Attenuating the rate of total body fat accumulation and alleviating liver damage by oral administration of vitamin D-enriched edible mushrooms in a diet-induced obesity murine model is mediated by an anti-inflammatory paradigm shift.

BACKGROUND: Hypovitaminosis D is associated with many features of the metabolic syndrome, including non-alcoholic fatty liver disease. Vitamin D-enriched mushrooms extracts exert a synergistic anti-inflammatory effect. The aim of the present study is to determine the immunomodulatory effect of oral administration of vitamin D-enriched mushrooms extracts on high-fat diet (HFD) animal model of non-alcoholic steatohepatitis (NASH). METHODS: C57BL/6 mice on HFD were orally administered with vitamin D supplement, Lentinula edodes (LE) mushrooms extract, or vitamin D-enriched mushrooms extract for 25 weeks. Mice were studied for the effect of the treatment on the immune system, liver functions and histology, insulin resistance and lipid profile. RESULTS: Treatment with vitamin D-enriched LE extracts was associated with significant attenuation of the rate of total body fat accumulation, along with a decrease in hepatic fat content as measured by an EchoMRI. Significant alleviation of liver damage manifested by a marked decrease in ALT, and AST serum levels (from 900 and 1021 U/L in the control group to 313 and 340; 294 and 292; and 366 and 321 U/L for ALT and AST, in Vit D, LE and LE + Vit D treated groups, respectively). A corresponding effect on hepatocyte ballooning were also noted. A significant decrease in serum triglycerides (from 103 to 75, 69 and 72 mg/dL), total cholesterol (from 267 to 160, 157 and 184 mg/dL), and LDL cholesterol (from 193 mg/dL to 133, 115 and 124 mg/dL) along with an increase in the HDL/LDL ratio, and improved glucose levels were documented. These beneficial effects were associated with a systemic immunomodulatory effect associated with an increased CD4/CD8 lymphocyte ratio (from 1.38 in the control group to 1.69, 1.71 and 1.63), and a pro- to an anti-inflammatory cytokine shift. CONCLUSIONS: Oral administration of vitamin-D enriched mushrooms extracts exerts an immune modulatory hepato-protective effect in NASH model.

Runx2 (Cbfa1) inhibits Shh signaling in the lower but not upper molars of mouse embryos and prevents the budding of putative successional teeth.

Heterozygous mutations in the RUNX2 (CBFA1) gene cause cleidocranial dysplasia, characterized by multiple supernumerary teeth. This suggests that Runx2 inhibits successional tooth formation. However, in Runx2 knockout mice, molar development arrests at the late bud stage, and lower molars are more severely affected than upper ones. We have proposed that compensation by Runx3 may be involved. We compared the molar phenotypes of Runx2/Runx3 double-knockouts with those of Runx2 knockouts, but found no indication of such compensation. Shh and its mediators Ptc1, Ptc2, and Gli1 were down-regulated only in the lower but not the upper molars of Runx2 and Runx2/Runx3 knockouts. Interestingly, in front of the mutant upper molar, a prominent epithelial bud protruded lingually with active Shh signaling. Similar buds were also present in Runx2 heterozygotes, and they may represent the extension of dental lamina for successional teeth. The results suggest that Runx2 prevents the formation of Shh-expressing buds for successional teeth.

Inhibitory effects of stress on postprandial gastric myoelectrical activity and vagal tone in healthy subjects.

