Mucocutaneous inflammation in the Ikaros Family Zinc Finger 1-keratin 5-specific transgenic mice.

BACKGROUND: Our genomewide association study documented an association between cold medicine-related Stevens-Johnson syndrome/toxic epidermal necrolysis (CM-SJS/TEN) and Ikaros Family Zinc Finger 1 (IKZF1). Few studies examined biological and pathological functions of IKZF1 in mucosal immunity. We hypothesized that IKZF1 contributes to the mucocutaneous inflammation. METHODS: Human skin and conjunctival tissues were obtained for immunohistological studies. Primary human conjunctival epithelial cells (PHCjECs) and adult human epidermal keratinocytes (HEKa) also used for gene expression analysis. We also generated K5-Ikzf1-EGFP transgenic mice (Ikzf1 Tg) by introducing the Ik1 isoform into cells expressing keratin 5, which is expressed in epithelial tissues such as the epidermis and conjunctiva, and then examined them histologically and investigated gene expression of the epidermis. Moreover, Ikzf1 Tg were induced allergic contact dermatitis. RESULTS: We found that human epidermis and conjunctival epithelium expressed IKZF1, and in PHCjECs and HEKa, the expression of IKZF1 mRNA was upregulated by stimulation with polyI:C, a TLR3 ligand. In Ikzf1 Tg, we observed dermatitis and mucosal inflammation including the ocular surface. In contact dermatitis model, inflammatory infiltrates in the skin of Ikzf1 Tg were significantly increased compared with wild type. Microarray analysis showed that Lcn2, Adh7, Epgn, Ifi202b, Cdo1, Gpr37, Duoxa1, Tnfrsf4, and Enpp5 genes were significantly upregulated in the epidermis of Ikzf1 Tg compared with wild type. CONCLUSION: Our findings support the hypothesis that Ikaros might participate in mucocutaneous inflammation.

Increased systemic inflammatory interleukin-1ß and interleukin-6 during agitation as predictors of Alzheimer's disease.

OBJECTS: Identification of biomarkers for Alzheimer's disease (AD) is important for its early diagnosis and prevention and a key in advancing our understanding of its pathophysiology. The aim of this study was to determine whether systemic inflammatory interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) as well as hypertension (HT), diabetes mellitus (DM), and body mass index (BMI) are predictors of AD. METHODS: We performed a 10-year follow-up study on 133 elderly who were institutionalized in a nursing home. The associations of IL-1ß and IL-6 at both rest and agitation, as well as HT, DM, and BMI at baseline, were analyzed with the incidences of vascular dementia (VD) and AD during a 10-year follow-up period. RESULTS: The Kaplan-Meier method with log-rank test and Cox regression analyses for the total of 133 subjects showed significantly higher incidences of both VD and AD in subjects with DM or HT at baseline. Resting IL-1ß or IL-6 value, or agitation score, was not significantly associated with the subsequent development of VD or AD. The analyses of 40 subjects who had shown agitation at least once in the previous 3 months demonstrated that IL-1ß and IL-6 values at the agitation stage were significantly associated with AD, but not with VD. CONCLUSION: Our results indicate that systemic inflammatory IL-1ß and IL-6 at the agitation stage are risk factors for the development of AD, but not VD. Inflammatory mechanisms for AD seem to be causal and specific to the development of AD.

Association between altered systemic inflammatory interleukin-1beta and natural killer cell activity and subsequently agitation in patients with Alzheimer disease.

OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disorder that is the most common cause of dementia in the elderly and is frequently accompanied by emotional disorder, including agitation. Although evidence of neuroendocrine immune and inflammatory functions during emotional changes has been accumulated, the pathogenic mechanisms in the development of agitation accompanied by AD remain to be elucidated. METHODS: To clarify the involvement of neuroendocrine and immune and inflammatory systems in agitation in AD, we examined agitation levels, circadian rhythms of behavior, cortisol, interleukin-1beta (IL-1beta), and natural killer cell activity (NKCA) in controls without dementia and 16 AD patients who were recognized to be easily agitated in their nursing homes. These behavioral and blood indicators were assessed according to the progress of the stage of agitation in 16 AD patients (stable, pre-agitation, and agitation stages). RESULTS: Elevations in night behavior and blood cortisol, IL-1beta and an reduced blood NKCA level in the evening were observed not only in the agitation stage, but also when stable in AD patients as compared to the control. Increased IL-1beta and decreased NKCA occurred in both the morning and evening in pre-agitation and agitation stages in AD. CONCLUSIONS: The increased IL-1beta and decreased NKCA with the progress of agitation in AD suggest that inflammation produces agitation and aggravates AD.

