomized to receive either placebo (n = 75), or 50 mg (n = 82), 100 mg (n = 74), 200 mg (n = 77) or 300 mg canagliflozin (n = 75). At week 12, significant reductions in HbA1c were observed in all canagliflozin groups relative to placebo (-0.61, -0.80, -0.79 and -0.88% for 50, 100, 200 and 300 mg, respectively, versus +0.11% for placebo; all, p < 0.01). FPG and postprandial glycaemic parameters improved significantly in the canagliflozin groups. Body weight was significantly decreased by canagliflozin. No deaths or drug-related serious AEs were reported. There was no dose-dependent increase in the incidence of AEs in the canagliflozin groups. The incidence of hypoglycaemia was low; episodes were not severe or dose dependent. Canagliflozin did not affect serum creatinine levels or the urinary albumin/creatinine ratio. CONCLUSIONS: Treatment with canagliflozin for 12 weeks significantly improved glycaemic control and reduced body weight in Japanese patients with T2DM. Canagliflozin was well tolerated.

Clinicopathological analysis of GATA3-positive breast cancers with special reference to response to neoadjuvant chemotherapy.

BACKGROUND: The aim of this study was to investigate the clinicopathological characteristics of GATA binding protein 3 (GATA3)-positive breast cancers as well as the association of GATA3 expression with response to chemotherapy. PATIENTS AND METHODS: Tumor specimens obtained before neoadjuvant chemotherapy [paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide)] from breast cancer patients (n = 130) were subjected to immunohistochemical and mutational analysis of GATA3 and DNA microarray gene expression analysis for intrinsic subtyping. RESULTS: Seventy-four tumors (57%) were immunohistochemically positive for GATA3. GATA3-positive tumors were significantly more likely to be lobular cancer, estrogen receptor (ER)-positive, progesterone receptor (PgR)-positive, Ki67-negative, and luminal A tumors. Somatic mutations were found in only three tumors. Pathological complete response (pCR) was observed in 8 (11%) GATA3-positive tumors and in 22 (39%) GATA3-negative tumors. multivariate analysis showed that tumor size, human epidermal growth factor receptor 2 (her2), and gata3 were independent predictors of pcr. CONCLUSIONS: GATA3-positive breast cancers showed luminal differentiation characterized by high ER expression and were mostly classified as luminal-type tumors following intrinsic subtyping. Interestingly, GATA3 was an independent predictor of response to chemotherapy, suggesting that GATA3 might be clinically useful as a predictor of a poor response to chemotherapy.

Recombinant high molecular weight-glutenin subunit-specific IgE detection is useful in identifying wheat-dependent exercise-induced anaphylaxis complementary to recombinant omega-5 gliadin-specific IgE test.

BACKGROUND: Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a special form of food allergy typically induced by exercise after ingestion of wheat products. We identified wheat omega-5 gliadin and high molecular weight-glutenin subunit (HMW-glutenin) as major allergens for WDEIA and clarified that simultaneous detection of serum IgE binding to synthetic epitope peptides of these allergens identifies more than 90% of WDEIA patients. However, the short synthetic peptides are not suitable for CAP-fluorescent enzyme-immunoassay (CAP-FEIA), which is widely utilized for detecting allergen-specific IgE. OBJECTIVE: In this study, we constructed a CAP-FEIA with recombinant HMW-glutenin, and evaluated its usefulness in identifying the patients with WDEIA. METHODS: Recombinant HMW-glutenin was expressed as histidine-tag protein in E. coli and purified by histidine-tag affinity column. Wheat, gluten, recombinant omega-5 gliadin, epitope peptide of HMW-glutenin, native and recombinant HMW-glutenin specific IgE in the sera from 48 patients with WDEIA, 16 patients with atopic dermatitis (AD) who had no immediate allergic reaction after wheat ingestion and 12 healthy controls were determined by using CAP-FEIA method. RESULTS: In 16 AD patients without wheat allergy 12 of them (75%) had positive results for native HMW-glutenin test in contrast to epitope peptide of HMW-glutenin (12.5%) and recombinant HMW-glutenin test (12.5%). These results indicate the native HMW-glutenin test has low specificity. Sensitivity and specificity of the IgE test with recombinant HMW-glutenin were 16.7% and 92.9%. These are well compatible with results obtained by using epitope peptide of HMW-glutenin. However, sensitivity and specificity reached to 93.8% and 92.9%, when the test was combined to the test with recombinant omega-5 gliadin. CONCLUSIONS AND CLINICAL RELEVANCE: We demonstrated that recombinant HMW-glutenin is best for CAP-FEIA system in point of stability and specificity and confirmed that detection of specific IgE against recombinant HMW-glutenin is useful for diagnosis of WDEIA when combined with the CAP-FEIA (recombinant omega-5 gliadin) test.

