Modulation of tRNAAla identity by inorganic pyrophosphatase

A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3'-(32)P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA(Ala). The effect of tRNA(Ala) identity mutations on both aminoacylation efficiency (k(cat)/K(M)) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the k(cat)/K(M) value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNA(Ala) identity.     

Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport.

The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H(+)-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H(+)-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase has been identified and purified and cDNAs encoding it have been isolated and characterized, the minimal unit competent in pyrophosphate (PPi)-energized H+ translocation is not known. Here we address this question and show that heterologous expression of the cDNA (AVP) encoding the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase from the vascular plant Arabidopsis thaliana in the yeast Saccharomyces cerevisiae results in the production of vacuolarly localized functional enzyme active in PPi-dependent H+ translocation. Since the heterologously expressed pump is indistinguishable from the native plant enzyme with respect to PPi hydrolysis, H+ translocation, activation by potassium, and selective inhibition by calcium and 1,1-diphosphonates, it is concluded that all of the known catalytic functions of the enzyme map to the one subunit encoded by AVP.     

Afipia felis induces uptake by macrophages directly into a nonendocytic compartment

Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. A. felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A. felis-containing phagosomes is unknown. Here, we demonstrate that, in murine macrophages, most A. felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection. Established Afipia-containing phagosomes possess neither early endosomal marker proteins [early endosome antigen 1 (EEA1), Rab5, transferrin receptor, trytophane aspartate containing coat protein (TACO)] nor late endosomal or lysosomal proteins [cathepsin D, beta-glucuronidase, vacuolar proton-pumping ATPase, rab7, mannose-6-phosphate receptor, vesicle-associated membrane protein 8, lysosome-associated membrane proteins LAMP-1 and LAMP-2]. Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment, which remains negative for LAMP-1. The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment, which also subsequently acquires LAMP-1. Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A. felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing A. felis are disconnected from the endosome-lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.     

Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases

Two types of proteins that hydrolyze inorganic pyrophosphate (PPi), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping pyrophosphatases (sPPases and H(+)-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PPi releasing heat, whereas H+-PPases, so far unidentified in animal and fungal cells, couple the energy of PPi hydrolysis to proton movement across biological membranes. The budding yeast Saccharomyces cerevisiae has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (IPP1) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional S. cerevisiae mutant (named YPC-1) whose functional IPP1 gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H+-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H+-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PPi is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H+-PPases can mediate net hydrolysis of PPi in vivo. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure-function relationships of this type of proton pumps.     

Cloning a plant amino acid transporter by functional complementation of a yeast amino acid transport mutant.

Amino acids are transported across the plasma membrane of plant cells by proton-amino acid symports. We report here the successful cloning of a neutral amino acid carrier by functional complementation. A histidine transport deletion mutant of Saccharomyces cerevisiae was transformed with an Arabidopsis thaliana cDNA library constructed in a yeast expression vector. Forty transformants, out of 10(5), allowed growth on a histidine-limiting medium. The acquired ability to grow on low histidine was shown to be strictly dependent on the protein encoded by the expression plasmid. Histidine and alanine transport activity were 10- to 20-fold greater in the transformants. The transport kinetics, inhibitor sensitivity, and substrate specificity match those of neutral system II, a neutral amino acid carrier we previously described in plasma membrane vesicles isolated from leaf tissue. The cDNA insert is 1.7 kb with an open reading frame that codes for a protein containing 486 amino acids with a calculated molecular mass of 52.9 kDa and three sites of potential N-linked glycosylation. Hydropathy analysis of the deduced amino acid sequence suggests this is an integral membrane protein with 10-12 membrane-spanning alpha-helices. Overall, the sequence of this amino acid carrier is not closely related to any other protein sequences in the GenBank data base. Interestingly, however, there are small regions of sequence that exhibit significant levels of similarity with at least seven other integral membrane proteins.     

Evaluation of the partitioning of bound inorganic phosphate during medium and intermediate phosphate in equilibrium water oxygen exchange reactions of yeast inorganic pyrophosphatase.