The aim was to investigate gastric myoelectrical activity (GMA) and vagal activity in response to stress. The study was performed in 10 healthy subjects in three sessions (control, relaxation and stress). The control session was composed of 30-min recordings before and 30-min recordings after a test meal. The protocol of two other sessions was similar except that the fasting recording was extended to 60 min and the subjects were continuously watching a horror movie (stress) or guided meditation tape (relaxation) after the 30-min baseline. GMA was recorded using electrogastrography and heart rate variability (HRV) was derived from the electrocardiogram. Meal resulted in a postprandial increase in the dominant frequency (2.91 cpm vs 3.17 cpm, P < 0.007), dominant power (30.0 dB vs 32.5 dB, P < 0.05), and percentage of normal slow waves (79.8%vs 87.4%, P = 0.09). Similar responses were found in the relaxation session. Stress inhibited all these normal postprandial response and reduced the regularity of gastric slow waves (82.0%vs 66.0%, P < 0.01). In addition, spectral analysis of the HRV demonstrated an inhibition of postprandial vagal activity and an increase of postprandial sympathetic activity with stress. Stress has an inhibitory effect on postprandial GMA and this may involve both vagal and sympathetic pathway.

Inhibitory effect of white wine on gastric myoelectrical activity and the role of vagal tone.

Although extensively investigated throughout the gastrointestinal tract, the influence of alcohol on gastric motility is still unclear. Our aim was to investigate the effect of wine on gastric myoelectrical activity and vagal activity. Ten healthy subjects were studied in two sessions with the electrogastrogram (EGG) for 30 min at baseline, 30 min after ingesting the test liquid [white wine (12.5% alcohol) or matched juice], and 60 min after a standard test meal. Spectral analysis was performed to compute EGG parameters and their postprandial changes. The vagal activity was assessed based on spectral analysis of the heart rate variability (HRV) signal derived from the ECG recording. White wine preload significantly diminished the postprandial increase in EGG dominant power compared to juice preload (1.16 +/- 1.57 vs 5.48 +/- 1.01 dB, P < 0.001). A significant decrease in vagal activity was observed after wine (23.40 +/- 4.30 vs 17.43 +/- 3.40%, P < 0.005), which remained unchanged after the test meal (23.40 +/- 4.30 vs 16.77 +/- 4.40%, P < 0.05). This decrease was not noted in the juice session. A correlation was established between changes after wine consumption in EGG dominant power and in the percentage of the vagal activity (r = 0.89, P < 0.05). In conclusion, white wine preload inhibits the postprandial EGG dominant power, suggesting a possible inhibition of postprandial gastric contractions. This effect may be associated with diminished vagal activity.

The RUNX3 gene--sequence, structure and regulated expression.

The RUNX3 gene belongs to the runt domain family of transcription factors that act as master regulators of gene expression in major developmental pathways. In mammals the family includes three genes, RUNX1, RUNX2 and RUNX3. Here, we describe a comparative analysis of the human chromosome 1p36.1 encoded RUNX3 and mouse chromosome 4 encoded Runx3 genomic regions. The analysis revealed high similarities between the two genes in the overall size and organization and showed that RUNX3/Runx3 is the smallest in the family, but nevertheless exhibits all the structural elements characterizing the RUNX family. It also revealed that RUNX3/Runx3 bears a high content of the ancient mammalian repeat MIR. Together, these data delineate RUNX3/Runx3 as the evolutionary founder of the mammalian RUNX family. Detailed sequence analysis placed the two genes at a GC-rich H3 isochore with a sharp transition of GC content between the gene sequence and the downstream intergenic region. Two large conserved CpG islands were found within both genes, one around exon 2 and the other at the beginning of exon 6. RUNX1, RUNX2 and RUNX3 gene products bind to the same DNA motif, hence their temporal and spatial expression during development should be tightly regulated. Structure/function analysis showed that two promoter regions, designated P1 and P2, regulate RUNX3 expression in a cell type-specific manner. Transfection experiments demonstrated that both promoters were highly active in the GM1500 B-cell line, which endogenously expresses RUNX3, but were inactive in the K562 myeloid cell line, which does not express RUNX3.

Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis.