Risk factors for Salmonella prevalence in laying-hen farms in Japan.

Human salmonellosis cases, particularly those caused by Salmonella Enteritidis, have been closely linked to egg consumption. This epidemiological survey was conducted to determine the baseline Salmonella prevalence and identify the risk factors for Salmonella prevalence in laying-hen farms in Japan. Caecal excrement samples and dust samples were obtained from 400 flocks in 338 laying-hen farms. Salmonella was identified in 20.7% of the farms and 19.5% of the flocks. The prevalence of Salmonella was significantly higher in flocks reared in windowless houses than in those reared in open houses. In addition, the risk of Salmonella presence was significantly higher when the windowless house farms implemented induced moulting or in-line egg processing. Efforts to reduce human salmonellosis in Japan should continue to focus on the establishment of control measures in laying-hen farms, especially those with windowless houses implementing induced moulting and equipped with in-line egg processing.

Differential effects of estrogen receptors in the rostral ventrolateral medulla in Goldblatt hypertension.

Previous studies have shown that 17ß-estradiol plays a cardioprotective role in the central nervous system (CNS) of male rats. The aim of the present study was to determine the influence of 17ß-estradiol on sympathetic vasomotor activity and blood pressure in a renovascular hypertensive Goldblatt two-kidney one-clip (2K-1C) male rat model. We also determined the influence of angiotensin II AT1 receptor on the expression of estrogen receptors (ERα, ERß, and G protein-coupled ER (GPER)) in the rostral ventrolateral medulla (RVLM) of Goldblatt rats. Experiments were performed in Goldblatt and age-matched control rats six weeks after clipping of renal artery to induce hypertension. Microinjection of 17ß-estradiol into the RVLM led to a greater reduction in mean arterial pressure and renal sympathetic nerve activity in controls than in 2K-1C rats. Microinjection of the GPER agonist G-1 into the RVLM led to a significantly greater increase in mean arterial pressure and renal sympathetic nerve activity in 2K-1C rats. Expression levels of estrogen receptors GPER and ERα, but not ERß, were significantly higher in the RVLM of 2K-1C rats than in that of the control rats. Chronic treatment with losartan significantly reduced the expression levels of estrogen receptors in the RVLM of 2K-1C rats. Taken altogether, the data suggest that the imbalance of actions between ERα and GPER, particularly with the predominance of GPER in the RVLM, contributes to sympathetic overactivation in male rats with Goldblatt hypertension. AT1-Angiotensin II receptor in the RVLM upregulated estrogen receptor expression in male Goldblatt rats.

Increased serum levels of a proliferation-inducing ligand in patients with bullous pemphigoid.

BACKGROUND: B cells have been demonstrated to have critical roles in developing autoimmune bullous diseases. Recently identified tumor necrosis factor-like molecules, B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) are essential molecules for B cell development, survival, and proliferation. Although the functions of APRIL have not been fully evaluated, recent studies suggest that circulating levels of APRIL are increased in various autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. OBJECTIVES: To determine serum APRIL levels in patients with pemphigus vulgaris (PV) and bullous pemphigoid (BP), and compare those with clinical findings and laboratory findings. PATIENTS/METHODS: Sera from 15 PV patients, 43 BP patients, and 15 normal controls were subjected to ELISA assays to measure serum APRIL, BAFF, Dsg3, and BP180 levels. RESULTS AND CONCLUSIONS: Circulating APRIL levels were significantly elevated in BP patients but not in PV patients, and correlated with serum BAFF levels. Our study revealed that serum APRIL levels tended to be increased in the quite early stage of disease. In conclusion, circulating APRIL levels may be a useful marker for early activation of autoimmune diathesis, and furthermore, an effective therapeutic target molecule in patients with BP.

Clinical and experimental features of MuSK antibody positive MG in Japan.

