Regulation of insulin-like growth factor-binding protein 1 by hypoxia and 3',5'-cyclic adenosine monophosphate is additive in HepG2 cells.

Insulin-like growth factor-binding protein 1 (IGFBP-1) is important in regulating minute-to-minute IGF bioavailability in the circulation and is primarily an inhibitor of IGF action systemically and in most cellular systems. Understanding regulation of IGFBP-1 is, thus, important in understanding regulation of IGF actions. The IGFBP-1 promoter contains a cAMP response element, and cAMP stimulates IGFBP-1 gene expression at the transcriptional level. Recently, we have found three consensus sequences for the hypoxia response element in intron 1 of the IGFBP-1 gene. Herein, we have investigated the effects of hypoxia and a cAMP analog, 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP), on IGFBP-1 expression in HepG2 cells, a model system for IGFBP-1 gene regulation. HepG2 cells were exposed to normoxia (20% pO2) or hypoxia (2% pO2) for 24 h in the absence or presence of 8-Br-cAMP (0.1, 0.5, and 1 mM). Western ligand blotting revealed IGFBP-1 as the predominant IGFBP in HepG2-conditioned media, which increased in a dose-dependent manner after incubation with 8-Br-cAMP in normoxia and hypoxia (3-fold and 7-fold at 1 mM, respectively). Under hypoxic, compared with normoxic, conditions, IGFBP-1 protein and messenger RNA (mRNA) levels increased approximately 10-fold and 20-fold, respectively. In normoxia, 8-Br-cAMP stimulated IGFBP-1 protein and mRNA levels in a dose-dependent manner (7-fold and 10-fold at 1 mM). Hypoxia and 8-Br-cAMP showed additive stimulatory effects on IGFBP-1 protein and mRNA levels (35-fold and 50-fold at 1 mM) that were time and dose dependent. Primary transcripts of IGFBP-1 mRNA were increased concordantly with IGFBP-1 mRNA. The half-life of the IGFBP-1 mRNA was markedly increased (approximately 6-fold) by hypoxia, and cAMP minimally enhanced this effect. These results demonstrate that hypoxia and compounds that increase intracellular cAMP additively regulate IGFBP-1 gene expression by transcriptional and posttranscriptional mechanisms. Regulation of IGFBP-1 mRNA and protein by cAMP and hypoxia may be important for understanding the physiologic and pathophysiologic roles of IGFBP-1.

The life cycles of the temperate lactococcal bacteriophage phiLC3 monitored by a quantitative PCR method.

We present here a new and general approach for monitoring the life cycles of temperate bacteriophages which establish lysogeny by inserting their genomes site-specifically into the bacterial host chromosome. The method is based on quantitative amplification of specific DNA sites involved in various cut-and-join events during the life cycles of the phages (i.e. the cos, attP, attB, attL and attR sites) with the use of sequence-specific primers. By comparing the amounts of these specific DNA sites at different intervals, we were able to follow the development of the lytic and lysogenic life cycles of the temperate lactococcal bacteriophage phiLC3 after infection of its bacterial host Lactococcus lactis ssp. cremoris IMN-C18.

Regulation of insulin-like growth factor-binding protein-1 by nitric oxide under hypoxic conditions.

Nitric oxide (NO) is believed to play an important, but as yet undefined, role in regulating hypoxia inducible gene expression. Recently, we have reported evidence suggesting that the human insulin-like growth factor-binding protein-1 (IGFBP-1) gene is directly regulated by hypoxia through the hypoxia-inducible factor-1 pathway. The goal of the current study was to investigate NO regulation of hypoxic induction of IGFBP-1 gene expression using HepG2 cells, a model system of hepatic gene expression. We report that a NO generator, sodium nitroprusside, significantly diminishes hypoxic activation of IGFBP-1 protein and messenger ribonucleic acid expression. Furthermore, these effects are independent of guanylate cyclase/ cGMP signaling, as two different inhibitors, LY 83583, a specific inhibitor of guanylate cyclase, and KT 5823, a protein kinase G inhibitor, had no effect on IGFBP-1 induction by hypoxia. Hypoxic induction of a reporter gene containing four tandemly ligated hypoxia response elements was completely blocked by sodium nitroprusside, but not by 8-bromo-cGMP, an analog ofcGMP. These results suggest that NO blocks hypoxic induction of IGFBP-1 by a guanylate cyclase/ cGMP-independent pathway, possibly at the level of oxygen sensing. The impaired hypoxia regulation of IGFBP-1 by nitric oxide may play a key role in the hyperinduction of IGFBP-1 observed in pathophysiological conditions such as fetal hypoxia and preeclampsia where dysregulation of NO has been observed.

