Characterization of erythrocyte carbonic anhydrase in an ancient fish,the longnose gar ( Lepisosteus osseus)

This study investigates the evolutionary history of vertebrate red blood cell carbonic anhydrase (CA) by characterizing the isozyme properties and nucleotide sequence of an ancient fish, the longnose gar ( Lepisosteus osseus). The inhibitor sensitivities of gar rbc CA closely resembled those for mammalian CA II, as well as those for CAs from more recently evolved fishes. The kinetic properties of gar rbc CA were not closely aligned with either mammalian CA I and CA II, but fit well into an emerging phylogenetic pattern for early vertebrates. Gar rbc CA cDNA was also amplified from mRNA using 5' and 3'-RACE and the open reading frame consisted of 786 bp. This sequence shares approximately 65% identity with the nucleotide and amino acid sequences of both mammalian CA I and CA II. When the amino acid sequences within the active site are compared, gar rbc CA differs from mammalian CA I, CA II and CA VII by 9, 4 and 3 of the 36 amino acids, respectively. Phylogenetic analyses suggest that gar rbc CA diverged before the amniotic CAs (CA I, CA II and CA III), but after CA V and CA VII.     

Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry

In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific ‘paints’ from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10–12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.     

Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K(+) channels

Dilatation of cerebral arterioles in response to arachidonic acid is dependent on activity of cyclooxygenase. In this study, we examined mechanisms that mediate dilatation of the basilar artery in response to arachidonate. Diameter of the basilar artery (baseline diameter = 216 +/- 7 micrometer) (means +/- SE) was measured using a cranial window in anesthetized rats. Arachidonic acid (10 and 100 microM) produced concentration-dependent vasodilatation that was not inhibited by indomethacin (10 mg/kg iv) or N(G)-nitro-L-arginine (100 microM) but was inhibited markedly by baicalein (10 micrometerM) or nordihydroguaiaretic acid (NDGA; 10 microM), inhibitors of the lipoxygenase pathway. Dilatation of the basilar artery was also inhibited markedly by tetraethylammonium ion (TEA; 1 mM) or iberiotoxin (50 nM), inhibitors of calcium-dependent potassium channels. For example, 10 microM arachidonate dilated the basilar artery by 19 +/- 7 and 1 +/- 1% in the absence and presence of iberiotoxin, respectively. Measurements of membrane potential indicated that arachidonate produced hyperpolarization of the basilar artery that was blocked completely by TEA. Incubation with [(3)H]arachidonic acid followed by reverse-phase and chiral HPLC indicated that the basilar artery produces relatively small quantities of prostanoids but large quantities of 12(S)-hydroxyeicosatetraenoic acid (12-S-HETE), a lipoxygenase product. Moreover, the production of 12-HETE was inhibited by baicalein or NDGA. These findings suggest that dilatation of the basilar artery in response to arachidonate is mediated by a product(s) of the lipoxygenase pathway, with activation of calcium-dependent potassium channels and hyperpolarization of vascular muscle.     

Chemical characterisation of cheese associated fungi

Recent work in our laboratory has demonstrated that the most common contaminating fungi on different types of cheese are;Penicillium commune, P. nalgiovense, P. solitum, P. discolor, P. roqueforti, P. crustosum, P. nordicum andAspergillus versicolor. On blue cheese a new speciesP. caseifulvum has been discovered as a surface contaminant. A large number of known and unknown metabolites have been described from the above mentioned cheese associated fungi from both synthetic media and real samples. Based on chemotaxonomy our laboratory has discovered thatP. roqueforti should be divided into three species:P. roqueforti (from cheese),P. carneum (from meat) andP. paneum (from bread). SimilarlyP. verrucosum should be divided intoP. verrucosum (from cereals) andP. nordicum (from cheese and meat products). Both species produce ochratoxins, however, only the former species produce citrinin.     

Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.     

Mutational spectrum in the cardioauditory syndrome of Jervell and Lange-Nielsen

Jervell and Lange-Nielsen syndrome (JLNS) is an autosomal recessive syndrome characterised by profound congenital sensorineural deafness and prolongation of the QT interval on the electrocardiogram, representing abnormal ventricular repolarisation. In a study of ten British and Norwegian families with JLNS, we have identified all of the mutations in the KCNQ1 gene, including two that are novel. Of the nine mutations identified in this group of 10 families, five are nonsense or frameshift mutations. Truncation of the protein proximal to the recently identified C-terminal assembly domain is expected to preclude assembly of KCNQ1 monomers into tetramers and explains the recessive inheritance of JLNS. However, study of a frameshift mutation, with a dominant effect phenotypically, suggests the presence of another assembly domain nearer to the N-terminus.     

Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.