We investigated the presence of antibodies (Abs) against muscle-specific tyrosine kinase (MuSK) in Japanese myasthenia gravis (MG) patients. MuSK Abs were found in 23 (27%) of 85 generalized seronegative MG (SNMG) patients but not in any of the ocular MG patients. MuSK Ab-positive patients were characterized as having female dominance (M:F, 5:18), age range at onset 18 to 72 (median 45) years old, and prominent oculobulbar symptoms (100%) with neck (57%) or respiratory (35%) muscle weakness. Limb muscle weakness was comparatively less severe (52%), thymoma absent. Most patients had good responses to simple plasma exchange and steroid therapy. MuSK IgG from all 18 patients was exclusively the IgG 4 subclass and bound mainly with the MuSK Ig 1-2 domain. Serial studies of 12 individuals showed a close correlation between the variation in MuSK Ab titers and MG clinical severity (P = 0.01 by Kruskal-Wallis). MuSK Ab titers were sharply decreased in patients who had a good response to early steroid therapy or simple plasma exchange, but there was no change, or a rapid increase on exacerbation after thymectomy. Measurement of MuSK Ab titers aids in the diagnosis of MG and the monitoring of clinical courses after treatment.

Alternative splicing variants of IkappaB beta establish differential NF-kappaB signal responsiveness in human cells.

To release transcription factor NF-kappaB into the nucleus, the mammalian IkappaB molecules IkappaB alpha and IkappaB beta are inactivated by phosphorylation and proteolytic degradation. Both proteins contain conserved signal-responsive phosphorylation sites and have conserved ankyrin repeats. To confer specific physiological functions to members of the NF-kappaB/Rel family, the different IkappaB molecules could vary in their specific NF-kappaB/Rel factor binding activities and could respond differently to activation signals. We have demonstrated that both mechanisms apply to differential regulation of NF-kappaB function by IkappaB beta relative to IkappaB alpha. Via alternative RNA processing, human IkappaB beta gives rise to different protein isoforms. IkappaB beta1 and IkappaB beta2, the major forms in human cells, differ in their carboxy-terminal PEST sequences. IkappaB beta2 is the most abundant species in a number of human cell lines tested, whereas IkappaB beta1 is the only form detected in murine cells. These isoforms are indistinguishable in their binding preferences to cellular NF-kappaB/Rel homo- and heterodimers, which are distinct from those of IkappaB alpha, and both are constitutively phosphorylated. In unstimulated B cells, however, IkappaB beta1, but not IkappaB beta2, is found in the nucleus. Furthermore, the two forms differ markedly in their efficiency of proteolytic degradation after stimulation with several inducing agents tested. While IkappaB beta1 is nearly as responsive as IkappaB alpha, indicative of a shared activation mechanism, IkappaB beta2 is only weakly degraded and often not responsive at all. Alternative splicing of the IkappaB beta pre-mRNA may thus provide a means to selectively control the amount of IkappaB beta-bound NF-kappaB heteromers to be released under NF-kappaB stimulating conditions.

Purification of senescence marker protein-30 (SMP30) and its androgen-independent decrease with age in the rat liver.

Age-associated changes in the soluble proteins from rat liver were examined by a newly developed two-dimensional cellulose acetate membrane electrophoresis (2D-CAME). We detected and isolated a novel rat liver protein, the amounts of which decreased androgen-independently with aging. We designated this protein, whose molecular mass was 30 kDa and pI value was 4.9, as senescence marker protein-30 (SMP30). The expression of SMP30 was not modified by castration or treatment with testosterone propionate after castration. Age-associated decrease of SMP30 level was also recognized in aged female. We noted another protein with a pI value of 7.3 that decreased with aging. Its expression seemed to be androgen-dependent and it was markedly expressed only in young and adult male rat livers. We then purified SMP30 and prepared an anti-serum to SMP30. Immunohistochemical analysis showed that the localization of SMP30 was restricted to liver and kidney among numerous organs tested. Although the amount of SMP30 in the liver was relatively large and the tissue distribution was characteristic, no known protein corresponds to this protein.

Isolation and characterization of genomic and cDNA clones encoding mouse senescence marker protein-30 (SMP30).

We isolated and characterized genomic and cDNA clones encoding mouse senescence marker protein-30 (SMP30), the protein amounts of which are known to decrease with aging in the livers of rats. This decrease in the expression of SMP30 is independent of androgen. SMP30 is a calcium binding protein also called regucalcin. The expression of SMP30 in aged mouse liver and 5' flanking sequence of the genome were also characterized. The cDNA contained an open reading frame encoding 299 amino acids with a calculated molecular weight of 33-404. The amino acid sequence of mouse SMP30 showed 94% similarity to rat SMP30 and 89% to human SMP30. Northern blot analysis specifically detected mouse SMP30 transcript in the liver and also confirmed its significant decrease with aging. Analysis of the murine genomic clone revealed that SMP30 was organized by seven exons and six introns, spanning approx. 17.5 kb. Primer extension analysis revealed that two major transcription initiation sites were localized at 101 bp and 102 bp upstream from ATG translation initiation codon. The nucleotide (nt) sequence of 5' flanking region showed a TATA-like sequence, a CAAT box, and SP-1 sites at nt -29, -72 and -169 in the promoter region, respectively. Interestingly, we found two classes of C/EBP sites which are highly and constantly expressed in the liver, in addition to AP-2, AP-1, GATA-1, AP-1/GRE and GAGA sites. These results provide important clues for understanding the regulatory mechanism of SMP30 gene expression and its relationship to aging.

