A comparative study of the expression patterns for vegf, vegf-b/vrf and vegf-c in the developing and adult mouse.

With the goal of better understanding the function and regulation of the different members of the VEGF family this study reports mapping of vegf, vegf-b and vegf-c mRNA expression in developing and adult mice. On embryonic day 14 (E14) there is a high expression of vegf and vegf-b, vegf-b being exceptionally high in heart and CNS. The vegf-c expression is lower with distinct signals in CNS and heart. Prior to birth (E17), vegf and vegf-b expression is moderately downregulated. Overlapping expression is present in intrascapular fat and heart. vegf dominates in thyroid and lung, while vegf-b appears to be the only VEGF member expressed at detectable levels in the CNS. In young adult mouse vegf and vegf-b show partly overlapping expression patterns particularly in kidney, heart and in the thymus, vegf displays higher levels in lung and liver, vegf-b appears to be dominating in brain, heart, testis and kidney. In brain the highest levels of vegf-b is present in the hippocampus. No vegf-c mRNA expression could be detected in the adult. Taken together, these results illustrate, in detail, the different regulations of the members of the VEGF gene family. There are at present at least three specific effectors of vascular proliferation with clear differences in their expression.

An evaluation of styrene genotoxicity using several biomarkers in a 3-year follow-up study of hand-lamination workers.

A study employing several biomarkers of styrene exposure and genotoxicity was carried out in a group of lamination (reinforced plastic) workers and controls, who had been repeatedly sampled during a 3-year period. Special attention will be paid to the last sampling (S.VI), reported here for the first time. Styrene concentration in the breathing zone, monitored by personal dosimeters, and urinary mandelic acid (MA) were measured as indicators of external exposure. Blood samples were assayed for styrene-specific O6-guanine adducts in DNA, N-terminal valine adducts of styrene in haemoglobin, DNA single-strand breaks (SSB), determined by use of the single cell gel electrophoresis (Comet) assay), and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutant frequencies (MF) in T-lymphocytes. O6-styrene guanine adduct levels were significantly higher in the exposed group (5.9 +/- 4.9 adducts/10(8) dNp) as compared to laboratory controls (0.7 +/- 0.8 adducts/10(8) dNp; P = 0.001). DNA adduct levels significantly correlated with haemoglobin adducts, SSB parameters and years of employment. Styrene-induced N-terminal valine adducts were detected in the lamination workers (1.7 +/- 1.1 pmol/g globin), but not in the control group (detection limit 0.1 pmol/g globin). N-terminal valine adducts correlated strongly with external exposure indicators, DNA adducts and HPRT MF. No significant correlation was found with SSB parameters. A statistically significant difference in HPRT MF was observed between the laminators (22.3 +/- 10.6/10(6)) and laboratory controls (14.2 +/- 6.5/10(6), P = 0.039). HPRT MF in the laminators significantly correlated with styrene concentration in air, MA and haemoglobin adducts, as well as with years of employment and age of the employees. No significant difference (P = 0.450) in MF between the laminators and the factory controls was observed. Surprisingly, we detected differences in MF between sexes. When data from all measurements were combined, women showed higher MF (geometric mean 15.4 vs. 11.2 in men, P = 0.020). The styrene-exposed group exhibited significantly higher SSB parameters (tail moment (TM), tail length (TL) and the percentage of DNA in the tail (TP)) than the control group (P < 0.001). SSB parameters correlated with indicators of external exposure and with O6-styrene guanine adducts. No significant correlation was found between SSB parameters and haemoglobin adducts or HPRT MF. The data encompassing biomarkers from repeated measurements of the same population over a 3-year period are discussed with respect to the mechanisms of genotoxic effects of styrene and the interrelationship of individual biomarkers.

Sequencing analysis of insulin receptor defects and detection of two novel mutations in INSR gene.

Mutations in the insulin receptor gene cause the inherited insulin resistant syndromes Leprechaunism and Rabson-Mendenhall syndrome. These recessive conditions are characterized by intrauterine and post-natal growth restrictions, dysmorphic features, altered glucose homeostasis, and early demise. The insulin receptor gene (INSR) maps to the short arm of chromosome 19 and is composed of 22 exons. Here we optimize the conditions for sequencing this gene and report novel mutations in patients with severe insulin resistance. METHODS: PCR amplification of the 22 coding exons of the INSR gene was performed using M13-tailed primers. Bidirectional DNA sequencing was performed with BigDye Terminator chemistry and M13 primers and the product was analyzed on the ABI 3100 genetic analyzer. Data analysis was performed using Mutation Surveyor software comparing the sequence to a reference INSR sequence (Genbank NC_000019). RESULTS: We sequenced four patients with Leprechaunism or Rabson-Mendenhall syndromes as well as seven samples from normal individuals and confirmed previously identified mutations in the affected patients. Three of the four mutations identified in this group caused premature insertion of a stop codon. In addition, the INSR gene was sequenced in 14 clinical samples from patients with suspected insulin resistance and one novel mutation was found in an infant with a suspected diagnosis of Leprechaunism. DISCUSSION: Leprechaunism and Rabson-Mendenhall syndrome are very rare and difficult to diagnose. Diagnosis is currently based mostly on clinical criteria. Clinical availability of DNA sequencing can provide an objective way of confirming or excluding the diagnosis.

Molecular characterization of two deletion events involving Alu-sequences, one novel base substitution and two tentative hotspot mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene in five patients with Lesch-Nyhan syndrome.

Mutations identified in the hypoxanthine phosphoribosyltransferase (HPRT) gene of patients with Lesch-Nyhan (LN) syndrome are dominated by simple base substitutions. Few hotspot positions have been identified, and only three large genomic rearrangements have been characterized at the molecular level. We have identified one novel mutation, two tentative hot spot mutations, and two deletions by direct sequencing of HPRT cDNA or genomic DNA from fibroblasts or T-lymphocytes from LN patients in five unrelated families. One is a missense mutation caused by a 610C-->T transition of the first base of HPRT exon 9. This mutation has not been described previously in an LN patient. A nonsense mutation caused by a 508C-->T transition at a CpG site in HPRT exon 7 in the second patient and his younger brother is the fifth mutation of this kind among LN patients. Another tentative hotspot mutation in the third patient, a frame shift caused by a G nucleotide insertion in a monotonous repeat of six Gs in HPRT exon 3, has been reported previously in three other LN patients. The fourth patient had a tandem deletion: a 57-bp deletion in an internally repeated Alu-sequence of intron 1 was separated by 14 bp from a 627-bp deletion that included HPRT exon 2 and was flanked by a 4-bp repeat. This complex mutation is probably caused by a combination of homologous recombination and replication slippage. Another large genomic deletion of 2969 bp in the fifth patient extended from one Alu-sequence in the promoter region to another Alu-sequence of intron 1, deleting the whole of HPRT exon 1. The breakpoints were located within two 39-bp homologous sequences, one of which overlapped with a well-conserved 26-bp Alu-core sequence previously suggested as promoting recombination. These results contribute to the establishment of a molecular spectrum of LN mutations, support previous data indicating possible mutational hotspots, and provide evidence for the involvement of Alu-mediated recombination in HPRT deletion mutagenesis.