Extinction of T cell receptor alpha-chain gene expression accompanied by loss of the lymphoid enhancer-binding factor 1 (LEF-1) in murine somatic cell hybrids.

To investigate the presence of a negative regulatory factor(s) suppressing T-cell receptor alpha-chain (TCR alpha) gene expression in non-T cells, 10 independent cell hybrid clones were generated between mouse T-cell lymphoma EL4 cells (TCR alpha+/beta+) and mouse fibroblast B82 cells. These cell hybrids showed a typical fibroblastic morphology and retained an approximate sum of chromosome numbers derived from both parental cells. No transcripts of the TCR alpha gene were detected in the cell hybrids, although the presence of the rearranged TCR alpha allele from EL4 cells was confirmed. The possibility of involvement of nuclear proteins responsible for the activity of the TCR alpha gene enhancer in the extinction of TCR alpha gene expression in the cell hybrids was examined. Nuclear proteins which bind to the lymphoid enhancer-binding factor 1 (LEF-1) binding motif present in EL4 cells disappeared in the hybrid clones, whereas no significant change was observed in DNA-binding activity of nuclear proteins to a consensus cyclic AMP response element (CRE) and the Ets-1 binding motif between the parental cells and the cell hybrids. No transcripts of the LEF-1 gene were detected in the cell hybrids, despite the retention of the LEF-1 gene and murine chromosomes 3, on which the LEF-1 allele is located, from both parental cells. These results suggest that a trans-acting negative regulatory factor(s) present in fibroblasts suppresses LEF-1 gene expression and that suppression of LEF-1 may lead to the extinction of TCR alpha gene expression in the cell hybrids.

Extinction of expression of the PU.1/Sfpi-1 putative oncogene encoding a B-cell- and macrophage-specific transcription factor in somatic cell hybrids.

Several examples of extinction of cell type-specific gene expression have been observed following fusion of different cell types. Possible mechanisms of the extinction include loss of transcriptional activators and acquisition of repressor factors responsible for cell type-specific gene expression. In this study, we demonstrated the extinction of expression of the PU.1/Sfpi-1 putative oncogene encoding a B-cell- and macrophage-specific transcription factor when plasmacytoma cells are fused with embryonal carcinoma (EC) cells. The hybrid cells retained most chromosome complements from both parental lines including chromosome 2 on which the PU.1 gene is located. Therefore, extinction of PU.1 gene expression in the hybrids is not likely the result of chromosome segregation but rather due to a transacting negative factor(s) present in EC cells. On the contrary, expression of the PU.1 mRNA in plasmacytoma cells was not extinguished upon cell fusion with T-lymphoma cells, although the parental T-lymphoma cells did not express PU.1 transcripts. Hence, T-lymphoma cells seemed to be permissive to PU.1 gene expression, while EC cells were repressive. These results suggest that PU.1 gene expression which positively regulates some B cell- and macrophage-specific gene expression is a target of negative regulatory mechanisms during cell differentiation, and the regulatory mechanisms repressing PU.1 gene expression is different between EC cells and T-cells.

New synthetic inhibitors of C1r, C1 esterase, thrombin, plasmin, kallikrein and trypsin.

p-Guanidinobenzoate derivates were prepared and their inhibitory effects on trypsin, plasmin, pancreatic kallikrein, plasma kallikrein, thrombin, C1r and C1 esterase were examined. Among the various inhibitors tested, 6'-amidino-2-naphthyl-4-guanidinobenzoate dihydrochloride, 4-(beta-amidinoethenyl)phenyl-4-guanidinobenzoate dimethanesulfonate and 4-amidino-2-benzoylphenyl-4-guanidinobenzoate dimethanesulfonate were the most effective inhibitors of trypsin, plasmin, pancreatic kallikrein. plasma kallikrein and thrombin and they strongly inhibited the esterolytic activities of C1r and C1 esterase, and then strongly inhibited complement-mediated hemolysis.