The human RUNX3/AML2 gene belongs to the 'runt domain' family of transcription factors that act as gene expression regulators in major developmental pathways. Here, we describe the expression pattern of Runx3 during mouse embryogenesis compared to the expression pattern of Runx1. E10.5 and E14.5-E16.5 embryos were analyzed using both immunohistochemistry and beta-galactosidase activity of targeted Runx3 and Runx1 loci. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory ganglia, epidermal appendages, and developing skeletal elements, their expression was confined to different compartments. These data provide new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross-regulation between them plays a role in embryogenesis.

Architecture and anatomy of the genomic locus encoding the human leukemia-associated transcription factor RUNX1/AML1.

The RUNX1 gene on human chromosome 21q22.12 belongs to the 'runt domain' gene family of transcription factors (also known as AML/CBFA/PEBP2alpha). RUNX1 is a key regulator of hematopoiesis and a frequent target of leukemia associated chromosomal translocations. Here we present a detailed analysis of the RUNX1 locus based on its complete genomic sequence. RUNX1 spans 260 kb and its expression is regulated through two distinct promoter regions, that are 160 kb apart. A very large CpG island complex marks the proximal promoter (promoter-2), and an additional CpG island is located at the 3' end of the gene. Hitherto, 12 different alternatively spliced RUNX1 cDNAs have been identified. Genomic sequence analysis of intron/exon boundaries of these cDNAs has shown that all consist of properly spliced authentic coding regions. This indicates that the large repertoire of RUNX1 proteins, ranging in size between 20-52 kDa, are generated through usage of alternatively spliced exons some of which contain in frame stop codons. The gene's introns are largely depleted of repetitive sequences, especially of the LINE1 family. The RUNX1 locus marks the transition from a ~1 Mb of gene-poor region containing only pseudogenes, to a gene-rich region containing several functional genes. A search for RUNX1 sequences that may be involved in the high frequency of chromosomal translocations revealed that a 555 bp long segment originating in chromosome 11 FLI1 gene was transposed into RUNX1 intron 4.1. This intron harbors the t(8;21) and t(3;21) chromosomal breakpoints involved in acute myeloid leukemia. Interestingly, the FLI1 homologous sequence contains a breakpoint of the t(11;22) translocation associated with Ewing's tumors, and may have a similar function in RUNX1.

Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms.

AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5' untranslated regions (5'UTRs) (D-UTR and P-UTR). Here we show that these 5'UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression.

Regulation of AML2/CBFA3 in hematopoietic cells through the retinoic acid receptor alpha-dependent signaling pathway.

AML2 is a member of the acute myelogenous leukemia, AML family of transcription factors. The biologic functions of AML1 and AML3 have been well characterized; however, the functional role of AML2 remains unknown. In this study, we found that AML2 protein expressed predominantly in cells of hematopoietic origin is a nuclear serine phosphoprotein associated with the nuclear matrix, and its expression is not cell cycle-related. In HL-60 cells AML2 expression can be induced by all three natural retinoids, all-trans-retinoic acid (RA), 13-cis-RA, and 9-cis-RA in a dose-dependent manner. A synthetic retinoic acid derivative, 4HPR, which neither activates RA receptor (RAR) alpha nor retinoic X receptor alpha was unable to induce the expression of AML2. A RAR-selective activator, TTNPB, induced AML2 expression similar to RA. Our study further showed that AGN193109, a potent RARalpha antagonist, suppressed AML2 expression induced by RA and that a retinoic X receptor pan agonist AGN194204 had no effect on its expression. Taken together, these studies conclusively demonstrated that the expression of AML2 in HL-60 cells is regulated through the RARalpha-specific signaling pathway. Our study further showed that after all-trans-retinoic acid priming, AML2 expression could be augmented by vitamin D(3). Based on these studies we hypothesize that AML2 expression is normally regulated by retinoid/vitamin D nuclear receptors mainly through the RARalpha-dependent signaling pathway and that it may play a role in hematopoietic cell differentiation.

Tannic acid and thiocarbohydrazide as structural reinforcement agents in the preparation of rabbit knee articular cartilage for the scanning electron microscope.