Isolation of cDNA clone encoding rat senescence marker protein-30 (SMP30) and its tissue distribution.

We have isolated and characterized two cDNA clones encoding senescence marker protein-30 (SMP30), the amounts of which are known to decrease androgen-independently with aging in the livers of rats. Of these cDNA clones, one consisted of 1588 bp nucleotides and the other of 1195 bp nucleotides generated by alternative polyadenylation. These two cDNA clones shared the same open reading frame, but the larger species had 393 bp nucleotides of 3' untranslated region in addition to the first polyadenylation site of smaller species. Northern hybridization analysis showed that two species of mRNA (1.7 kb and 1.4 kb) located in the liver and kidney were consistent with these short and long forms of cDNA. The open reading frame, 897 bp could encode 299 amino acids. The estimated molecular weight and pI of the deduced polypeptide were 33,387 and 5.1, respectively. Furthermore, immunohistochemical analysis confirmed that SMP30 was preferentially localized in the hepatocytes and renal proximal tubular epithelium. Genomic Southern hybridization analysis demonstrated that SMP30 was widely conserved among higher animals. A computer-assisted homology analysis of nucleic acid and protein databases revealed no remarkable homology with other known proteins. Therefore, SMP30 seems to be a novel protein. In addition, the existence of putative A-U rich mRNA degradation signals and protein degradation signals (PEST sequence) in the structure of SMP30 may suggest important regulatory function of this unique protein manifested by changes in its concentrations.

Involvement of calcium signaling in the fibronectin-stimulated macrophage recognition of oxidatively damaged erythrocytes.

Macrophages recognize oxidatively damaged autologous erythrocytes, and cell surface fibronectin of macrophages enhances the recognition (Beppu et al., FEBS Lett. 295 (1991) 135-140). In the present study, mechanisms of enhanced macrophage recognition of oxidatively damaged erythrocytes by fibronectin were investigated. Monolayers of thioglycollate-induced mouse peritoneal macrophages with cell surface fibronectin recognized autologous erythrocytes oxidized with an iron catalyst ADP/Fe(3+). The macrophage recognition of the oxidized erythrocytes was inhibited partially by pretreatment of the macrophage monolayers with a Ca(2+) channel blocker (diltiazem), calmodulin inhibitors (W-7, trifluoperazine, chlorpromazine and dibucaine), an inhibitor of myosin light chain kinase (ML-9), a microfilament formation inhibitor (cytochalasin B), phospholipase A(2) inhibitors (4-bromophenacyl bromide, mepacrine) and cyclooxygenase inhibitors (indomethacin and aspirin). Monolayers of macrophages depleted of fibronectin by trypsinization lost the ability of recognizing oxidized erythrocytes, but acquired the ability when stimulated with a fibronectin-coated coverslip. The recognition of fibronectin-stimulated trypsinized macrophages was also inhibited by the above inhibitors. On treatment with Ca ionophore A23187, trypsinized macrophages acquired the ability to recognize oxidized erythrocytes. The recognition of Ca ionophore-stimulated trypsinized macrophages was inhibited by the above inhibitors except the Ca(2+) channel blocker. These results indicate that the Ca(2+) signaling including Ca(2+) influx, calmodulin activation and myosin light chain phosphorylation are involved in the fibronectin stimulation of the recognition of macrophages for oxidized erythrocytes. Involvement of microfilament formation and arachidonate cascade in the fibronectin stimulation was also suggested.

Identification of soluble interleukin-4 receptor in rat glomerular epithelial cells(1).