Genome organization in Fusarium oxysporum: clusters of class II transposons.

Several families of transposable elements (TEs) are present in the genome of the phytopathogenic fungus Fusarium oxysporum. They are present in copy numbers ranging from just a few elements to tens or hundreds per genome. Sequence analysis of contiguous stretches of genomic DNA surrounding insertion sites of one family revealed that they are packed with repeated sequences. We have carried out a detailed study of the composition and arrangement of these repeats in three chromosomal regions. We found that they are essentially mixtures of several types of TEs, most of them being DNA transposons, different from those previously characterized. Some repeats are frequently reiterated and many of them are inserted into other elements. Parts of these regions are also duplications. These regions appear prone to rearrangement and transposition and are subject to rapid reorganization.

Loss of PTEN facilitates HIF-1-mediated gene expression.

In glioblastoma-derived cell lines, PTEN does not significantly alter apoptotic sensitivity or cause complete inhibition of DNA synthesis. However, in these cell lines PTEN regulates hypoxia- and IGF-1-induced angiogenic gene expression by regulating Akt activation of HIF-1 activity. Restoration of wild-type PTEN to glioblastoma cell lines lacking functional PTEN ablates hypoxia and IGF-1 induction of HIF-1-regulated genes. In addition, Akt activation leads to HIF-1alpha stabilization, whereas PTEN attenuates hypoxia-mediated HIF-1alpha stabilization. We propose that loss of PTEN during malignant progression contributes to tumor expansion through the deregulation of Akt activity and HIF-1-regulated gene expression.

Transposition of the autonomous Fot1 element in the filamentous fungus Fusarium oxysporum.

Autonomous mobility of different copies of the Fot1 element was determined for several strains of the fungal plant pathogen Fusarium oxysporum to develop a transposon tagging system. Two Fot1 copies inserted into the third intron of the nitrate reductase structural gene (niaD) were separately introduced into two genetic backgrounds devoid of endogenous Fot1 elements. Mobility of these copies was observed through a phenotypic assay for excision based on the restoration of nitrate reductase activity. Inactivation of the Fot1 transposase open reading frame (frameshift, deletion, or disruption) prevented excision in strains free of Fot1 elements. Molecular analysis of the Nia+ revertant strains showed that the Fot1 element reintegrated frequently into new genomic sites after excision and that it can transpose from the introduced niaD gene into a different chromosome. Sequence analysis of several Fot1 excision sites revealed the so-called footprint left by this transposable element. Three reinserted Fot1 elements were cloned and the DNA sequences flanking the transposon were determined using inverse polymerase chain reaction. In all cases, the transposon was inserted into a TA dinucleotide and created the characteristic TA target site duplication. The availability of autonomous Fot1 copies will now permit the development of an efficient two-component transposon tagging system comprising a trans-activator element supplying transposase and a cis-responsive marked element.

Identification of fifteen novel mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in European patients with severe hypophosphatasia.

Hypophosphatasia is an inherited disorder characterised by defective bone mineralisation and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterisation of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 13 European families affected by perinatal, infantile or childhood hypophosphatasia. Eighteen distinct mutations were found, only three of which had been reported previously in North American and Japanese populations. Most of the 15 new mutations were missense mutations, but we also found two mutations affecting donor splice sites and a nonsense mutation. A missense mutation in the last codon of the putative signal peptide probably affects the final maturation of the protein. Despite extensive sequencing of the gene and its promotor region, only one mutation was identified in two cases, one of which was compatible with a possible dominant effect of certain mutations and the putative role of polymorphisms of the TNSALP gene. In 12 of the 13 tested families, genetic diagnosis was possible by characterisation of the mutations or by use of polymorphisms as genetic markers. Hypophosphatasia diagnosis was assigned in two families where clinical, laboratory and radiographic data were unclear and prenatal diagnosis was performed in one case. The results also show that severe hypophosphatasia is due to a very large spectrum of mutations in European populations with no prevalent mutation and that genetic diagnosis of the disease must be performed by extensive analysis of the gene.