Isolation, characterization, and expression in Escherichia coli of a cDNA encoding rat heme oxygenase-2

In a recent study (Cruse, I., and Maines, M.D. (1988) J. Biol. Chem. 263, 3348-3353), we reported the isolation of a small cDNA fragment encoding a portion of heme oxygenase-2 through immunological screening of a rat testis cDNA library in lambda gt11. We have now used this 274-base pair (bp) cDNA fragment as a hybridization probe for rescreening of the same library, and have thereby recovered a number of additional positive isolates. Of these, three candidates of approximately 900, 1100, and 1300 bp, respectively, were subsequently subcloned and sequenced. Although differing in length, the sequences of these clones were found to be otherwise identical. Moreover, the length of isolate 18B, 1284 bp, corresponded well with that of the single mRNA species (approximately 1300-1350 nucleotides) detected through Northern blot hybridization analysis of rat testis total and poly(A)+RNA. This full- or near full-length cDNA encodes a 315-amino acid protein with a molecular weight of 35,757, in good agreement with the 36,000 estimated molecular weight of heme oxygenase-2. When expressed in Escherichia coli, cDNA encodes a protein that cross-reacts with heme oxygenase-2 antiserum (as assayed by Western immunoblotting) and yields high levels of heme oxygenase activity in bacterial soluble cell extracts. Finally, computer analysis of the heme oxygenase-2 cDNA sequence indicates that the predicted amino acid sequence and hydropathy profile of the heme oxygenase-2 protein exhibit similarity with heme oxygenase-1.     

Regulation of N-acetylglucosaminyltransferase V activity. Kinetic comparisons of parental, Rous sarcoma virus-transformed BHK, and L-phytohemagglutinin-resistant BHK cells using synthetic substrates and an inhibitory substrate analog

Baby hamster kidney (BHK) cells transformed with Rous sarcoma virus, RS-BHK cells, demonstrate a 2.5-fold increase in the activity of N-acetylglucosaminyltransferase V (GlcNAc-T V, EC 2.4.1.155), and this increase in activity appears to be specific for this enzyme. By contrast, a lectin-resistant BHK cell line selected for its ability to grow in high levels of L-phytohemagglutinin, LP3.3, is characterized by a specific decrease in its GlcNAc-T V activity. To test if these alterations in the apparent Vmax of GlcNAc-T V are due to changes in the efficiency of populations of enzymes in RS-BHK and LP3.3 cells compared to the parental BHK cells, we have compared the kinetic properties of the enzymes from these three sources. The Km constants observed for both the sugar nucleotide donor (UDP-GlcNAc) and two synthetic trisaccharide acceptors were indistinguishable. The Vmax values toward three synthetic acceptors were also determined first for the BHK GlcNAc-T V, and they varied by over 5-fold. When these values were measured for the variant and transformed cell enzymes, however, similar 5-fold differences were still observed, although the absolute values for these acceptors were all higher or lower for the RS-BHK and LP3.3 enzymes, respectively. In addition, we have synthesized a deoxygenated analog of the specific GlcNAc-T V acceptor, beta GlcNAc(1,2) alpha Man(1,6) beta ManOR, where the reactive 6'-OH group has been removed, and the resulting trisaccharide was found to be a competitive inhibitor of the enzyme. The Ki for this inhibitor was near 70 microM for the GlcNAc-T V from all three sources. These kinetic comparisons demonstrate that the enzymes from the three cell types have kinetically indistinguishable active sites. These results suggest that the differences in the apparent Vmax values among the cell types are most likely due to alterations in the number of active molecules rather than in the modulation of either their catalytic activities or specificities.     

Molecular cloning and expression of the rat beta 1-adrenergic receptor gene.

