Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the four biotin-dependent carboxylases in human cells. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS is targeted to the mitochondria and cytoplasm. We isolated 21 human HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complementation of an Escherichia coli birA mutant defective in biotin ligase. Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragment of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid. The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759. Northern blot analysis revealed the presence of a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of poly(A)+ RNA in human tissues. Human HCS shows specific regions of homology with the BirA protein of E. coli and the presumptive biotin ligase of Paracoccus denitrificans. Several forms of HCS mRNA are generated by alternative splicing, and as a result, two mRNA molecules bear different putative translation initiation sites. A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG codon to direct its translation. We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitochondria remain to be confirmed.     

Two-dimensional mapping and microsequencing of lysosomal proteins from human placenta

Lysosomes degrade a wide range of macromolecules to yield monomer products which are exported out of the lysosome by a series of transporters. In addition, lysosomes perform a range of other functions which are cell or tissue specific. In order to gain insight into the tissue specific role of lysosomes, carrier-ampholyte two-dimensional electrophoresis (2-DE) was used in combination with N-terminal sequencing to identify the major proteins present in both the membrane and luminal space of placental lysosomes. From the 45 N-terminal peptide sequences generated, 14 luminal and five membrane proteins were identified while three other sequences were novel. The sequenced proteins were a mixture of lysosomal and non-lysosomal proteins. The lysosomal proteins consisted of gamma-interferon-inducible protein (IP-30), Saposin D, cathepsins B and D, beta-hexosaminidase, palmitoyl protein thioesterase, alpha-glucosidase, and LAMP-1. The non-lysosomal proteins were serum albumin, serotransferrin, haemoglobin gamma G chain, alpha-1-antitrypsin, placental lactogen, endoplasmin, peptide binding protein 74, p60 lymphocyte protein, p450 side chain cleavage enzyme and placental alkaline phosphatase. The 2-DE maps obtained in this study are the first to identify the major proteins in both the lumen and membrane of placental lysosomes through sequence analysis, and thus provide the basis upon which to build a complete 2-DE database of the lysosome. Furthermore, the identities of the proteins sequenced from the placental lysosomes suggest a role for lysosomes in the transport of nutrients across the trophoblastic layer.     

Endoglin (CD 105) expression in human lymphoid organs and placenta

Endoglin (CD 105) is a cell surface antigen widely expressed on vascular endothelium, syncytiotrophoblast, some tissue macrophages, certain culture cells (including early leukemic B-lineage) and some endothelial cell lines. Though its relation to the transforming growth factor-beta (TGF-beta) receptor system is well documented, its function and detailed pattern of expression still remain to be clarified. We examined the differential tissue distribution of endoglin in human lymphoid organs and placenta with several anti-CD 105 monoclonal antibodies (mAbs) using an indirect immunoperoxidase technique, and performed semi-quantitative measurements using an image-analyzing system for comparison. Arterial, venous and capillary endothelia in these organs were reactive with anti-CD 105 mAbs at varying intensities. Interestingly, a distinctly stronger staining pattern was observed in the high endothelial venules (HEVs) which may indicate a special role for endoglin in lymphocyte trafficking. Syncytiotrophoblast expressed endoglin strongly on their apical cell membrane. Extravillous trophoblasts at certain locations selectively expressed endoglin on their cell membranes, suggesting a special role for this surface antigen during trophoblast differentiation.     

Identification of glutathione S-transferase isozymes and gamma-glutamylcysteine synthetase as negative acute-phase proteins in rat liver

Because acute infection and inflammation affect drug metabolism and drug-metabolizing enzymes, the effect of the acute-phase response on the expression of glutathione S-transferase (GST) isoenzymes, glutathione synthesis, and several antioxidant enzymes was investigated. Hepatic expression of GST isozymes, positive and negative acute-phase reactants, and antioxidant enzymes were determined by Northern blotting and hybridization with gene-specific oligonucleotide probes after lipopolysaccharide treatment of rats. Lipopolysaccharide caused the expected acute-phase response as judged by the increased expression of positive and decreased expression of negative acute-phase proteins. The messenger RNA (mRNA) expression of the major hepatic rat GST isozymes A1, A2, A3, M1, and M2 was decreased 50% to 90%. Total hepatic GST activity toward 1-chloro-2,4-dinitrobenzene was also significantly decreased. mRNA expression of gamma-glutamylcysteine synthetase (GCS) large subunit and catalase was reduced by approximately 60%. GCS enzyme activity was also decreased, resulting in a 35% decrease in the hepatic content of reduced glutathione 4 days after lipopolysaccharide challenge. Mn-Superoxide dismutase expression was increased 13-fold, and thioredoxin level was elevated 3-fold after lipopolysaccharide challenge. The expression of all parameters determined returned to near control levels 7 days after treatment. Together, these data show that GSTs and GCS are negative acute-phase proteins and that decreased GCS activity results in a decrease in hepatic glutathione content. Thus, in addition to the phase I drug-metabolizing enzymes known to be decreased during the acute-phase response, some phase II enzymes involved in the elimination of xenobiotics and carcinogens are also decreased.     