The role of Ets family transcription factor PU.1 in hematopoietic cell differentiation, proliferation and apoptosis.

The PU.1 gene encodes an Ets family transcription factor which controls expression of many B cell- and macrophage-specific genes. Expression of the gene is critical for development of lymphoid and myeloid cell lineages, since PU.1-deficient mice exhibit defects in the development of these cell lineages. The PU.1 gene is identical to the Spi-1 gene isolated from common proviral integration sites in Friend virus-induced murine erythroleukemia (MEL), and deregulated expression of the gene is believed to be an essential step of the disease. We recently demonstrated that overexpression of PU.1 inhibits erythroid differentiation of MEL cells induced with the differentiating agent DMSO. We also noticed unexpectedly that overexpression of PU.1 together with DMSO induces marked growth arrest and apoptosis in MEL cells, supporting the notion that some oncogenes induce growth inhibition and apoptosis rather than cell proliferation and transformation under specific circumstances as shown with the c-myc gene. In this review, the role of PU.1 in hematopoietic cell differentiation, proliferation and apoptosis is described and the possible molecular mechanisms of PU.1-induced effects in MEL cells are discussed.

Augmentation of ouabain sensitivity of rat liver Na/K-ATPase by in vivo adenovirus-mediated expression of the Na/K-ATPase alpha2 subunit.

These are the first experiments to study the effect of in vivo expression of the Na/K-ATPase alpha2 subunit which serves as a receptor for cardiac glycosides. The alpha2 subunit is not normally expressed in rat liver, so hepatocytes which lack endogenous alpha2 protein are a logical first target to study the effects of alpha2 expression on membrane Na/K-ATPase activity. At 3 days after alpha2 adenovirus vector infusion, Wistar rat livers contained alpha2 DNA, alpha2 mRNA, and alpha2 protein. Rat liver membrane ouabain binding activity and the sensitivity of Na/K-ATPase activity to ouabain significantly increased. Total membrane Na/K-ATPase was regulated at a constant level while expressed alpha2 activity represented 10% of the total active Na/K-ATPase sites in alpha2 transduced rat liver. These studies are the first to establish a paradigm in which an endogenous drug receptor is expressed to alter cellular pharmacologic sensitivity.

Detection of intermediary Clr with complete active site, using a synthetic proteinase inhibitor.

The synthetic proteinase inhibitor, FUT-175 (6-amidino-2-naphthyl-4-guanidinobenzoate), strongly suppressed activation of Clr at 37 degrees C, causing 50% inhibition at 0.03 mM. To clarify whether the inhibitor was incorporated into the active site of intermediary Clr formed during the incubation, determination of the active site was tried using this inhibitor. Consequently, release of amidinonaphthol equimolar with the amount of Clr used was observed in the early period of incubation, in which the activation to Clr- was about 5%. These results indicate that intermediary Clr already has a complete active site.

Cell type specific patterns of mRNA splicing in hepatoma cells transfected with the mutated albumin minigene of Nagase analbuminemic rats.

We constructed an SV40-derived expression vector containing a mutated albumin minigene of Nagase analbuminemic rats (NAR), and introduced it into cultured cells. Transient expression of the minigene mRNA was determined by RT-PCR. Three kinds of aberrant mRNAs were expressed by the non-hepatic cells COS-1, and the undifferentiated human hepatoma cells HLE, transfected with the minigene. Their predominant mRNA lacked exon H (delta H), while mRNAs lacking exons H and I, or exons G and H were less abundant. This pattern of the mRNAs was similar to that of albumin mRNAs in the liver of old NAR. In contrast, a differentiated type of human hepatoma cell line, HepG2, expressed only delta H mRNA, like young NAR. These findings indicate that the expression system of the mutated minigene in cultured hepatoma cells is useful for understanding two-exon-skipping of albumin pre-mRNA of NAR.