Using the sequence homology approach for cloning related genes within the G-protein-coupled receptor gene family, we have cloned the gene for the rat beta 1-adrenergic receptor (beta 1-AR). The rat beta 1-adrenergic receptor gene was isolated from a lambda EMBL3 rat genomic DNA library using the hamster beta 2-adrenergic receptor (beta 2-AR) coding sequence as a probe under low stringency hybridization conditions. The rat beta 1-AR gene encodes a protein of 466 amino acids that contains one consensus site for N-linked glycosylation (Asn-15) and three consensus sites for cAMP-dependent protein kinase phosphorylation (Ser-296, Ser-301, and Ser-401). The encoded rat beta 1-AR is 98 and 91% similar at the amino acid level with the human beta 1-AR in the transmembrane domains and in the overall sequence, respectively. Genomic Southern blot and gene dosage analyses indicate that the rat beta 1-AR gene is a single copy gene. The tissue distribution of the rat beta 1-AR mRNA was highest in the pineal gland with other brain regions and peripheral tissues, including the heart, expressing the mRNA at moderate levels. The bacteriophage clone containing the rat beta 1-AR gene with its natural promoter was co-transfected with the selectable marker (pRSVneo) conferring neomycin resistance into beta 1-AR-deficient mouse L cells. Analyses of the selected transfectant demonstrates efficient expression of the beta 1-AR gene and functional receptor. 125I-Labeled iodocyanopindolol bound transfectant membranes with an affinity of KD = 24 pm; the beta 1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a Ki approximately 140 times lower than that for the beta 2-AR-selective antagonist ICI 118,551. In addition, in the transfectant cell line, adenylylcyclase was stimulated by beta-adrenergic receptor agonists with the rank order of potency of isoproterenol greater than norepinephrine = epinephrine, consistent with properties expected of the beta 1-AR subtype.     

r14 syndrome without major dysmorphism

An 8-year-old boy, mentally retarded and epileptic since the age of six months, was found carrier of ring 14 chromosome. A dystrophy of the eye fundi was observed (whitish puncta of the macula); except for the "almond shaped eyes", there was no obvious dismorphism.     

Chemotaxonomy of the genus Talaromyces

Species of the ascomycetous genus Talaromyces have been examined for profiles of secondary metabolites on TLC. The greatest number of specific metabolites were produced on oatmeal-, malt extract- and yeast-extract sucrose agars. Profiles of intracellular secondary metabolites produced on oatmeal agar were specific for each species and provided a means of simple differentiation of the taxa. Examination of the most important species using high performence liquid chromatography (HPLC) allowed to solve some taxonomic problems. Known mycotoxins are produced by T. stipitatus (duclauxin, talaromycins, botryodiploidin), T. stipitatus chemotype II (emodin), T. panasenkoi (spiculisporic acid), T. trachyspermus (spiculisporic acid), T. trac macrosporus (duclauxin) and T. wortmannii (rugulosin). Wortmannin is produced by an atypical strain of T. flavus but not T. wortmannii. Several other secondary metabolites were discovered for the first time in the following species: Glauconic acid is produced by T. panasenkoi, T. ohiensis and T. trachyspermus; vermiculine by T. ohiensis; duclauxin by T. flavus var. macrosporus and the mitorubrins by T. flavus and T. udagawae. The profiles of secondary metabolites support the established taxonomy of the species based on morphology, showing the genetic stability of profiles of secondary metabolites in Talaromyces. Two new taxa are proposed: T. macrosporus comb. nov. (stat. anam. Penicillium macrosporum stat. nov.), and Penicillium vonarxii, sp. nov. for the anamorph of T. luteus.     

Alteration of cell surface carbohydrates associated with ordered and disordered proliferation of oral epithelia: a lectin histochemical study in oral leukoplakias, papillomas and carcinomas