Identification of cGMP-dependent protein kinase anchoring proteins (GKAPs)

To promote both efficiency and selectivity, many protein kinases and phosphatases are maintained in specific subcellular microenvironments through their association with anchoring proteins. In this study, we describe a new class of proteins, called GKAPS, that specifically bind the Type II cGMP-dependent protein kinase (PKG). GKAPs were detected in rat aorta, brain, and intestine using a protein overlay technique. The PKG binding proteins were distinct from AKAPs, proteins known to bind the cAMP-dependent protein kinase (PKA). Furthermore, a synthetic peptide that blocks association of PKA with AKAPs did not affect the PKG-GKAP interaction. Deletion mutagenesis was used to map the GKAP binding determinants within PKG to the N-terminal regulatory region. While most GKAPs were tissue-specific, a ubiquitous PKG-binding protein was detected and identified as myosin. Analysis of myosin fragments revealed that PKG binds within Subfragment 2. The results define a novel class of anchoring proteins that may target PKG for specific functional roles.     

Identification of actin as an estradiol 17-beta-stimulated protein in the human placenta

Addition of estradiol 17-beta to first trimester human placental minces resulted in an increased synthesis of a protein of apparent molecular weight 45 kDa. The specific involvement of estrogen in the stimulation of this protein was established by demonstrating a reduction in the level of this protein by the addition of CGS 16949 A, an inhibitor of aromatase, a key enzyme in the biosynthesis of estradiol 17-beta and ICI 182,780, an estrogen receptor antagonist. The protein was purified to homogeneity and N-terminal sequencing of two of the internal peptides obtained by enzymatic digestion of the protein, as well as the absence of a free N-terminal indicated that it could be actin. This was confirmed by Western blotting using commercially available actin antiserum. The role of estradiol 17-beta in the stimulation of actin synthesis in human placenta was also established by monitoring the quantitative inhibition of DNase I by actin.     

Identification of the human ltk gene product in placenta and hematopoietic cell lines

Two different monoclonal antibodies (MAbs) were raised against an extracellular domain and a C-terminal portion of the human ltk protein which is a receptor-type protein tyrosine kinase. Western blot analysis showed that these MAbs specifically immunoprecipitated a 100 kDa ltk protein which was transiently expressed in COS-1 cells transfected with a human ltk cDNA. By an in vitro immune complex kinase assay using these MAbs, a 100 kDa phosphoprotein was detected in human placenta and hematopoietic cell lines. These data indicate that the ltk gene product expressed in human placenta and hematopoietic cells shows tyrosine kinase activity. This is the first detection of native ltk protein naturally expressed in human cells.     

Purification and properties of the chloroplastic form of biotin holocarboxylase synthetase from Arabidopsis thaliana overexpressed in Escherichia coli

Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes. In a previous study, we demonstrated that, in plants, HCS activity is localised in cytosol, chloroplasts and mitochondria. We also described the cloning and sequencing of a full-length cDNA encoding an Arabidopsis thaliana HCS isoform with a putative organelle-transit peptide. In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated using an efficient three-step purification procedure. Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localisation of this protein. Immunodetection carried out by Western blotting of isolated pea leaf subcellular compartments specifically revealed a single polypeptide that was ascribed to the chloroplast compartment. Immunocytochemistry of thin-cut sections from tobacco leaves, transformed by the complete coding sequence of A. thaliana HCS cDNA via Agrobacterium tumefaciens, confirmed that the enzyme encoded by this cDNA is the chloroplastic isoform. Moreover, physicochemical, biochemical and kinetic properties of the pure recombinant HCS were determined. The native recombinant enzyme is a 37-kDa monomer. In contrast to the major part of HCS activity measured in leaf extracts, the recombinant chloroplastic enzyme did not require addition of Mg2+ to be fully active, but was substantially inhibited by EDTA. This suggested that the chloroplastic HCS may contain a tightly-bound divalent cation required for enzyme activity. The recombinant enzyme was able to biotinylate efficiently apo-biotin carboxyl carrier protein (BCCP) from E. coli and apo-methylcrotonoyl-CoA carboxylase (MCCase) from A. thaliana. Apparent Km values for the enzyme substrates D-biotin, ATP and apo-MCCase were found to be 130 nM, 4.4 microM and 32 microM, respectively. Steady-state kinetic analyses of the HCS-catalysed reaction were investigated with respect to reaction mechanism and inhibition by AMP, one of the end-products of the enzyme-catalysed reaction. Substrate interaction and product inhibition patterns indicated that ATP and D-biotin bind sequentially, in an ordered manner, to the enzyme and that ATP or D-biotin and apo-BCCP bind in ping-pong fashion.     

