Aldehyde dehydrogenase class 3 expression: Identification of a cornea-preferred gene promoter in transgenic mice

Aldehyde dehydrogenase class 3 (ALDH3) constitutes 20–40% of the total water-soluble proteins in the mammalian cornea. Here, we show by Northern blot analysis that ALDH3 expression in the mouse is at least 500-fold higher in the cornea than in any other tissue examined, with very low levels of expression detected in the stomach, urinary bladder, ocular lens, and lung. Histochemical localization reveals that this exceptional level of expression in the mouse cornea occurs in the anterior epithelial cells and that little ALDH3 is present in the keratocytes or corneal endothelial cells. A 13-kbp mouse ALDH3 promoter fragment containing >12 kbp of the 5′ flanking sequence, the 40-bp untranslated first exon, and 29 bp of intron 1 directed cat reporter gene expression to tissues that express the endogenous ALDH3 gene, except that transgene promoter activity was higher in the stomach and bladder than in the cornea. By contrast, when driven by a 4.4-kbp mouse ALDH3 promoter fragment [1,050-bp 5′ flanking region, exon 1, intron 1 (3.4 kbp), and 7 bp of exon 2] expression of the cat reporter gene was confined to the corneal epithelial cells, except for very low levels in the liver, effectively reproducing the corneal expression pattern of the endogenous ALDH3 gene. These results indicate that tissue-specific expression of ALDH3 is determined by positive and negative elements in the 5′ flanking region of the gene and suggests putative silencers located in intron 1. We demonstrate regulatory sequences capable of directing cornea-specific gene expression, affording the opportunity for genetic engineering in this transparent tissue.

d-Ribulose-5-Phosphate 3-Epimerase: Cloning and Heterologous Expression of the Spinach Gene, and Purification and Characterization of the Recombinant Enzyme1

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding d-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by dl-α-glycerophosphate or ethanol and destabilized by d-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} acts as a phosphate analog in proteins phosphorylated on aspartate: Structure of a BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} complex with phosphoserine phosphatase

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Å resolution the x-ray crystal structure of the complex of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅PSP and BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

Two mitochondrial group I introns in a metazoan, the sea anemone Metridium senile: one intron contains genes for subunits 1 and 3 of NADH dehydrogenase.

Mitochondrial genes for cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) of the sea anemone Metridium senile (phylum Cnidaria) each contain a group I intron. This is in contrast to the reported absence of introns in all other metazoan mtDNAs so far examined. The ND5 intron is unusual in that it ends with A and contains two genes (ND1 and ND3) encoding additional subunits of NADH dehydrogenase. Correctly excised ND5 introns are not circularized but are precisely cleaved near their 3' ends and polyadenylylated to provide bicistronic transcripts of ND1 and ND3. COI introns, which encode a putative homing endonuclease, circularize, but in a way that retains the entire genome-encoded intron sequence (other group I introns are circularized with loss of a short segment of the intron 5' end). Introns were detected in the COI and ND5 genes of other sea anemones, but not in the COI and ND5 genes of other cnidarians. This suggests that the sea anemone mitochondrial introns may have been acquired relatively recently.

Status assessment of toxic chemicals : tris(2,3-dibromopropyl) phosphate /

Mode of access: Internet.

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Triscitrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P< 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa ( P< 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol ( P< 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.