IGF2 is parentally imprinted during human embryogenesis and in the Beckwith-Wiedemann syndrome.

The phenomenon of parental imprinting involves the preferential expression of one parental allele of a subset of chromosomal genes and has so far only been documented in the mouse. We show here, by exploiting sequence polymorphisms in exon nine of the human insulin-like growth factor 2 (IGF2) gene, that only the paternally-inherited allele is active in embryonic and extra-embryonic cells from first trimester pregnancies. In addition, only the paternal allele is expressed in tissues from a patient who suffered from Beckwith-Wiedemann syndrome. Thus the parental imprinting of IGF2 appears to be evolutionarily conserved from mouse to man and has implications for the generation of the Beckwith-Wiedemann syndrome.

Epigenetic regulation in obesity.

The availability to the DNA strand and the activity of the transcription machinery is crucial for the cell to use the information in the DNA. The epigenetic mechanisms DNA methylation, modification of histone tails, other chromatin-modifying processes and interference by small RNAs regulate the cell-type-specific DNA expression. Epigenetic marks can be more or less plastic perpetuating responses to various molecular signals and environmental stimuli, but in addition apparently stochastic epigenetic marks have been found. There is substantial evidence from animal and man demonstrating that both transient and more long-term epigenetic mechanisms have a role in the regulation of the molecular events governing adipogenesis and glucose homeostasis. Intrauterine exposure such as poor maternal nutrition has consistently been demonstrated to contribute to a particular epigenotype and thereby developmental metabolic priming of the exposed offspring in animal and man. Epigenetic modifications can be passed not only from one cell generation to the next, but metabolic disease-related epigenotypes have been proposed to also be transmitted germ-line. Future more comprehensive knowledge on epigenetic regulation will complement genome sequence data for the understanding of the complex etiology of obesity and related disorder.

Comparison of two twice-daily doses of budesonide/formoterol maintenance and reliever therapy.

The aim of this study was to compare two budesonide/formoterol maintenance doses within the budesonide/formoterol maintenance and reliever therapy concept and to identify possible patient characteristics at baseline which would predict a better response to a higher than standard maintenance dose. A total of 8,424 patients with symptomatic asthma when using an inhaled corticosteroid (ICS) with or without a long-acting ß(2)-agonist were randomised to budesonide/formoterol 160/4.5 µg, one (1 × 2) or two (2 × 2) inhalations b.i.d. Patients used the same inhaler as needed for symptom relief. The primary outcome variable was time to first severe asthma exacerbation. In the total study population, the time to first severe asthma exacerbation was prolonged by 18% with 2 × 2 versus 1 × 2 (hazard ratio 0.82; p = 0.03). Lung function (peak expiratory flow) was the only statistically significant predictor of a better response to 2 × 2. The mean daily ICS doses were 737 and 463 µg in the 2 × 2 and 1 × 2 groups, respectively. In a real-life setting, budesonide/formoterol maintenance and reliever therapy at the 2 × 2 maintenance dose did prolong time to first severe exacerbation but at a higher medication load. Patients with low lung function benefited most from the higher maintenance dose.

Effect of formoterol with or without budesonide in repeated low-dose allergen challenge.

The use of combination therapy in mild asthma is debated. The current authors evaluated the effects of formoterol alone and a formoterol/budesonide combination inhaler on asthma deterioration induced by repeated low-dose allergen exposure. In total, 15 subjects with intermittent allergic asthma inhaled low doses of allergen on seven consecutive weekdays in a three-period, crossover, double-blind, double-dummy comparison between formoterol 4.5 microg Turbuhaler, budesonide 160 microg/formoterol 4.5 microg Turbuhaler and placebo, each taken as two puffs 30 min after allergen dosing. The outcome variables were: provocative dose of methacholine causing a 20% fall in forced expiratory volume in one second (PD(20)), exhaled nitric oxide fraction (F(eNO)), sputum eosinophils and prostaglandin D(2), and diary card recordings of symptoms (on a scale of 0-10), short-acting beta(2)-agonist use and evening forced expiratory volume in one second (FEV(1)). With placebo treatment, allergen exposure caused significant increases in airway hyperresponsiveness (geometric mean (coefficient of variation) PD(20): 397 (98) microg before versus 168 (82) microg after), F(eNO) (mean+/-sd 46+/-31 ppb before versus 73+/-46 ppb after) and asthma symptom score (mean+/-sd 0.39+/-0.55 before versus 0.68+/-0.67 after). Budesonide/formoterol abolished these changes and significantly improved baseline FEV(1). Formoterol alone, while providing symptom relief, was no better than placebo in protecting against the allergen-induced increase in airway inflammation. Signs of deteriorating asthma, provoked by low-dose allergen, are prevented by short-term use of budesonide/formoterol but not by temporary use of formoterol alone.