Placental lactogen-like proteins in the rabbit placenta

Rabbit placentae and embryos at days 11 and 12 were analyzed by two-dimensional gel electrophoresis and by light microscopic histology for the presence of placental lactogen-like proteins. Immunoblotting and immunohistochemistry were performed by using a goat anti-human placental lactogen serum as well as a monoclonal mouse anti-human prolactin immunoglobulin; the results were similar. In the gel electrophoresis of placental tissue, three protein spots at pH 5.6 and 43, 39, and 35 kDa were immunostained; they were absent in the embryo. Immunoresponse was restricted to the cytotrophoblast. Immunofluorescent cells were mainly found on the proximal parts of the placental trabeculae.     

Detection and cellular localization of plasma membrane-associated and cytoplasmic fatty acid-binding proteins in human placenta

The aim of this study was to investigate location and the types of membrane-associated and cytoplasmic fatty acid-binding proteins in human placental trophoblasts using monospecific polyclonal antibodies. Western blot analysis demonstrated the presence of multiple membrane and cytoplasmic fatty acid transport/binding proteins in human placenta. In addition to previously reported placental membrane fatty acid-binding (p-FABPpm, 40 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transport protein (FATP, 62 kDa) were detected in both microvillous and basal membranes of the human placenta. Among the cytoplasmic proteins, heart (H) and liver (L) type FABP were detected in the cytosol of the human placental primary trophoblasts as well as in human placental choriocarcinoma (BeWo) cells. The immunoreactivity of epidermal type (E)-FABP was not detected in trophoblasts or BeWo cells despite its presence in human placental cytosol. Location of FAT and FATP on the both sides of the bipolar placental cells may favour transport of free fatty acids (FFA) pool in both directions i.e. from the mother to the fetus and vice versa. However, p-FABPpm, because of its exclusive location on the microvillous membranes, may favour the unidirectional flow of maternal plasma long-chain polyunsaturated fatty acids present in the FFA pool to the fetus, due to binding specificity for these fatty acids. Although the roles of these proteins in placental fatty acid uptake and metabolism are yet to be understood fully, their complex interaction may be involved in the uptake of maternal FFA by the placenta for delivery to the fetus.     

Identification of acute-phase proteins (APP) in circulating immune complexes (CIC) in esophageal cancer patients'' sera

OBJECTIVES: We describe the attitude and views of general practitioners towards the menopause and hormone replacement therapy (HRT) in metropolitan Brisbane, Australia. METHODS: A total of 216 general practitioners were nominated by a random sample of urband-welling women aged 45-54 years who formed the Brisbane Women's health study. A 20-30 minute face-to-face questionnaire with the general practitioners was administered and analysed by demographic characteristics. RESULTS: There was a 93% response rate. Management of the menopause and HRT was routinely undertaken by general practitioners for their own patients. After deciding to initiate HRT, > 60% of general practitioners ordered five investigations or more. They may have confused the risk of thrombo-embolism from oestrogens used in the post-menopause with that for contraception. There were differences between male and female practitioners in some areas. Male general practitioners, in particular, reported more difficulty with tailoring and adjusting regimes. CONCLUSIONS: Specific areas for further education are explored to meet the educational needs of general practitioners.     

Localization of histidyl-tRNA synthetase (Jo-1) in human laryngeal epithelial carcinoma cell line (HEp-2 cells)

The present study was designed to determine the subcellular localization of histidyl-tRNA synthetase (Jo-1) in human laryngeal epithelial carcinoma cell line (HEp-2 cells). Indirect immunofluorescence using commercial HEp-2 cells with human serum and human-affinity-purified anti-Jo-1 antibodies was performed using confocal microscopy. Anti-histidyl-tRNA-synthetase-positive sera showed distinct nuclear and cytoplasmic granular staining in HEp-2 cells. Affinity purified anti-Jo-1 produced an identical pattern to the whole serum, whereas the serum fraction that did not bind to the affinity column was negative by immunofluorescence on HEp-2 cells. Two commercial human anti-Jo-1-positive control sera and seven anti-Jo-1-positive sera from patients with myositis reproduced the nuclear and cytoplasmic granular pattern. We conclude that Jo-1 is present in cytoplasm and in intact nuclei from HEp-2 cells. The presence of tRNA synthetases in intact nuclei suggests that they have an unsuspected function in the nucleus.     