High efficiency prokaryotic expression and purification of a portion of the hepatitis C core protein and analysis of the immune response to recombinant protein in BALB/c mice.

Hepatitis C virus (HCV) produces chronic persistent liver infection in 1-2% of the U.S. population and is the leading cause of end stage liver disease in patients presenting for liver transplantation at our center. Efforts to cure persistent HCV infection are frequently unsuccessful, so the development of a HCV vaccine is a high priority. HCV envelope proteins are hypervariable so production of a recombinant surface antigen vaccine such as is available for hepatitis B is not likely to confer widespread, high level protective immunity. As the most highly conserved structural protein in the HCV genome, the core protein is one reasonable target for vaccine production. Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression. The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 and 163 amino acids of the core region. Deletion of the 23 amino acids (aa) from aa141-163 led to a marked increase in the efficiency of protein production from < 1 to 3-4 mg/liter for both systems studied. Protein purification was accomplished using affinity chromatography (MBP) or inclusion body isolation (GST) as determined by SDS-PAGE gels and immunotransblot with HCV core protein-specific monoclonal antibody. Finally, the immune response to recombinant protein was assessed in BALB/c mice using a MBP HCV core fusion protein and an ELISA developed using GST HCV core protein as a target. In all mice of this strain, serum anti-HCV core antibody titer increased to 10(-4), two logs above background, following immunization in conjunction with Freund's complete adjuvant. These results represent an encouraging first step toward production of a core protein vaccine. Recombinant core protein is a useful tool to study the immune response to core protein and may be useful to further study the epidemiology and biology of the HCV virus.

A sensitive colorimetric assay for various proteases using naphthyl ester derivatives as substrates.

A convenient and highly sensitive colorimetric assay for various proteases, such as trypsin, chymotrypsin, plasmin, thrombin, and urokinase is described. The substrates used are alpha-naphthyl ester derivatives of N alpha-tosyl-L-lysine, N alpha-acetylglycyl-L-ination of alpha-naphthol released from them. Use of these alpha-naphthyl ester derivatives made the method more sensitive than the use of the corresponding methyl or ethyl ester derivatives. The minimum detectable concentrations of trypsin, chymotrypsin, plasmin, thrombin and urokinase in this method were about 0.002 micrograms, 0.01 microgram, 0.002 CU, 0.01 IU, and 2 IU, respectively. The Km values of trypsin and thrombin for TLNE were 0.11 mM and 0.15 mM while those for TLME were 2.5 mM and 6.7 mM, respectively; the Km values of chymotrypsin for ATNE and ATEE were 0.18 mM and 0.7 mM, respectively; and the Km values of urokinase for AGLNE and AGLME were 0.17 mM and 4 mM, respectively. Zymograms of various proteases were easy to prepare using these alpha-naphthyl ester substrates, and zymograms of trypsin and chymotrypsin were made with TLNE and ATNE, respectively, as substrates.

The effect of phlebotomy on serum erythropoietin levels in normal healthy subjects.

We evaluated endogenous serum erythropoietin (Epo) levels in 14 normal subjects (eight males and six females) after a single 400-ml phlebotomy. The subjects were followed up for 56 days. The hemoglobin (Hb) values of both males and females decreased to a nadir on days 3 to 7 post-phlebotomy. Hb values gradually increased, but did not completely recover to pre-phlebotomy levels by day 56. Serum Epo levels increased after 6 h post-phlebotomy, to 20.1 +/- 5.4 (mU/ml) in males and 20.7 +/- 7.0 in females, from the pre-phlebotomy levels of 14.6 +/- 4.0 in males and 13.4 +/- 4.1 in females, respectively. Epo levels continued to increase to peak levels of 25.5 +/- 6.3 in males and 28.7 +/- 11.5 in females on days 7 to 14 and thereafter decreased until day 56. Thus, the Epo response to a 400-ml phlebotomy was relatively small in magnitude and was not sufficient to initiate a significant increase in erythropoiesis. This finding suggests that the administration of recombinant human erythropoietin (rHu-Epo) may be effective for the prompt correction of anemia induced by autologous blood donation and for increasing the volume of predonated autologous blood.

