The initiation of a calcium signal in Xenopus oocytes

Clinical and radiographic healing observations were categorized into four patterns: rapid, typical, delayed, and adverse. While considerable overlap of characteristics was noted between the categories, singular factors or combinations of factors enabled pattern identification. The factor primarily associated with the rapid healing pattern was the appearance of bone in the former defect adjacent to the membrane at removal. In contrast, the adverse healing pattern depicted surface necrosis or loss of tissue height at membrane removal. One hundred random sites were evaluated, revealing 13% rapid healing patterns, 76% typical healing patterns, 8% delayed healing patterns, and 3% adverse healing patterns. With favorable patient compliance with oral hygiene and follow-up care, the rapid and typical healing patterns became clinically successful cases. The level of clinical success varied with the delayed healing pattern; the adverse pattern failed to achieve the therapeutic objective.     

The 3'-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3'-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5'-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3'-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5'-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3' end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.     

Molecular cloning and functional expression of a cDNA for rat phospholipid hydroperoxide glutathione peroxidase: 3'-untranslated region of the gene is necessary for functional expression

A full-length cDNA clone for phospholipid hydroperoxide glutathione peroxidase (PHGPx) was isolated from a rat brain. The cDNA was 0.761 kb in length and encoded 170 amino acids, which included a TGA-encoded selenocysteine at residue 46. The protein has a calculated molecular mass of 19,473 Da. We succeeded in the transient functional expression of a full-length cDNA for PHGPx, which includes the 3'-UTR, in COS-7 cells at the first attempt. Deletion of the 3'-UTR prevented the expression of the PHGPx activity and the incorporation of [75Se]selenious acid into the monomeric 19.7 kDa PHGPx protein. Thus, the 3'-UTR of the cDNA for PHGPx was required for the functional expression of PHGPx. Northern blot analysis demonstrated that the mRNA for PHGPx was widely expressed in normal rat tissues, especially in the testis. The mRNA levels of PHGPx in the cultured cells such as hepatomas, neuronal cells, nephroblastoma, and mammary myo-epithelial cells were higher than those of the tissues. The ratio of PHGPx to cytosolic glutathione peroxidase (cGPx) was significantly high in the testis and relatively high in the cultured cells. The mRNA levels of PHGPx in tissues were lower than those of cGPx.     

Functional expression of the parathyroid cell calcium receptor in Xenopus oocytes

Various studies suggest the existence of a plasma membrane receptor on parathyroid cells that senses changes in the concentration of extracellular Ca2+. To test this hypothesis, Xenopus laevis oocytes were injected with poly(A)(+)-enriched mRNA from bovine parathyroid cells and examined for their ability to respond to increases in the concentration of extracellular Ca2+ or other polycations. Cytosolic Ca2+ concentrations were measured indirectly by recording Cl- currents through the endogenous, cytosolic Ca(2+)-activated Cl- channel. Increasing the concentration of extracellular Ca2+ (from 0.7 to 5 mM) or Mg2+ (from 0.8 to 10 mM) elicited oscillatory increases in the Cl- current. Responses to either divalent cation were not observed in oocytes injected with water or with mRNA prepared from HL-60 cells or rat liver. Responses elicited by extracellular Mg2+ persisted when extracellular Ca2+ was reduced to low micromolar levels. La3+, Gd3+, or neomycin B also evoked oscillatory increases in the Cl- current in oocytes under conditions of low extracellular Ca2+ levels. These extracellular polycations all cause the mobilization of intracellular Ca2+ in oocytes injected with parathyroid cell mRNA like they do in intact parathyroid cells. The injection of parathyroid cell mRNA thus confers on oocytes the ability to detect and respond to changes in the concentration of extracellular polycations. The data provide compelling evidence for the existence of a cell surface Ca2+ receptor protein(s) on parathyroid cells that regulates cellular function.     

Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3'-untranslated region

The stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is regulated by oxygen tension in the pheochromocytoma-derived PC12 cell line. We previously identified a pyrimidine-rich 27-base-long protein-binding sequence in the 3'-untranslated region of TH mRNA that is associated with hypoxia-inducible formation of a ribonucleoprotein complex (hypoxia-inducible protein-binding site (HIPBS)). In this study, we show that HIPBS is an mRNA stabilizing element necessary for both constitutive and hypoxia-regulated stability of TH mRNA. The mutations within this sequence that abolish protein binding markedly decrease constitutive TH mRNA stability and ablate its hypoxic regulation. A short fragment of TH mRNA that contains the wild-type HIPBS confers the increased mRNA stability to the reporter chloramphenicol acetyltransferase mRNA. However, it is not sufficient to confer hypoxic regulation. The HIPBS element binds two isoforms of a 40-kDa poly(C)-binding protein (PCBP). Hypoxia induces expression of the isoform 1, PCBP1, but not the isoform 2, PCBP2, in PC12 cells.     