Samples from seven sectors of the rabbit knee articular cartilage were shaved and prepared for the scanning electron microscope using either tannic acid, thiocarbohydrazide or nothing (control). Surface morphology was found to be more typical to a given sector and less so to a specific preparation procedure. Rough areas were recorded from load-bearing sectors, while smooth areas appeared on load-free ones. However, fibrillations were discerned on control load-bearing sectors only, and pits and humps were never detected. Tannic acid and thiocarbohydrazide may have exerted their structural reinforcing effect on the tissue preservation by enhancing the binding of osmium tetroxide to it, possibly along with that of other soluble tissue constituents.

Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors.

The mammalian AML/CBFalpha runt domain (RD) transcription factors regulate hematopoiesis and osteoblast differentiation. Like their Drosophila counterparts, most mammalian RD proteins terminate in a common pentapeptide, VWRPY, which serves to recruit the corepressor Groucho (Gro). Using a yeast two-hybrid assay, in vitro association and pull-down experiments, we demonstrate that Gro and its mammalian homolog TLE1 specifically interact with AML1 and AML2. In addition to the VWRPY motif, other C-terminal sequences are required for these interactions with Gro/TLE1. TLE1 inhibits AML1-dependent transactivation of the T cell receptor (TCR) enhancers alpha and beta, which contain functional AML binding sites, in transfected Jurkat T cells. LEF-1 is an additional transcription factor that mediates transactivation of TCR enhancers. LEF-1 and its Drosophila homolog Pangolin (Pan) are involved in the Wnt/Wg signaling pathway through interactions with the coactivator beta-catenin and its highly conserved fly homolog Armadillo (Arm). We show that TLE/Gro interacts with LEF-1 and Pan, and inhibits LEF-1:beta-catenin-dependent transcription. These data indicate that, in addition to their activity as transcriptional activators, AML1 and LEF-1 can act, through recruitment of the corepressor TLE1, as transcriptional repressors in TCR regulation and Wnt/Wg signaling.

Impaired postprandial gastric slow waves in patients with functional dyspepsia.

The aim of this study was to investigate gastric myoelectrical activity in patients with functional dyspepsia. Thirteen healthy subjects and 14 patients with functional dyspepsia participated in the study. The electrogastrogram (EGG) recording was made in each subject for 30 min in the fasting state and 120 min after a standard test meal of 475 calories. Spectral analysis methods were applied to derive quantitative EGG parameters. There was no difference in the EGG between the patients and controls in the fasting state. However, abnormalities in the postprandial EGG were found in the patients. The percentage of 2-4 cpm waves was significantly lower (74.4+/-4.0% vs 85.7+/-1.6%, P < 0.03) and the postprandial increase in EGG dominant power was significantly less (-0.52+/-0.92 dB vs 2.24+/-0.88 dB, P < 0.03) in patients than in controls. It was also found that the percentage of postprandial 2-4 cpm waves could be used to differentiate the patients with functional dyspepsia from the healthy controls with a specificity of 100% and a sensitivity of 43%. It was concluded that a subset of patients with functional dyspepsia have impaired gastric myoelectrical activity in the fed state.

Articular cartilage of the rabbit knee after synovectomy: a scanning electron microscopy study.

We sought to study the effect of synovectomy on the surface morphology of articular cartilages of the rabbit knee using scanning electron microscopy (SEM). Fifteen rabbits were surgically synovectomised and allowed to regenerate their synovia during time intervals ranging from 3 to 44 wk. Cartilage specimens were shaved from 5 distinct articular sectors of synovectomised, contralateral and sham-operated knees and prepared for SEM using tannic acid. Applying structural reinforcement by tannic acid was found to secure the in vivo surface morphology of the cartilages and thus production of surface irregularities during preparation was excluded. The surface morphology of cartilages both from the synovectomised and contralateral joints was found to differ from that of intact healthy rabbits. A 'chaotic' nature of the altered cartilaginous morphology persisted as late as 44 wk postsynovectomy. Cartilages from sham-operated joints did not differ detectably from normal cartilages.