Cell surface carbohydrates in healthy oral mucosa (n = 15), leukoplakias without (n = 48) and with (n = 62) dysplasia, oral papillomas (n = 6) and squamous cell carcinomas (SCCs) (n = 40) were examined using the lectins peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEA I), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), and Griffonia simplicifolia agglutinin I (GS I-B4). Binding of these lectins in formalin-fixed, paraffin-embedded tissues was demonstrated using either the peroxidase-anti-peroxidase (PAP) method or the avidin-biotin method. Healthy oral epithelia revealed binding sites for these lectins mostly in the suprabasal keratinocytes with occasional PNA binding also in their basal cells. Unlike healthy mucosa, a number of leukoplakias without and with dysplasia revealed receptor sites for UEA I also in their basal layer. Only those keratinocytes undergoing squamoidal differentiation exhibited SBA binding. Staining patterns of UEA I and SBA did not vary significantly between either leukoplakias without and with dysplasia or papillomas and SCCs. Conversely, a reduction or lack of binding sites for PNA (Gal beta 1-3GalNAc), HPA (D-GalNAc alpha) and GS I-B4 (alpha D-Gal) was observed more frequently in leukoplakias with dysplasia and SCCs contrasting their counterparts lacking epithelial dysplasia. Cell surface glycosyl residues play an important role in the regulation of cell proliferation and epithelial growth. Aberrant glycosylation in oral dysplastic leukoplakias and carcinomas leading to the lack of the relevant terminal sugar residues from their cell surface carbohydrates is probably a major reason for the hyper-/disordered proliferation.     

Response analysis of vertebrate retina

In a recent work (Ouztöreli, 1980) a mathematical model for studying the neural activities in a vertebrate retina has been investigated, where the basic network contains five interconnected neurons: a receptor cell, a bipolar cell, a horizontal cell, an amacrine cell, and a retinal ganglion cell. More recently, in (Ouztöreli and O'Mara, 1980) the basic network has been extended to a larger network containing twelve neurons. In both of these works, the performances of the basic and extended models were discussed under different structural and processing conditions with constant inputs by using the results of one of our earlier work (Ouztöreli, 1979). In the present paper we investigate by simulations the responses of the basic retinal network to piecewise constant and periodic inputs. The step and frequency responses of the extended retinal network will be discussed in a forthcoming paper.This work was partially supported by the Natural Sciences and Engineering Research Council of Canada under Grant A-4345 through the University of alberta     

The Roberts syndrome

Summary Four siblings with the autosomal recessive Roberts syndrome are reported, and we discuss the phenotypic overlap of this syndrome with other similar radial, aplasia syndromes.     

Stereological Analysis of Floral Development and Quantitative Histochemistry of Nucleic Acids in Fertile and Base-sterile Varieties of Wheat

The speltoid series of mutations provides a genetic tool forinvestigating the control of flower development. In fertileGabo wheat and in St₂ and St₃ basal sterile speltoids, the changesin RNA staining patterns have been followed histochemicallyas a marker of cellular activity. Sterile floret sites are characterizedby a reduced RNA content. Use of dual-wavelength microspectrophotometryhas provided evidence that RNA content is similar in lemma primordiasubtending presumptive fertile and sterile primordia, but isstrikingly different in the floral meristems in the two types.The total nucleic acid content of the floral primordia has beenrelated to the number of epidermal cells in the subtending lemmaprimordium as a marker of development, using a linear modelanalysis of covariance. There is a linear increase in nucleicacid content during fertile floret development, but no significantincrease in RNA content in sterile St₃ primordia. The rate constantis only 1/10th to 1/20th of that of Gabo. A stereological analysisshows that cell number in the floral meristems of Gabo increasesexponentially during development. In contrast, in St₃, whilethere is a significant increase in cell number, it is at a drasticallyreduced rate. The intercept values are close to zero, indicatingthat only one or two cells initiate floral meristem development.The fertility-controlling alleles exert their effect prior tothe appearance of a visible floral primordium, and probablyafter initiation of the lemma. Triticum aestivum, wheat, floral development, histochemistry, nucleic acids, speltoids     

Plasmids of serogroup 1 strains ofLegionella pneumophila

Twelve strains ofLegionella pneumophila were tested for the presence of plasmid DNA. Three strains, belonging to serogroup 1, had large plasmids of 83.8×10⁶ daltons, as determined by electron microscopy. A fourth strain, also from serogroup 1, had a similar large plasmid in addition to a smaller plasmid. Restriction analysis of plasmid DNA isolated from the strains with a single size plasmid indicated that the plasmids were structurally very similar. The biologic functions of these plasmids are yet to be determined.