Identification of the reaction products of (2''-5'')oligoadenylate synthetase in the marine sponge

A new type of affinity cross-linking strategy has been developed in which His6-tagged proteins can be cross-linked to their binding partners in the presence of unmodified proteins (D. Fancy, K. Melcher, S. A. Johnston, and T. Kodadek, 1996, Chem. Biol. 3, 551-559). The chemistry involves the addition of Ni(II) to the His6 tag, followed by oxidation of the metal with a peracid. It is shown here that, in addition to the His6 tag, a tyrosine residue placed in close proximity to the metal-binding site can strongly stimulate the yield of cross-linked product. This finding has important practical implications in the use of the His6-Ni-based cross-linking reaction for the analysis of multiprotein complexes.     

Interaction of D2-dopamine receptor with two pertussis toxin sensitive G proteins in human placenta

We have demonstrated the presence in human placenta of D2 dopamine receptors (D2R) which inhibit human placental lactogen (hPL) release. This inhibitory effect of dopamine (DA) was sensitive to pertussis toxin (PTX) indicating that it may be mediated by the Gi/Go family of G proteins. However, nothing is known on this G proteins/D2R interaction in human placenta. In this study, we demonstrate that DA (10(-4) M) inhibits by 39% the ADP-ribosylation by PTX of two G proteins of 40 and 41 kDa. This inhibition is receptor specific since it is reversed by spiperone, a D2R antagonist. Moreover we show that bromocriptine, a D2 agonist, inhibited the labeling of these two proteins in a dose-dependent manner with a maximal inhibition of 37% at a concentration of 10(-6) M. In order to understand the role of D2R in placental endocrinology, we have analyzed the interactions of these two PTX-sensitive G proteins with D2R in normal and abnormal pregnancies. The autoradiographs of both PTX ADP-ribosylated placental proteins of 40 and 41 kDa showed differential labeling during normal pregnancy. Thus, the relative levels of ADP-ribosylation by PTX of both proteins were 2.5 and 3.0 fold lower at term than those observed during first and second trimester whereas no difference was observed between the first and second trimester. Also, no significant change in the level of inhibition by DA was observed between 7-9 weeks and 18-40 weeks of pregnancies (35-45% inhibition). However, we observed a maximal inhibition between 10 to 17 weeks of pregnancy (64% inhibition). In placentas from preeclamptic pregnancies, the levels of ADP-ribosylation were similar to those observed in normal pregnancy, while the DA inhibition was increased by 24%. The levels of ADP-ribosylation in molar placentas reached 20% of normal values, while no difference in DA inhibition was observed. This study demonstrates that two distinct PTX-sensitive G proteins are coupled to human placental D2R. The physiological significance of the variations in these ADP-ribosylated-G proteins/D2R interaction during normal and preeclamptic pregnancies remains to be investigated.     

Identification and classification of 16 new kinesin superfamily (KIF) proteins in mouse genome

KIF (kinesin superfamily) proteins are microtubule-dependent molecular motors that play important roles in intracellular transport and cell division. The extent to which KIFs are involved in various transporting phenomena, as well as their regulation mechanism, are unknown. The identification of 16 new KIFs in this report doubles the existing number of KIFs known in the mouse. Conserved nucleotide sequences in the motor domain were amplified by PCR using cDNAs of mouse nervous tissue, kidney, and small intestine as templates. The new KIFs were studied with respect to their expression patterns in different tissues, chromosomal location, and molecular evolution. Our results suggest that (i) there is no apparent tendency among related subclasses of KIFs of cosegregation in chromosomal mapping, and (ii) according to their tissue distribution patterns, KIFs can be divided into two classes-i.e., ubiquitous and specific tissue-dominant. Further characterization of KIFs may elucidate unknown fundamental phenomena underlying intracellular transport. Finally, we propose a straightforward nomenclature system for the members of the mouse kinesin superfamily.     

Identification of proteins that are abnormally regulated in differentiated cultured human keratinocytes

The inhabitants living in the neighbourhood of a deserted mercury-contaminated industrial site are subjected to an age-group differentiated mercury exposure assessment based on a scenario-linked calculation. Analytical input data for the calculation procedure are provided for from soil, air and plants in a large number. The most sensitive group are small children being mainly exposed by soil ingestion which makes up nearly 80% of the ADI, followed by inhalation of mercury contaminated indoor air. On the other hand, inhalation of indoor air has a predominant impact on youth and adults.     

Fc gamma receptors in human placenta

This review summarizes the status of our knowledge on the structure, expression and function of Fc gamma R in the placenta. The discovery in syncytiotrophoblast of an MHC class I--related FcR, of the type responsible for intestinal uptake of milk IgG in suckling rats and mice is also described.