Molecular identification of a putative human hyaluronan synthase

To identify the putative mammalian hyaluronan synthase, we cloned a human cDNA that is related to the Streptococcus hyaluronan synthase (HasA) and the Xenopus developmental protein DG42 which has been shown to have chitin synthase activity. The cDNA, for which we propose the name Has2, encodes a novel protein with a predicted molecular mass of 63.6 kDa. Has2 shows 55% amino acid identity with Xenopus DG42 and 52% identity with the mouse HAS protein, another putative hyaluronan synthase recently reported by Itano and Kimata (Itano, N., and Kimata, K. (1996) J. Biol. Chem. 271, 9875-9878). The deduced primary structure revealed the presence of several hydrophobic stretches which can form multiple transmembrane domains. It also demonstrated the complete conservation of amino acid residues that are known to be critical for N-acetylglucosaminyltransferase activity of yeast chitin synthase. When the Has2 cDNA was transfected into human 293 and Chinese hamster ovary cells, the production of hyaluronan in the transfected cells increased up to 34- and 9-fold, respectively. Strong expression of Has2 mRNA was observed in exponentially proliferating human IMR-90 fibroblasts but not in growth-arrested IMR-90 cells. These results suggest that the Has2 protein is a crucial component of the human hyaluronan synthase system.     

Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria

Methionine synthase catalyzes the remethylation of homocysteine to methionine via a reaction in which methylcobalamin serves as an intermediate methyl carrier. Over time, the cob(I)alamin cofactor of methionine synthase becomes oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of functional enzyme requires reductive methylation via a reaction in which S-adenosylmethionine is utilized as a methyl donor. Patients of the cblE complementation group of disorders of folate/cobalamin metabolism who are defective in reductive activation of methionine synthase exhibit megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. Using consensus sequences to predicted binding sites for FMN, FAD, and NADPH, we have cloned a cDNA corresponding to the "methionine synthase reductase" reducing system required for maintenance of the methionine synthase in a functional state. The gene MTRR has been localized to chromosome 5p15.2-15.3. A predominant mRNA of 3.6 kb is detected by Northern blot analysis. The deduced protein is a novel member of the FNR family of electron transferases, containing 698 amino acids with a predicted molecular mass of 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase. The authenticity of the cDNA sequence was confirmed by identification of mutations in cblE patients, including a 4-bp frameshift in two affected siblings and a 3-bp deletion in a third patient. The cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme as a risk factor in hyperhomocysteinemia-linked vascular disease.     

Sequence of a mouse embryo cDNA clone encoding proteolipid subunit 9 (P1) of the mitochondrial H(+)-ATP synthase

A cDNA clone to an abundantly expressed mRNA in cleavage stage mouse embryos has been sequenced and identified as encoding subunit 9 (P1) of the mitochondrial H(+)-ATP synthase. The deduced amino acid sequence of the mature subunit 9 protein differs in a single residue from the corresponding rat, ovine, bovine and human subunits.     

Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg2+. In the presence of Mg2+ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.     

Biotin synthesis in higher plants: purification and characterization of bioB gene product equivalent from Arabidopsis thaliana overexpressed in Escherichia coli and its subcellular localization in pea leaf cells

Biotin synthase catalyses the final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli. To investigate the conversion of dethiobiotin to biotin in the plant kingdom, the cDNA encoding the bioB gene product equivalent from Arabidopsis thaliana was used to construct an E. coli overexpression strain. The purified A. thaliana bioB gene product is a homodimer (100 kDa) with a reddish color and has an absorbance spectrum characteristic of protein with [2Fe-2S] clusters. Its intracellular compartmentation in pea leaves discloses a unique polypeptide of 39 kDa within the matrix of mitochondria.     

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.     

Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 microM for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.     

Nitric oxide synthase regulation during embryonic implantation

It has previously been demonstrated that uterine nitric oxide synthase (NOS) activity increases before embryonic implantation in rats. The aim of the present work was to investigate the regulation and the physiological relevance of the nitric oxide (NO) system in ovoimplantation. The increase in NOS activity in early pregnancy was found to be independent of the presence of embryos in the uterus. Whereas the Ca2+-dependent isoform of NOS increased gradually in the preimplantation days, the Ca2+-independent isoform increased just at the beginning of implantation (Day 5, 1800 hours); then the activity of both isoforms declined. Oestradiol, whose concentration peaks before implantation, might be regulating NOS activity in the uterus, since treatment of rats with tamoxifen, a receptor antagonist, reduces the activity of both isoforms to preimplantation levels. Intraluminal injections of L-NAME (0.5 mg kg[-1]), a competitive inhibitor of NOS, reduced by 50% the number of implanted embryos; this suggests that the NO system plays a role during implantation. The data suggest that oestradiol might be a modulator of NOS activity during nidation and that NO production is necessary to achieve a successful embryo implantation.     

Therapists'' perceptions of pediatric occupational therapy interventions in self-care

A 1177 bp cDNA fragment encoding the human milk protein beta-casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase (mas1',2') promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human beta-casein cDNA. The presence of human beta-casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human beta-casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human beta-casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human beta-casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as beta-casein. The above experiments demonstrate the expression of human milk beta-casein as part of an edible food plant. These findings open the way for reconstitution of human milk in edible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children.     