Epigenetic modulation at birth - altered DNA-methylation in white blood cells after Caesarean section.

AIM: Delivery by C-section (CS) has been associated with increased risk for allergy, diabetes and leukaemia. Whereas the underlying cause is unknown, epigenetic change of the genome has been suggested as a candidate molecular mechanism for perinatal contributions to later disease risk. We hypothesized that mode of delivery affects epigenetic activity in newborn infants. METHODS: A total of 37 newborn infants were included. Spontaneous vaginal delivery (VD) occurred in 21, and 16 infants were delivered by elective CS. Blood was sampled from the umbilical cord and 3-5 days after birth. DNA-methylation was analyzed in leucocytes. RESULTS: Infants born by CS exhibited higher DNA-methylation in leucocytes compared with that of those born by VD (p < 0.001). After VD, newborn infants exhibited stable levels of DNA-methylation, as evidenced by comparing cord blood values with those 3-5 days after birth (p = 0.55). On postnatal days 3-5, DNA-methylation had decreased in the CS group (p = 0.01) and was no longer significantly different from that of VD (p = 0.10). CONCLUSION: DNA-methylation is higher in infants delivered by CS than in infants vaginally born. Although currently unknown how gene expression is affected, or whether epigenetic differences related to mode of delivery are long-lasting, our findings open a new area of clinical research with potentially important public health implications.

Functional disconnection between the visual cortex and right fusiform face area in schizophrenia.

Patients with schizophrenia show impairment in processing faces, including facial affect and face detection, but the underlying mechanisms are unknown. We used functional magnetic resonance imaging (fMRI) to characterize resting state functional connectivity between an independent component analysis (ICA)-defined early visual cortical network (corresponding to regions in V1, V2, V3) and a priori defined face-processing regions (fusiform face area [FFA], occipital face area [OFA], superior temporal sulcus [STS] and amygdala) using dual regression in 20 schizophrenia patients and 26 healthy controls. We also investigated the association between resting functional connectivity and neural responses (fMRI) elicited by a face detection paradigm in a partially overlapping sample (Maher et al., 2016) that used stimuli equated for lower-level perceptual abilities. Group differences in functional connectivity were found in right FFA only; controls showed significantly stronger functional connectivity to an early visual cortical network. Functional connectivity in right FFA was associated with (a) neural responses during face detection in controls only, and (b) perceptual detection thresholds for faces in patients only. The finding of impaired functional connectivity for right FFA (but not other queried domain-specific regions) converges with findings investigating face detection in an overlapping sample in which dysfunction was found exclusively for right FFA in schizophrenia during face detection.

Molecular cloning of mouse acetylcholinesterase: tissue distribution of alternatively spliced mRNA species.

We have isolated cDNA clones encoding acetylcholinesterase from mouse muscle and brain. The polymerase chain reaction was used to amplify cDNA clones from C2 myotubes encoding the entire open reading frame and large segments of the 5' and 3' untranslated regions. The muscle cDNA clones were used to isolate clones from a brain library encoding the same mRNA species. The mouse clones encode a catalytic subunit containing a C-terminal sequence similar to that of the hydrophilic species of Torpedo. The mouse acetylcholinesterase sequence shares approximately 88% and 61% amino acid identity with bovine and Torpedo acetylcholinesterases, respectively, but only 52% identity with mouse butyrylcholinesterase, the sequence of which we have also deduced by molecular cloning. Northern blot and RNAase protection analyses indicate that the cDNA clones were derived from the acetylcholinesterase transcript that predominates in most expressing tissues. In contrast, erythroid cells are enriched in an mRNA species whose sequence diverges from that of the cDNA in the region encoding the C-terminus of the enzyme.

Single gene encodes glycophospholipid-anchored and asymmetric acetylcholinesterase forms: alternative coding exons contain inverted repeat sequences.

Polymorphic forms of acetylcholinesterase are tethered extracellularly either as dimers membrane-anchored by a glycophospholipid or as catalytic subunits disulfidelinked to a collagen tail that associates with the basal lamina. Genomic clones of acetylcholinesterase from T. californica revealed that individual enzyme forms are encoded within a single gene that yields multiple mRNAs. Each enzyme form is encoded in three exons: the first two exons, bases -22 to 1502 and 1503 to 1669, encode sequence common to both forms, while alternative third exons encode a hydrophobic C-terminal region, to which a glycophospholipid is added upon processing, and a nonprocessed C-terminus, yielding a catalytic subunit that disulfide-links with a collagen-like structural unit. The 3' untranslated region of each alternative exon contains tandem repeat sequences that are inverted with respect to the other exon. This may either dictate alternative exon usage by formation of cis stem-loops or affect the abundance of translatable mRNA by trans-hybridization between the alternative spliced mRNA species.