Interleukin (IL)-4, a pleiotropic cytokine involved in many glomerular diseases, is regulated positively by membrane-bound IL-4R (mIL-4R) and negatively by soluble IL-4R (sIL-4R). Because natural sIL-4R has been documented only in mice, we undertook this study in rats to determine whether they, too, express sIL-4R, particularly in kidney cells. A pair of IL-4R primers was designed for this purpose and used in the polymerase chain reaction. As a result, sIL-4R was found not only in rats spleen cells but also in their glomerular epithelial cells (GEC). Sequence analysis revealed that the mRNA of rat sIL-4R has a 75-bp insert sequence. This insert generated a termination TGA codon upstream from the transmembrane region, resulting in formation of the sIL-4R. Subsequent screening of the kidney cDNA library enabled us to obtain the whole 3605-bp cDNA of sIL-4R; the full-length 3530-bp mIL-4R cDNA was also identified as a much longer sequence than previously published. Among the total 39 clones positive for IL-4R, two were confirmed as sIL-4R, and 37 clones were positive for mIL-4R. Next, the translated portion of sIL-4R cDNA was constructed into an expression vector, enabling us to obtain a recombinant sIL4R-myc fusion protein. By using this recombinant sIL-4R, we proved that sIL-4R can antagonize the IL-4-induced proliferation of spleen cells. Present study demonstrated that sIL-4R is expressed in kidney cells and antagonistically functional.

ATF-2-binding regulatory element is responsible for the Ly49A expression in murine T lymphoid line, EL-4.

To understand the mechanism of Ly49A-expression and its significance in T-cell differentiation, we analyzed the 5'-flanking region of the Ly49A gene in a search for the Ly49A-regulatory element. Since very few known regulatory elements have been found in this region, presumably a novel regulatory sequence(s) could exist. Accordingly, we defined the 13-bp regulatory element, 5'-ATGACGAGGAGGA-3', restricted to Ly49A-expression in EL-4 cells in comparison with two other representative cell lines tested. This element, designated as EL13, proved to be previously undiscovered by homology search and is highly homologous with several virus DNAs. Using EL13 as a probe we have cloned a cDNA encoding a binding protein to EL13. Its deduced nucleotide sequence revealed that EL13-binding protein is almost identical with rat ATF-2. Although ATF-2 is known to bind to cyclic AMP responsive element (CRE), EL13 shares five out of eight nucleotides with this consensus sequence. Our results suggested that ATF-2 may play an important role via binding to EL13 for the expression of Ly49A. These data will provide useful information for understanding T-cell and NK-cell differentiation in murine immune system.

Isolation of cDNA clone encoding human homologue of senescence marker protein-30 (SMP30) and its location on the X chromosome.

We have isolated and characterized a cDNA clone encoding human homologue of senescence marker protein-30 (SMP30), a calcium binding protein also called regucalcin (RC). This clone (pHSMP6) has 1356 base pairs (bp) and contains an open reading frame of 897 bp, which encodes 299 amino acids. The estimated molecular weight of the deduced polypeptide is 33,250 and pI is 5.836. The homology of amino acid sequences between human homologue and rat SMP30 is 88.6%. Using pHSMP6 as a probe, the chromosomal location of the human homologue of SMP30 gene was determined. The results of regional mapping using a panel of 11 rodent-human somatic hybrids indicated that the gene is located in the p11.3-q11.2 segment of the X chromosome. This gene thus could be a candidate for one of the X-linked diseases mapped to this regions.

The strain-dependent constitutive expression of murine serum amyloid-P component is regulated at the transcriptional level.

The strain-dependent expression of murine serum amyloid P-component (SAP) has been known to be linked to the Sap locus. We have quantified the SAP mRNA in several inbred strains including DBA/2 and C57BL/6 mice which represent high and low producers of SAP at resting state, respectively, and found that the mRNA levels correlated well with the amount of SAP protein. Interestingly, the SAP mRNA level of F1 mouse between DBA/2 and C57BL/6 was low and similar to that of C57BL/6. Primer extension and ribonuclease (RNAase) protection analyses demonstrated that a single type of transcript was generated from the SAP gene and that the cap sites were identical regardless mouse strains tested under unstimulated and stimulated (by lipopolysaccharide (LPS) or interleukin-6 (IL-6)) conditions. To investigate possible structural difference of the SAP gene including 5' flanking region, we have cloned, sequenced and compared the SAP genes from DBA/2 and C57BL/6 mice. Sequence analyses revealed that the 5' flanking regulatory regions, as well as the coding regions, were well-conserved between the two strains. These results demonstrate that the strain-dependent SAP expression occurs at the transcriptional level but seems to be affected by neither different type of the transcripts nor structural difference of the 5' flanking and coding regions of the SAP gene. It was suggested that a possible transcription factor with suppressive activity, which is encoded by a gene linked to Sap, may be involved.