Site-directed mutagenesis supports a three-dimensional model of the runt domain.

The "runt domain" (RD) is a 128 amino acid region of the Drosophila pair-rule gene runt. This highly conserved region delineates the DNA-binding domain of a new family of transcription factors; the RD proteins. The family includes genes from Drosophila, chicken and mammals that are involved in a wide range of developmental processes, from sex determination and neurogenesis in Drosophila to hematopoiesis and osteoblast differentiation in mouse and human. The RD confers DNA binding ability and mediates the interaction of mammalian RD proteins with the beta-subunit (CBFbeta), which enhances the DNA binding. The primary sequence of RD shows no similarity to other known DNA-binding motifs and its three-dimensional (3D) structure is not known. We employed molecular modeling-based mutagenesis to generate a 3D model of RD. Fold recognition programs identified the palm subdomain of rat DNA polymerase beta as the most likely fold for RD. In the predicted model, the RD region which interacts with DNA contains two arginine residues, R130 and R135, which appear to be in close contact with the major groove of the DNA and to interact with the three essential guanine bases of the core DNA motif PyGPyGGT. We mutated these two R residues and demonstrated that mutations markedly reduced the binding of RD to DNA with no effect on RD interaction with CBFbeta. The data provide important clues about the possible 3D structure of the RD and its interaction with the core DNA motif.

Efficiency and efficacy of the electrogastrogram.

The efficiency and efficacy of the electrogastrogram (EGG) involve a few practical factors, including recording length, sample size, and the characteristics of subjects. The aim of this study was to investigate the effect of these factors on the accuracy of EGG analysis. Gastric myoelectrical activity was recorded using electrogastrography in 24 subjects (ages 22-91 years) for 1 hr in the fasting state and 2 hr after a test meal. Computerized spectral analysis was performed to compute EGG parameters, including dominant frequency, dominant power, and the percentage of 2-4 cycles per minute (cpm) slow waves. A parameter called misinterpretation was defined to investigate the effect of recording length. The results were as follows: (1) Using the recording length of 1 hr in each state as a gold standard, the misinterpretation for the recording length of 30 min was 27% for the dominant frequency and 17% for the dominant power. When the recording length was reduced to 15 min, the misinterpretation increased to 61% for the dominant frequency and 38% for the dominant power. (2) With a sample size of 10 subjects and a recording length of 60 min, a statistically significant postprandial increase was observed in the dominant frequency and power, and a trend in the postprandial increase of the regularity of the EGG was noted. When the sample size increased to 24 subjects, a significant postprandial increase was found in all these parameters. (3) None of the EGG parameters exhibited any significant difference between the younger and older subjects or between men and women. In conclusion, a recording length of 30-60 min seems to be appropriate and produces reliable and predictable results. Age and gender do not affect any of the EGG parameters.

Effects of meal volume and composition on gastric myoelectrical activity.

The absence of a standard meal in electrogastrography may limit its clinical significance. Different meals may fail to produce the expected postprandial motility pattern. The aim of this study was to investigate the effect of meal volume and composition on postprandial myoelectrical activity. Fourteen healthy subjects were given four meals that differed from a "reference meal" in one single parameter (volume, calorie, or fiber content). Gastric myoelectrical activity was measured using surface electrogastrography. Spectral and statistical analyses were performed to investigate the effect of food properties on electrogastrogram (EGG) parameters. It was found that the reference meal produced a postprandial increase in the dominant frequency (P < 0.007), dominant power (P < 0.04), and percentage of normal 2-4 cycle/min gastric slow waves (P > 0.05). Similar changes were observed with the low-volume and high-fiber meals but not with the reduced-calorie meal. Fasting EGG parameters in all four sessions showed no significant difference. It was concluded that low-calorie meals do not result in expected postprandial physiological responses and thus are not appropriate for EGG tests. A volume reduction of down to one-half the volume of a regular meal does not affect postprandial changes of the EGG; thus a condensed test meal may be recommended for symptomatic patients.