Methylation of the p16INK4A promoter is associated with malignant behavior in abdominal extra-adrenal paragangliomas but not pheochromocytomas.

Pheochromocytomas and abdominal extra-adrenal paragangliomas are related to endocrine tumors of the sympathetic nervous system. Studies in animal models have shown that inactivation of the products of the cyclin dependent kinase inhibitor 2A (CDKN2A) gene locus, p16INK4A and p14ARF, promotes the development of pheochromocytoma, especially in malignant form. The present study evaluated the involvement of CDKN2A in human pheochromocytomas and abdominal extra-adrenal paragangliomas from 55 patients. Promoter methylation was assessed using quantitative Pyrosequencing and methylation-specific PCR, and mRNA expression was measured by quantitative real-time PCR. For p16, western blot analysis and sequencing were also performed. succinate dehydrogenase complex subunit B (SDHB) sequencing analysis included extra-adrenal paragangliomas, all tumors classified as malignant, and cases diagnosed at 30 years or younger. The p16INK4A promoter was heavily methylated in a subset of paragangliomas, and this was significantly associated with malignancy (P<0.0043) and SDHB mutation (P<0.002). p16INK4A mRNA expression showed moderate suppression in malignant cases (P<0.05). In contrast, very little p14ARF promoter methylation was seen and there was no significant difference in p14ARF expression between tumors and normal samples. The p16 protein expression was reduced in 16 tumors, and sequence variations were observed in four tumors including the missense mutation A57V and the single nucleotide polymorphism (SNP) A148T. The results suggest that p16INK4A, and not p14ARF, is a subject of frequent involvement in these tumors. Importantly, hypermethylation of the p16INK4A promoter was significantly associated with malignancy and metastasis, and SDHB gene mutations. This finding suggests an etiological link and could provide a clinical utility for diagnostic purposes.

Disrupted IGF2 promoter control by silencing of promoter P1 in human hepatocellular carcinoma.

Previous investigations have supported or indicated a stimulatory role of the insulin-like growth factor II gene (IGF2) in hepatocarcinogenesis. We have studied the transcript levels, promoter usage, and imprinting status of the ICF2 gene and its relationship to H19 in human hepatocellular carcinomas (HCCs) and liver tumor cell lines. The activity of the IGF2 promoter P1 was lost in about 70% of the cases (18 of 25). This is the most prominent abnormality regarding the IGF2 regulation in this study. Total IGF2 as well as promoter P3 transcription were up-regulated in a small group of the tumors. Twenty genetically informative cases were obtained from 26 cases, thus excluding the probability of loss of heterozygosity of the IGF2 gene. Among these, nine showed abnormal monoallelic expression of IGF2. One HCC and one HCC cell line proved loss of functional imprinting of IGF2. H19 and IGF2 were regulated in parallel, and expression levels were variable. Taken together, the disruption of the IGF2 promoter regulation, particularly the loss of P1 activity, is a common feature of human HCCs. The loss of P1 activity explains the frequent loss of biallelic IGF2 expression and may potentially be used as a diagnostic or monitoring marker for human HCC.

Impaired visual cortical processing of affective facial information in schizophrenia.

Facial emotion perception impairment in schizophrenia is currently viewed as abnormal affective processing. Facial emotion perception also relies on visual processing. Yet, visual cortical processing of facial emotion is not well understood in this disorder. We measured perceptual thresholds for detecting facial fear and happiness in patients (n=23) and controls (n=23), and adjusted emotion intensity of facial stimuli (via morphing between images of neutral and emotive expressions) for each subject. We then evaluated activations of the visual cortex and amygdala during the performance of perceptually-equated facial emotion detection tasks. Patients had significantly lower fear- and happiness-induced activations in the visual cortex and amygdala. Activations between the visual cortex and amygdala were largely correlated, but the correlations in patients occurred abnormally early in response time course during fear perception. In schizophrenia, visual processing of facial emotion is deficient and visual and affective processing of negative facial emotion may be prematurely associated.

Deficient cortical face-sensitive N170 responses and basic visual processing in schizophrenia.