During the rapid exchange of oxygens of Pi with water catalyzed by yeast inorganic pyrophosphatase, Pi at the catalytic site may either dissociate or undergo reversible loss of an oxygen to water. The effective partitioning of bound Pi during exchange starting with medium Pi containing 18O in all four oxygens has been evaluated by mass spectral analysis of the change in the distribution of Pi species containing zero to four 18O oxygens per Pi. This analysis indicates that the rate of Pi release from the enzyme is only 1.4 times faster than the rate of reformation of the anhydrous intermediate. A similar partitioning of bound Pi is observed during PPi hydrolysis, indicating that hydrolysis and medium exchange have common intermediates. The approach should be applicable to study of related phosphate oxygen exchanges.     

An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells

Transferred DNA (T-DNA) transfer from Agrobacterium tumefaciens into eukaryotic cells is the only known example of interkingdom DNA transfer. T-DNA is a single-stranded segment of Agrobacterium's tumor-inducing plasmid that enters the plant cell as a complex with the bacterial virulence proteins VirD2 and VirE2. The VirE2 protein is highly induced on contact of A. tumefaciens with a plant host and has been reported to act in late steps of transfer. One of its previously demonstrated functions is binding to the single-stranded (ss) T-DNA and protecting it from degradation. Recent experiments suggest other functions of the protein. A combination of planar lipid bilayer experiments, vesicle swelling assays, and DNA transport experiments demonstrated that VirE2 can insert itself into artificial membranes and form channels. These channels are voltage gated, anion selective, and single-stranded DNA-specific and can facilitate the efficient transport of single-stranded DNA through membranes. These experiments demonstrate a VirE2 function as a transmembrane DNA transporter, which could have applications in gene delivery systems.     

Group translocation as a mechanism for sucrose transfer into vacuoles from sugarcane cells

Isolated vacuoles from sugarcane cells took up uridine diphosphate glucose (UDP-Glc) from the surrounding medium at a rapid rate. After a 7-min incubation of vacuoles with UDP-[14C]Glc, sucrose and sucrose phosphate were identified in the vacuole extract. UDP-Glc in the incubation medium was converted to hexose phosphates, sucrose, and glucose, with very little UDP-Glc remaining. Fructose 6-phosphate was not required for UDP-Glc uptake nor was [¹⁴C]fructose 6-phosphate taken up even in the presence of UDP-Glc. Glucose 6-phosphate and glucose 1-phosphate also were not taken up into vacuoles. UDP-Glc uptake showed saturation kinetics with a K_m of 0.7 mM and a V_max of 11.1 nmol/min per 10⁶ vacuoles. The optimum pH for UDP-Glc uptake was between 6.5 and 7.0. Uptake of UDP-Glc could be inhibited by p-chloromercuriphenylsulfonic acid, UDP, and GDP, and to a lesser extent by carbonyl cyanide m-chlorophenylhydrazone. The UDP-Glc binding site was specific for UDP-Glc; adenosine diphosphate glucose was not taken up, and guanosine diphosphate glucose did not compete with UDP-Glc for the binding site. The results suggest that sucrose transfer into vacuoles from sugarcane is via a group translocation mechanism, probably involving five tonoplast-bound enzymes.     

Malate transport into heart mitochondria.

The oxidation of l-Malate to carbon dioxide was employed as a measure of malate transport into mitochondria. Conditions of incubation were optimized with regard to osmolarity, pH, phosphate concentration, and substrate concentrations. Under these conditions the rate of malate oxidation to carbon dioxide was 17.1 ± 2.1 μmol/min/g mitochondrial protein at 30°C. Previous workers have found that maximum oxidation of glucose by the isolated perfused rat heart is from 2.4 to 3.0 μmol/min/g dry weight of tissue which would require the transport of 22 to 28 μmol of malate/min/g mitochondrial protein. Since the perfused heart experiments were carried out at 37°C, it is apparent that malate transport into mitochondria can account for most, if not all, of the reducing equivalents generated during glycolysis.     