A sensitive colorimetric assay for human urinary kallikrein.

L-Prolyl-L-phenylalanyl-L-arginine-alpha-naphthylester (Pro-Phe-Arg-NE) was synthesized as a new substrate for use in the assay of kallikreins. An assay was developed based on the colorimetric determination of alpha-naphthol released by the enzyme reaction. With Pro-Phe-Arg--NE as substrate, the minimum detectable concentration of human urinary kallikrein, was about 0.001 KU. Thus use of Pro-Phe-Arg-NE provides a highly sensitive method for detection of human urinary kallikrein. Kallikrein could be determined with a 25-microliter sample of human urine. Zymograms of human urinary kallikrein were prepared using Pro-Phe-Arg-NE as substrate. Six bands were separated by polyacrylamide disc gel isoelectrophoresis.

A sensitive colorimetric assay for thrombin, prothrombin and antithrombin III in human plasma using a new synthetic substrate.

Benzoyl-L-leucyl-L-alanyl-L-arginine-alpha-naphthylester (Bz-Leu-Ala-Arg-NE) was synthesized as a new substrate for use in the assay of thrombin. In the assay alpha-naphthol released by the enzyme reaction was measured colorimetrically. With Bz-Leu-Ala-Arg-NE as substrate, the minimum detectable concentration of human thrombin was 0.0025 U. This assay using Bz-Leu-Ala-Arg-NE is a highly sensitive method for detecting prothrombin, thrombin and antithrombin III in human plasma. Prothrombin could be determined with 0.2 microliter of human plasma using Echis carinatus venom (ECV) as activator. Antithrombin III activity could be determined with 2 microliter of human plasma using human thrombin and heparin as cofactor. A zymogram of human prothrombin was prepared with Bz-Leu-Ala-Arg-NE as substrate. The preparation gave one band (pI 4.9) on polyacrylamide disc gel isoelectrophoresis.

Effects of restricted food access on diurnal fluctuation of behaviors and biochemical functions in hereditary microphthalmic rats.

The characteristics in circadian rhythms of spontaneous locomotor activity, and some metabolic properties were examined in microphthalmic mutant rats of the Donryu strain under ad lib or restricted food access conditions. The growth of microphthalmic rats was retarded compared to that of normal-sighted rats from the same strain. Under a 12:12-h light:dark (LD) cycle with free access to food, normal-sighted rats showed basically nocturnal patterns of the locomotor activity rhythms, but most of microphthalmic rats manifested free-running rhythms and a few of them showed arrhythmic. When food access was restricted only for 6 h in the light period of the LD cycle, the normal and hereditary blind rats generated gradually new patterns of the locomotor activities in which the animals showed to be more active in the light period. Plasma glucose concentration in normal rats showed a peak after food consumption, but microphthalmic mutants exhibited no periodic changes of the glucose levels. Responses of the biochemical parameters of protein and mineral metabolism to restricted food access in the mutants did not differ from those in normal rats. These results suggest that microphthalmic mutant rats show the free-running circadian rhythm of locomotor activity due to a complete lack of their optic nerve and visual input to the circadian clock, but the mutants maintained the ability to shift their circadian phase induced by restricted food access similar to that in control rats, and also that the mutants have almost normal properties of biochemical and physiological functions except for glucose metabolism.

Germinated barley foodstuff increases fecal volume and butyrate production at relatively low doses and relieves constipation in humans.