Expression of AML1-d, a short human AML1 isoform, in embryonic stem cells suppresses in vivo tumor growth and differentiation.

The human AML1 gene encodes a heterodimeric transcription factor which plays an important role in mammalian hematopoiesis. Several alternatively spliced AML1 mRNA species were identified, some of which encode short protein products that lack the transactivation domain. When transfected into cells these short isoforms dominantly suppress transactivation mediated by the full length AML1 protein. However, their biological function remains obscure. To investigate the role of these short species in cell proliferation and differentiation we generated embryonic stem (ES) cells overexpressing one of the short isoforms, AML1-d, as well as cells expressing the full length isoforms AML1-b and AML2. The in vitro growth rate and differentiation of the transfected ES cells were unchanged. However, overexpression of AML1-d significantly affected the ES cells' ability to form teratocarcinomas in vivo in syngeneic mice, while a similar overexpression of AML1-b and AML2 had no effect on tumor formation. Histological analysis revealed that the AML1-d derived tumors were poorly differentiated and contained numerous apoptotic cells. These data highlight the pleiotropic effects of AML1 gene products and demonstrate for the first time an in vivo growth regulation function for the short isoform AML1-d.

Patient satisfaction after 5.0-mm photorefractive keratectomy for myopia.

PURPOSE: To examine patient satisfaction following photorefractive keratectomy (PRK) in a large number of subjects. METHODS: We used a questionnaire to examine overall patient satisfaction in 173 patients (173 first-operated eyes) at least 1 year after they had undergone PRK. Mean preoperative spherical equivalent refraction was -7.05 +/- 3.73 D (range, -1.50 to -15.00 D). Fifty-one patients had unilateral surgery and 122 had bilateral surgery, 60 of whom had follow-up of 1 year for both eyes. Visual and refractive results, the use of corrective lenses, and subjective side effects were also studied. RESULTS: Eighty percent of 173 patients reported they were satisfied or very satisfied with the surgical outcome, while 19.7% reported they were dissatisfied. The average satisfaction score was 7.92 +/- 2.22 out of a possible 10. After PRK, 77.5% reported improvement or great improvement in their general quality of life; 16.8% were very disturbed by subjective visual symptoms. Of the 51 bilateral patients, 85% required no corrective lenses after surgery. Patients who had bilateral operations were more satisfied than those who had unilateral ones. Statistically significant associations were found between patient satisfaction and initial refraction: as preoperative refraction increased, percentage of satisfied or very satisfied patients decreased. CONCLUSION: Patient satisfaction after PRK was generally high, but subjective visual symptoms remain a problem.

Influence of patient age on photorefractive keratectomy for myopia.

BACKGROUND: To determine whether the outcomes of photorefractive keratectomy (PRK) for myopia are age-dependent. METHODS: The influence of age on PRK outcomes was analyzed for one eye of each of 72 patients divided into two groups: 39 patients (18 to 26 years) and 33 patients (35 to 54 years). The influence of the amount of preoperative myopia (low myopia less than -4.00 diopters (D); moderate to high myopia -4.00 or more D) was also evaluated. All patients were followed for at least 1 year, and all underwent cycloplegic refractions. RESULTS: One year after PRK, the average refraction was -0.15 D in the younger group and +0.38 D in the older group. The achieved refraction was higher than the attempted by +1.00 D or more only in eyes with moderate to high myopia (-4.00 to -10.00 D); 11.54% (3 eyes) of the younger group and in 34.78% (8 eyes) of the older group. CONCLUSIONS: After PRK for myopia, patients between the ages of 35 and 54, with moderate to high myopia, obtained more refractive change with the same intended dioptric correction compared to younger patients with moderate to high myopia. Attempted correction should be adjusted according to age and amount of myopia.