Potassium stimulation of aldosterone secretion in vivo is reversed by nisoldipine, a calcium transport antagonist

The effects of a novel calcium transport antagonist, nisoldipine, on K-stimulated aldosterone secretion have been examined in vivo. Direct KCl infusion into the adrenal gland stimulated aldosterone secretion. Infusion of nisoldipine, concomitantly with KCl at two rates, abolished the stimulation of aldosterone independently of its effects on K transport. The results suggest that K stimulation of aldosterone secretion in vivo is at least in part mediated by alterations in transmembrane Ca flux.     

Inhibition of intestinal sodium-dependent inorganic phosphate transport by fibroblast growth factor 23

The mechanisms by which fibroblast growth factor 23 (FGF23) alters inorganic phosphate (Pi) homeostasis is not entirely clear. In the present study, we examined the effect of FGF23 on intestinal sodium-dependent Pi transport in mice. Injection of FGF23(R179Q) markedly reduced serum Pi and 1,25(OH)2D3 levels in normal mice. Those animals show the reduction of intestinal sodium-dependent Pi transport activity and the amount of type IIb sodium-dependent Pi cotransporter (type IIb NaPi) protein in the brush border membrane vesicles. In vitamin D receptor null mice (VDR-/-), FGF23(R179Q) had no effect on intestinal sodium-dependent Pi transport activity and type IIb NaPi protein levels. The present study suggests that FGF23(R179Q) reduces intestinal sodium-dependent Pi transport activity and type IIb NaPi protein levels by a mechanism that is dependent on VDR.     

Behavior of genes directly injected into the rat heart in vivo.

Gene transfer can be achieved in the adult rat heart in vivo by direct injection of plasmid DNA. In this report we define the spatial and temporal limits of reporter gene expression after a single intracardiac injection. pRSVCAT (100 micrograms), in which the Rous sarcoma virus long terminal repeat is fused to the chloramphenicol acetyltransferase reporter gene, and p alpha MHCluc (100 micrograms), in which the alpha-cardiac myosin heavy chain promoter is fused to the firefly luciferase gene, were injected into hearts, and reporter gene activities were assayed at various times. Both chloramphenicol acetyltransferase and luciferase were detectable in 100% of the rats from 1 to 7 days, in 60% of the rats from 17 to 23 days, and in 30% of the rats from 38 to 60 days after injection. Reporter gene activity was largely limited to a 1-2-mm region of the ventricle surrounding the injection site. Closed circular DNA was far more effective than linear DNA in transfecting cells in vivo. The relative strengths of three different promoters, Rous sarcoma virus long terminal repeat, alpha-myosin heavy chain, and alpha 1-antitrypsin, all fused to the luciferase reporter gene were determined. The constitutive viral promoter was approximately 20-fold more active than the cardiac-specific cellular promoter, and the liver-specific cellular promoter was not active at all in the cardiac environment. Thus, direct injection of genes into the heart offers a simple and powerful tool with which to assess the behavior of genes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine triphosphate pyrophosphohydrolase and neutral inorganic pyrophosphatase in pathologic joint fluids. Elevated pyrophosphohydrolase in calcium pyrophosphate dihydrate crystal deposition disease

Adenosine triphosphate pyrophosphohydrolase (ATPPPH) and neutral inorganic pyrophosphatase activities were assayed in synovial fluids (SF) from 37 patients with a variety of arthropathies. ATPPPH activity was detected in all fluids, but was highest in patients with chronic chondrocalcinosis; its activity in patients with osteoarthritis was higher than that in patients with rheumatoid arthritis, gout, or pseudogout. ATPPPH activity correlated positively with SF pyrophosphate concentration and negatively with SF white blood cell count. Pyrophosphatase activity did not correlate with diagnosis, pyrophosphate level, or white blood cell count.     