Germinated barley foodstuff (GBF), derived from the aleurone layer, scutellum and germ of germinated barley, contains a large quantity of fermentable dietary fibers, especially hemicellulose. GBF was given to 9 healthy volunteers in a dose of 9 g of GBF per day for 10 consecutive days, and subsequently 18 g of GBF for another 10 days. As a control, no GBF was given for 3 days before administration of GBF (control period). Fecal weight, water content and short chain fatty acid content were measured before and during the last 3 days of each experimental period. Feeding of GBF significantly increased the fecal butyrate content as well as fecal weight at both dose-levels (9 and 18 g/day), compared with those during the control period. Daily administration of 9 g GBF induced the maximum level of defecation in humans. Relatively mild but chronic constipated volunteers (n = 16) were administered 9 g of GBF daily for 14 days. In this experiment, the condition of defecation (frequency, volume) was estimated by a questionnaire survey. GBF significantly improved defecation within a short period without severe adverse effects. No major abnormalities in laboratory findings were found in hematologic and urinary analyses. In conclusion, daily administration of 9 g GBF was effective for improving defecation in healthy but constipated humans. GBF is a highly safe and effective foodstuff for improving defecation.

Increased growth of Bifidobacterium and Eubacterium by germinated barley foodstuff, accompanied by enhanced butyrate production in healthy volunteers.

Germinated barley foodstuff (GBF) derived from the aleurone and scutellum fractions of germinated barley mainly consists of low-lignified hemicellulose and glutamine-rich protein. GBF improves the proliferation of intestinal epithelial cells and defecation, through the bacterial production of short chain fatty acids (SCFA), especially butyrate. In this study we investigated the mechanism of production of butyrate by microflora in humans and in vitro. Daily administration of 9 g GBF for 14 successive days significantly increased fecal butyrate content. Fecal Bifidobacterium and Eubacterium were also significantly increased by GBF administration in healthy volunteers. Ten anaerobic micro-organisms selected from intestinal microflora were cultured in vitro in the medium containing GBF as a sole carbon source (GBF medium). After a 3-day incubation, 7 strains (Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Bacteroides ovatus, Clostridium butyricum, and Eubacterium limosum) lowered the medium pH producing SCFA. Eubacterium grown together with Bifidobacterium in GBF medium efficiently produced butyrate. On the other hand, GBF changed the intestinal microflora and increased probiotics such as Bifidobacterium in the intestinal tract. As a result, butyrate was produced by the mutual action of Eubacterium and Bifidobacterium. This butyrate is considered to enhance the proliferation of colonic epithelial cells.

Contribution of the calcineurin signaling pathway to overload-induced skeletal muscle fiber-type transition.

Skeletal muscle is highly adaptable, being capable of undergoing changes in its structural and functional properties in response to physiological stimuli. The fast-to-slow muscle fiber-type transition is evoked by increased motor nerve activity. Recently, the calcineurin (CaN) signaling pathway has been implicated in the transcriptional regulation of slow muscle fiber genes. Here we investigated the effect of treatment with a CaN-specific inhibitor, FK506, on skeletal muscle fiber-type transition in functionally overloaded muscles. The overloaded plantaris muscle showed fast-to-slow muscle fiber type transition, i.e., a decrease in myosin heavy chain (MHC) IIb, an increase in MHCIIa+d/x, and new expression of MHCI. In the FK506-administered group, however, overload-induced muscle fiber-type transition was completely prevented. We have demonstrated, therefore, that the CaN signaling pathway is required for fast-to-slow skeletal muscle fiber-type transition. Furthermore, we also confirmed that the protein expression levels of downstream effectors of CaN signaling exhibit a transient increase in the early phase of the overloaded condition.

Genetic variation in hypoxia-inducible factor 1alpha and its possible association with high altitude adaptation in Sherpas.

Hypoxic stress at high altitude requires adaptations in several physiological functions to ensure the optimal oxygenation of all cells. Several lines of evidence suggested that high-altitude native populations such as Sherpas have been genetically adapted to their stressful environment. We investigated the genetic variation in the hypoxia-inducible factor (HIF)-1alpha gene in Sherpas as compared with Japanese, native lowlanders, and found a novel dinucleotide repeat polymorphism in intron 13 of the HIF-1alpha gene. GT15 allele was more frequent in Japanese than in Sherpas with statistical significance, while GT14 allele was significantly more frequent in Sherpas as compared with Japanese. A possible genetic variation in the HIF-1alpha gene might function in adaptation to living at high altitude. Because the activity of HIF-1 is regulated by multiple steps including the transcriptional level, the effect of the polymorphism in intron 13 on the cellular hypoxic responses remains to be elucidated.