Cloning, expression and characterisation of the cDNA encoding human hepatic squalene synthase, and its relationship to phytoene synthase

The reaction catalysed by squalene synthase (SQS) shows many similarities to that performed by another polyisoprene synthase, phytoene synthase (PhS). By identifying sequences conserved between yeast SQS (ySQS) and PhS, we have cloned a 2-kb cDNA (hSQS) encoding human SQS, a protein of 417 amino acids with a predicted M(r) of 48,041, which has only limited homology to ySQS. When expressed in E. coli, the hSQS cDNA directed the production of active enzyme. Two hSQS mRNA species of 2.0 and 1.55 kb have been identified which differ in their 3' untranslated sequences. The two mRNAs are present in roughly equal amounts in heart, placenta, lung, liver, kidney and pancreas, but the 2-kb mRNA predominates in brain and skeletal muscle. In HepG2 cells, both mRNAs are induced 2-4-fold by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin. In contrast, Northern blot analysis of rat tissues reveals only a 2.0-kb mRNA, which is considerably up-regulated in vivo by lovastatin.     

cDNA cloning and expression of a novel family of enzymes with calcium-independent phospholipase A2 and lysophospholipase activities

Previous studies have suggested that activation of calcium-independent PLA2 (CaIPLA2) is an early event in cell death after hypoxic injury in proximal tubule cells. An approximately 28-kD CaIPLA2 with preferential activity toward plasmalogen phospholipids has been recently purified from rabbit kidney cortex (D. Portilla and G. Dai, J Biol Chem 271, 15,451-15,457, 1996). Their report describes the cloning of a full-length rat cDNA encoding CaIPLA2, using sequences derived from the purified rabbit kidney cortex enzyme. In addition, cDNA from rabbit kidney that encode the rabbit homologue of the enzyme and a closely related isoform were isolated. The rat cDNA is predicted to encode an approximately 24-kD protein, and each cDNA contains the sequence G-F-S-Q-G, which fits the active site consensus sequence G-X-S-X-G of carboxylesterases. Several lines of evidence (DNA sequence comparison, Southern blot analysis, and examination of the expressed sequence tag database) show that CaIPLA2 enzymes are encoded by a multigene family in rats, mice, rabbits, and humans. Northern analysis of various tissues from the rat indicated that the CaIPLA2 gene is ubiquitously expressed, with highest mRNA abundance observed in the kidney and small intestine. The rat CaIPLA2 cDNA, when expressed in a baculovirus expression system, and the purified rabbit kidney cortex protein exhibit both CaIPLA2 and lysophospholipase activities. The cloned CaIPLA2 cDNA are expected to aid in understanding the role of CaIPLA2 in cell death after hypoxic/ischemic cell injury.     

Identification of a UPTG inhibitor protein from maize endosperm: high homology with sucrose synthase protein

A thermolabile UPTG inhibitor protein (IP) was isolated and purified from a developing maize endosperm preparation. High homology of two internal peptides of IP with known plant sucrose synthase (SS) sequences suggested that IP might be related somehow with SS. IP and SS activities were found in the same preparation and showed thermolability between 60-65 degrees C. IP and SS activities presented the same ionic charge and molecular mass in native conditions (Mono Q and Superose-12 columns chromatographies). Western blot experiments with an anti-SS antibody as well as with an anti-IP antibody showed a single 80 kDa polypeptide band where IP and SS activities were present. Anti-SS antibody can neutralize SS as well as IP activities in a neutralization assay. It was found that in the maize mutant shrunken-1, lacking SS1 protein, the UPTG activity was not inhibited. Furthermore, the solubilized preparation of the sh1 endosperm is unable of inhibiting UPTG activity from potato tuber. The high correlation between IP and SS properties suggests that IP might be in fact a form of SS. Moreover, the relation between IP and the SS1 isoform is discussed. So, a new biological activity of SS is suggested.     

The effects of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on intracellular adenosine 3'',5''-monophosphate (cAMP) levels and GH secretion in ovine and rat somatotrophs

Human A431 epidermoid carcinoma cells express 12-lipoxygenase enzymatic activity. However, the isoform identity based on cDNA sequence data is not known. Further, the simultaneous characterization of the intracellular distribution of 12-lipoxygenase protein and activity is lacking. Here we report that the cDNA sequence from RT-PCR-amplified 12-lipoxygenase mRNA is identical with the platelet-type 12-lipoxygenase isoform, and the leukocyte-type isoform of 12-lipoxygenase is not expressed in A431 cells. The predominant amount (78%) of 12-lipoxygenase protein resides in the cytosol. In contrast, the predominant (98%) 12-lipoxygenase activity is localized in the membrane fraction. Western blot and immunofluorescence data demonstrate that epidermal growth factor increases total cellular 12-lipoxygenase protein and enhances the association of 12-lipoxygenase protein with perinuclear or nuclear membrane sites. In addition, epidermal growth factor stimulates 12-lipoxygenase activity resulting in generation of 12(S)-hydroxyeicosatetraenoic acid from cellular arachidonate. In contrast, both 12-lipoxygenase protein and activity decrease approximately 80% within 24 h during serum starvation. The recovery of 12-lipoxygenase expression in serum-deprived cells can be induced by readdition of epidermal growth factor or serum. Further, the basal expression of 12-lipoxygenase depends on signal pathways requiring protein tyrosine kinase activity, since genistein, herbimycin A, and tyrphostin 25 reduce the expression of 12-lipoxygenase protein in A431 cells.