Occupational exposure to inorganic lead in a printing plant in Mexico City

OBJECTIVE: To describe occupational lead exposure and its biological indicators in workers in a printing company. MATERIAL AND METHODS: An epidemiological and industrial hygiene research was undertaken. Lead was measured in the air of work environment and on the hands of the participants; additionally, subjects underwent a venous blood samples for the determination of whole blood lead by atomic absorption spectrophotometry; and a bone lead measurement using a spot-source 109Cd K-X-ray fluorescence instrument. Also, a standardized questionnaire was applied. We obtained information on demographic and life styles factors, work history, type of work, position and activity within the company. RESULTS: Of the 209 workers, 117 agreed to participate and 90 (83.3% males and 16.7% females) completed all phases of the study. The average lead concentrations were: in air samples, of 0.94 microgram/m3; in hands before washing, of 6,802 micrograms/m2; in hands after washing, of 194 micrograms/m2; in whole blood, of 12.3 micrograms/dl; and in tibia and fibula, of 25.9 and 43.3 micrograms Pb/g of bone mineral, respectively. Important variations in these measurements were observed according to the workers post. CONCLUSIONS: Worldwide, lead exposure levels have been used to evaluate acute exposures being in the workplace. The higher lead levels find in the bone of the workers in this study are similar to other occupational studies in the United States of America and suggest that the accumulated metal in bone may be an important endogenous exposure source, and here its measurement importance. However in developing countries because its high costs per application, the measurements in bone lead become limited to epidemiological research, although not as an instrument for occupational epidemiological surveillance. In Mexico, there are no reliable studies of occupational lead exposure, which would allow the establishment of both, maximum permissible ambient and biological levels. This study is intended to contribute to blood lead standard setting, which is being discussed in Mexico.     

Characterization of oligonucleotide transport into living cells.

Addition of antisense oligonucleotides to cell cultures has been used to specifically inhibit gene expression. We have investigated the mechanism by which oligonucleotides enter living cells. These compounds are taken up by cells in a saturable, size-dependent manner compatible with receptor-mediated endocytosis. Polynucleotides of any length are competitive inhibitors of oligomer transport, providing they possess a 5'-phosphate moiety. Using oligo(dT)-cellulose for affinity purification, we have identified an 80-kDa surface protein that may mediate transport. Knowledge of the oligonucleotide transport mechanism should facilitate the design of more effective synthetic antisense oligomers as potential clinical agents.     

Resistance to parathyroid hormone-induced inhibition of inorganic phosphate transport in opossum kidney cells cultured in low inorganic phosphate medium.

Renal resistance to the phosphaturic action of parathyroid hormone (PTH) is observed during dietary deprivation of inorganic phosphate (Pi) in vivo. In the present work, the influence of short (3 h)- or long (72 h)-term deprivation of Pi on the effect of bovine PTH (bPTH(1-34)) on both Na-dependent Pi transport and cyclic AMP(cAMP) production was examined in cultured opossum kidney epithelium. Na-dependent Pi transport increased by 100% in cells exposed to low Pi medium containing no Pi (LPM) for 3 h, as compared with transport in high Pi medium containing 2 mmolPi/l (HPM). In response to a submaximal dose (1 nmol/l) of bPTH(1-34), Na-dependent Pi transport was similarly inhibited by about 40% in LPM and HPM. This inhibition was preceded by increased cAMP production which was identical in LPM and HPM. In opossum kidney cells exposed for 72 h to LPM, Na-dependent Pi transport was also increased by 100% compared with that in HPM. However, bPTH(1-34) added at 1 nmol/l did not induce any significant change in Na-dependent Pi transport or cAMP. Stimulation of cAMP could only be elicited at bPTH(1-34) concentrations higher than 1 nmol/l. Such a reduced cAMP response was also observed with forskolin in cells incubated for 72 h in LPM. The cellular resistance to the generation of cAMP was associated with a significantly lower level of ATP in cells cultured for 72 h in LPM compared with ATP levels in HPM.(ABSTRACT TRUNCATED AT 250 WORDS)