Emx2 in adult neural precursor cells.

Emx2 is a vertebrate homeobox gene involved in the control of the central nervous system development. In the formation of cerebral cortex, Emx2 expression is restricted mainly to the germinal ventricular zone fading away in the first postmitotic neurons. This expression pattern, the severe impairment of cortex organization and the size in mutant mice suggest a role of Emx2 in the control of proliferation and migration of neural precursor cells. The observed persistence of Emx2 expression in adult neurogenic areas in vivo is here confirmed at later stages. We also find that Emx2 is expressed at high levels in adult neural stem cells (ANSCs) in vitro and is down modulated upon differentiation. Overexpression of Emx2 gene in ANSCs has an anti-proliferative effect but it does not influence a particular differentiation pathway. Our results suggest that Emx2 may act promoting an asymmetric mode of cell division thereby increasing the size of a transit amplifying population.

5,10-Methylenetetrahydrofolate reductase polymorphism and early organ damage in primary hypertension.

Hyperhomocyst(e)inemia is a known risk factor for the development of atherosclerotic vascular damage. Plasma homocyst(e)ine levels are influenced by nutritional and hereditary factors. A point mutation (cytosine to thymidine substitution; C677T) in the gene encoding 5,10-methylenetetrahydrofolate reductase (MTHFR) makes the enzyme thermolabile and has been associated with elevated homocyst(e)ine levels in homozygous carriers (TT genotypes). We evaluated the relationship between the T allele encoding for the thermolabile variant of MTHFR and several biochemical risk factors and early signs of hypertensive and atherosclerotic organ damage in 206 untreated patients with primary hypertension. The MTHFR genotype was evaluated by polymerase chain reaction. Albuminuria was measured as albumin-to-creatinine ratio in three nonconsecutive first morning urine samples (negative urine culture). Persistent Mi (Alb+) was defined as an average albumin-to-creatinine ratio between 2.38 and 19 (men) and 2.96 and 20 (women). Left ventricular (LV) mass index (LVMI) was assessed by M-B mode echocardiography (LV hypertrophy, LVH = LVMI > or = 125 g/m2), carotid geometry by high-resolution ultrasound scan, and retinal vascular changes by direct ophthalmoscopy (Keith-Wagener classification). The prevalence of Mi, LVH, and retinopathy was 14%, 45%, and 42%, respectively. The prevalence of carotid plaque was 25%. Allele frequencies for C (wild-type allele) and T allele (mutant allele) were 56% and 44%, respectively. Genotype frequencies were CC 29%, CT 54%, TT 17% according to Hardy Weinberg equilibrium. There were no differences as for age, sex, body mass index, blood pressure levels, lipid profile, smoking habits, and alcohol intake, and LVMI and urinary albumin excretion on the basis of MTHFR genotype. Patients with TT polymorphism showed a higher prevalence of retinal vascular changes (TT, 61% v CT + CC, 38%; P < .02) and carotid plaque (TT, 42% v CT + CC, 21%; P < .05) compared to patients with CC and CT polymorphism. Moreover, patients with T allele showed increased carotid artery size as demonstrated by intima plus media thickness (IT, 0.79 +/- 0.05 mm v CT + CC, 0.67 +/- 0.02 mm; P < .02), relative wall thickness (TT, 0.23 +/- 0.01 mm v CT + CC, 0.20 +/- 0.005 mm; P < .02), and surface area (TT, 19 +/- 1.9 mm2 v CT + CC, 15 +/- 0.55 mm2; P < .05). Multiple linear regression analysis demonstrated that MTHFR genotype and systolic blood pressure independently influence intima-media thickness and together account for about 11% of its variations (r2 = 0.11, F = 9.7, dF = 1-205, P < .0001). Homozygosity for the T allele of the MTHFR gene is an independent risk factor for the development of early atherosclerotic organ damage in hypertensive patients.

Induction of heme oxygenase 1 in liver of spontaneously diabetic rats.

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified beta-actin mRNA. The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.

OTX2 homeodomain protein binds a DNA element necessary for interphotoreceptor retinoid binding protein gene expression.

Transcription of the human interphotoreceptor retinoid binding protein (IRBP) gene is strictly tissue specific, being restricted to retinal photoreceptors and pinealocytes. We have previously demonstrated that a sequence named A element, in the IRBP promoter is essential for IRBP gene transcription in vivo. Here we demonstrate that the human homeodomain protein OTX2 is present in nuclear extracts of IRBP expressing cells and specifically interacts with the IRBP A promoter element in vitro. OTX2, as well as CRX, a homeodomain protein very similar to OTX2, activates the human IRBP promoter in co-transfection experiments.

Intron 16 insertion of the angiotensin converting enzyme gene and transcriptional regulation.

BACKGROUND: The insertion/deletion (I/D) polymorphism in intron 16 of angiotensin converting enzyme (ACE) is associated with circulating and tissue enzymatic levels, around two-fold higher in homozygous D than homozygous I individuals. The mechanism underlying this quantitative difference is unknown and the hypothesis that the deletion removes a transcriptional silencer located within the intron has been proposed. METHODS: We have set up an assay based on constructs carrying fragments of intron 16, either in the I or in the D form, fused to the Herpes simplex virus thymidine kinase heterologous promoter driving the expression of the CAT reporter gene. These constructs have been used in transfection experiments in ACE expressing cells. RESULTS: Plasmids containing either intronic fragments from I and D subjects or an artificial D fragment derived from an I intron did not show significant difference between I and D in affecting the heterologous promoter activity. The finding of a yet undescribed polymorphism in intron 16 is also reported. CONCLUSION: The above results suggest that the intron 16 deletion by itself has no effect in regulating transcription in our assay system.

Toxic action of advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance to diabetic retinopathy.

The toxic effects of advanced glycation end products (AGEs) on bovine retinal capillary pericytes (BRP) and endothelial cells (BREC) were studied. AGE-modified bovine serum albumin (AGE-BSA) was toxic to BRP. At a concentration of 500 micrograms/ml it reduced the BRP number to 48 +/- 3% (p < 0.05) of untreated controls, as determined by cell counting with haemocytometer. AGE-BSA was also toxic to bovine aortic endothelial cells (BAEC) reducing cell number to 84 +/- 3.1% of untreated controls. Under similar conditions, low concentrations (62.5 micrograms/ml) of AGE-BSA were mitogenic to BREC increasing the cell proliferation to 156 +/- 11% (p < 0.05) above that of untreated controls. At a higher dose of 500 micrograms/ml AGE-BSA decreased the proliferation of BREC to 85 +/- 6% of untreated controls. Immunoblot analysis demonstrated that BRP and BREC express the p60 AGE-receptor. Retinal capillary bed from the human also stained positively for the p60 AGE-receptor. Addition of 0.25 micrograms/ml of p60 AGE-receptor antibody was able to block the effects of AGE-BSA on BRP and BREC. The level of binding of [125I]-labelled AGE-BSA to the cell surface was small but significant among the three cell types. There was also an increase in the internalized pool of radioligand in BRP and BREC but this was very much lower than in BAEC. In all the cell types the internalized pool of [125I]-labelled AGE-BSA was much larger than the amount associated with the cell surface. Degradation products were not detected in the media over the 24-h incubation of the cells with [125I]AGE-BSA. The binding of [125I]-labelled AGE-BSA to the cell surface was prevented by the addition of p60 AGE-receptor. These results suggest that the interaction of AGE-modified proteins with the membrane-bound AGE-receptor may play an important role in the pathogenesis of diabetic retinopathy.