BACKGROUND: Face detection, an ability to identify a visual stimulus as a face, is impaired in patients with schizophrenia. It is unclear whether impaired face processing in this psychiatric disorder results from face-specific domains or stems from more basic visual domains. In this study, we examined cortical face-sensitive N170 response in schizophrenia, taking into account deficient basic visual contrast processing. METHODS: We equalized visual contrast signals among patients (n=20) and controls (n=20) and between face and tree images, based on their individual perceptual capacities (determined using psychophysical methods). We measured N170, a putative temporal marker of face processing, during face detection and tree detection. RESULTS: In controls, N170 amplitudes were significantly greater for faces than trees across all three visual contrast levels tested (perceptual threshold, two times perceptual threshold and 100%). In patients, however, N170 amplitudes did not differ between faces and trees, indicating diminished face selectivity (indexed by the differential responses to face vs. tree). CONCLUSION: These results indicate a lack of face-selectivity in temporal responses of brain machinery putatively responsible for face processing in schizophrenia. This neuroimaging finding suggests that face-specific processing is compromised in this psychiatric disorder.

Greater sensitivity of the cortical face processing system to perceptually-equated face detection.

Face detection, the perceptual capacity to identify a visual stimulus as a face before probing deeper into specific attributes (such as its identity or emotion), is essential for social functioning. Despite the importance of this functional capacity, face detection and its underlying brain mechanisms are not well understood. This study evaluated the roles that the cortical face processing system, which is identified largely through studying other aspects of face perception, play in face detection. Specifically, we used functional magnetic resonance imaging (fMRI) to examine the activations of the fusifom face area (FFA), occipital face area (OFA) and superior temporal sulcus (STS) when face detection was isolated from other aspects of face perception and when face detection was perceptually-equated across individual human participants (n=20). During face detection, FFA and OFA were significantly activated, even for stimuli presented at perceptual-threshold levels, whereas STS was not. During tree detection, however, FFA and OFA were responsive only for highly salient (i.e., high contrast) stimuli. Moreover, activation of FFA during face detection predicted a significant portion of the perceptual performance levels that were determined psychophysically for each participant. This pattern of result indicates that FFA and OFA have a greater sensitivity to face detection signals and selectively support the initial process of face vs. non-face object perception.

Expression, promoter usage and parental imprinting status of insulin-like growth factor II (IGF2) in human hepatoblastoma: uncoupling of IGF2 and H19 imprinting.

We have studied the promoter utilization and parental imprinting status of human IGF2 in three genetically informative hepatoblastomas from patients ranging in age from 9 months to 3 years. In all three cases, there is a downregulation of promoter P1 in the tumor tissues while the P2 and P3 promoters are upregulated compared to the normal liver. One of three patients displayed loss of imprinting (LOI) of IGF2 in the tumor tissue. We also investigated the expression of the H19 gene in all three cases and the methylation pattern in H19 from the patient with LOI of IGF2. The expression of H19 was greatly reduced in all tumors. Monoallelic H19 expression however, was retained even in the case which showed LOI of IGF2. Unlike the situation in Wilms' tumor, no differences in the methylation pattern between the normal liver and tumor tissues were observed in the H19 promoter or 3' region, using HpII analysis. We show here, that in contrast to the situation in Wilms' tumor, H19 expression is not a prerequisite for maintaining a monoallelic IGF2 expression.

Developmental-dependent DNA methylation of the IGF2 and H19 promoters is correlated to the promoter activities in human liver development.

We have previously shown that the four promoters of the IGF2 gene are under a tight but dynamic control during human liver development, whereby P3 and P1 are reciprocally active before and after birth respectively while the P2 and P4 promoters are constitutively active at a relatively lower level. In this study, we investigated the methylation status of the promoters P1 and P3 of IGF2 and the promoter region of the H19 gene in developing human livers ranging from fetal to late adult. A region of about 300 bp immediately upstream of the IGF2 exon 5 was found to be subjected to a developmental-specific methylation and this may correlate to the P3 promoter activity. The P1 domain of IGF2 was also found to be methylated in a developmentally-specific pattern. The promoter region of the H19 gene displayed different methylation patterns in different development stages showing decreased general methylation with increase of age. Therefore, regional- and developmental-specific DNA methylation is displayed in the promoter regions of the IGF2 and H19 genes. This may be an important factor involved in gene regulation in the developing human liver.

Acute and short-term effects of growth hormone on insulin-like growth factors and their binding proteins: serum levels and hepatic messenger ribonucleic acid responses in humans.

We investigated the acute (4-5 h) and short-term (5 days) effects of GH treatment on hepatic messenger RNA (mRNA) levels of the genes for the insulin-like growth factors (IGFs), insulin-like growth factor binding protein-1, -2, and -3 (IGFBPs), and the acid labile subunit (ALS), as well as serum levels of these proteins in humans. At the mRNA level, we observed an increase in IGF-1 transcription (+173%) following GH treatment in the acute group, which remained elevated in the short-term treatment group. IGFBP-2 mRNA decreased after short-term GH treatment, without changes in IGFBP-1 or -3 expression. The ALS transcript level increased after 5 days. In serum, we found increased levels of IGF-I and insulin, and decreased levels of IGF-II, in the short-term treatment group. IGFBP-1 decreased in both treatment groups, whereas IGFBP-2 was reduced after 5 days treatment. ALS increased in the short-term group. We observed increased IGFBP-3 serum levels after 5 days of GH treatment, likely due to increased formation of the ternary complex. Our results show that the metabolic effects by GH on the IGF axis are complex. In addition to a direct stimulation of IGF-I and ALS expression, GH inhibits IGFBP-1 serum levels and IGFBP-2 expression in an indirect manner, possibly facilitating enhanced IGF bioavailability to target tissues.

Biosynthesis of dolichol and dolichylphosphate in rat hepatocytes in vivo.

The biosynthesis of dolichol and dolichylmonophosphate in rat liver was studied using [3H]mevalonate as precursor. The radioactive precursor was either injected into the portal vein of the rat or added to the incubation medium containing isolated hepatocytes, followed by the isolation of microsomes and mitochondria from the liver or the hepatocytes. In both systems dolichol in microsomes was highly labeled after a short labeling period followed by a rapid decrease. During this period the labeling of mitochondrial dolichol was low. The specific radioactivity of dolichyl-P in microsomes of both systems was higher in the initial phase than in dolichol and increased further with time. The mitochondrial labeling was also increased but was at a much lower level.

Differentiation associated modulation of K-FGF expression in a human teratocarcinoma cell line and in primary germ cell tumours.

The human teratocarcinoma cell line Tera 2 can be induced to differentiate in vitro after exposure to retinoic acid. We show in this paper that whereas the K-FGF oncogene is expressed in undifferentiated cells, addition of retinoic acid rapidly (less than 60 min) downregulates the expression of this gene. However, when cells are cultured in RA for an extended period of time (greater than 15 days) K-FGF transcripts reappear. We also report that K-FGF is expressed in approximately one-third of primary human germ cell tumours but not in the corresponding normal testicular tissue.

Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein and connexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells.

Human glioblastoma cell cultures were established and the expression of glial fibrillary acidic protein (GFAP) and the gap-junction protein connexin 43 (Cx43) was confirmed by Western blot. Following treatment with 4-phenylbutyrate (4-PB), increased concentrations of non-phosphorylated GFAP were seen, while phosphorylated isoforms remained intact. Immunocytochemical staining of glioblastoma cells revealed an intracellular redistribution of GFAP. In addition to cytoplasmic immunostaining, GFAP immunoreactivity was also associated with the nucleus and/or the nuclear membrane. Phosphorylated and non-phosphorylated Cx43 proteins were increased 2- to 5-fold following 4-PB treatment, and were redistributed to areas of the cell surface, participating in cell-to-cell contacts. In addition, functional gap-junction coupling was amplified, as indicated by increased fluorescent dye transfer, and elevated levels of Cx43 protein were detected in parallel with enhanced gap-junction communication. Induced cell differentiation, with improved functional coupling of tumour cells, may be of importance for therapeutic strategies involving intercellular transport of low molecular-weight compounds.

Mammary tumours in Sprague-Dawley rats after initiation with DMBA followed by exposure to 50 Hz electromagnetic fields in a promotional scheme.

In order to test whether a transient-producing intermittent magnetic field (MF) acts as a promoter in rat mammary tumour development, a study of a 50 Hz sinusoidal MF with flux densities of 0.25 and 0.5 mT was performed on female Sprague-Dawley rats. A single administration of 7 mg of 7,12-dimethyl-benz[a]anthracene (DMBA) was given by gavage to 52-day-old animals. After 1 week, exposure to an intermittent (15 s on/15 s off) transient-associated magnetic field was started. MF exposure was performed for 19 or 21 h per day up to 25 weeks, when the study was terminated. Twice a week a careful examination with palpation for tumours was done. Tumour incidence, the number of tumours per animal, tumour volume and tumour weight were recorded. A total of 70% of MF-exposed animals developed tumours. For animals exposed to DMBA alone the correspondent figure was 71.7%. No statistical differences were seen either for tumour-bearing animals or for the total number of tumours. A slightly larger total tumour weight and tumour volume was seen for animals exposed to 0.25 mT MF. These differences were not statistically significant.