Regulation of calcium spiking in mammalian oocytes through a combination of inositol trisphosphate-dependent entry and release

In the group of 36 patients with acute ischemic stroke the study of the influence of arterial hypertension on the red cells and fibrinogen interaction was carried out in the aspect of the fibrinogen molecules contribution to this phenomenon, respecting the role of other plasma biochemical factors. Patients were divided into two clinical groups: with and without arterial hypertension. The quantitative fibrinogen molecules contribution to the inter-red cells connections in patients with arterial hypertension was lower than in the group without this accompanying disease (statistical significance was indicated for 80% and 60% of plasma dilution). The first group of patients was also characterized by a significant increase of IgA level (in comparison with the control group), whereas the red cells and fibrinogen interaction measured as a value of YSS was similar in both analyzed clinical groups.     

Amyloid precursor protein mRNA stability is controlled by a 29-base element in the 3'-untranslated region

In the accompanying paper (Zaidi, S. H. E., Denman, R., and Malter, J. S. (1994) J. Biol. Chem. 269, 24000-24006) we demonstrate that in tumor and normal cells, multiple cytosolic proteins interact with a 29-base sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. These data suggested that APP gene expression may be modulated by regulated APP mRNA decay. We have investigated this prediction by measuring the decay rates of APP mRNA in resting and mitogen-treated peripheral blood mononuclear cells and H4 and K562 tumor cell lines. In resting peripheral blood mononuclear cells, APP mRNA decayed with a half-life of 4 h. Under these conditions, the activity of APP mRNA-binding proteins was not detectable. After activation, binding protein activities were induced, and APP mRNA decay was blocked with a half-life of > 12 h. In log phase neuronal or lymphoid tumor cell lines, binding activity was constitutively present and APP mRNA displayed a half-life of > 12 h. Protein synthesis inhibition by cycloheximide had no effect on APP mRNA decay in normal or tumor cells. Transfected wild type or mutant APP mRNAs that lacked the 29-base region were stable (t1/2 > 10 h) in K562 tumor cells. Therefore, we conclude that the 29-base region functions in cis to destabilize APP mRNA in resting, normal cells. Upon activation APP mRNA-binding proteins are induced, interact with the 29-base region, and likely participate in stabilization of the mRNA.     

A possible mechanism underlying corymine inhibition of glycine-induced Cl- current in Xenopus oocytes

We previously reported that corymine, an alkaloid extracted from the leaves of Hunteria zeylanica native to Thailand, inhibited glycine-induced chloride current using a receptor expression model of Xenopus oocytes. In this study, we investigated the mechanism underlying the inhibitory action of this alkaloid on glycine current using the same model. Corymine inhibited glycine current in a noncompetitive fashion. Co-application with strychnine, a competitive glycine receptor antagonist, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, corymine decreased the ED50 value of strychnine, but did not change that of DIDS. Moreover, the inhibitory effects of corymine and either strychnine or DIDS were additive. The desensitization phase of glycine current showed two exponentials and corymine preferentially inhibited the fast component, whereas strychnine affected both of them to the same extent and DIDS preferentially inhibited the slow component. When these drugs were applied repeatedly, the inhibitory effects of corymine and strychnine were not use-dependent and reversible, while the effect of DIDS was use-dependent and irreversible. The inhibitory effect of corymine on gamma-aminobutyric acid (GABA) current was less potent than the effect on glycine current, while this alkaloid failed to affect acetylcholine and serotonin currents. These results demonstrate that corymine inhibits glycine-gated CI- channels by interacting with the site different from that of DIDS.     

Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes

Lysophosphatidic acid (1-acyl-2-lyso-snglycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl- currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5-11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl- current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.     

Selection of a cDNA clone for chicken high-mobility-group 1 (HMG1) protein through its unusually conserved 3'-untranslated region, and improved expression of recombinant HMG1 in Escherichia coli

Screening of cDNA libraries for the homologous vertebrate proteins high mobility group (HMG) 1 and 2 using DNA probes based on the coding sequences is likely to result in isolation of both HMG1 and HMG2 clones, as well as pseudogenes, which may be transcribed at low levels. However, the 3'-untranslated regions (UTRs) of HMG1 and 2 are quite distinct, and unusually conserved across species. We have used this property to select the true chicken HMG1 cDNA clone from a chicken lymphocyte cDNA library in lambdagt11, using a probe based on the 3'-UTR of rat HMG1 cDNA. The chicken HMG1 cDNA clone is very similar to all the complete HMG1 cDNA clones isolated so far. We suggest that the sequence designated chicken HMG1 in the GenBank Data Library (Accession number D14314) is, in fact, that of HMG2a [and moreover that the recently reported mouse clone (Accession number AF022465), proposed to encode a new HMG protein, HMG4, is also likely to encode an HMG2a, based on the translated amino-acid sequence and 3'-UTR]. We also report much improved expression of intact recombinant HMG1 in Escherichia coli by the use of chloramphenicol rather than ampicillin selection and conditions that limit cell growth. This should be general for all members of the HMG1 (and 2) family which may be toxic to cells (possibly because of the long acidic tail), and may also prove useful in the production of other such proteins.     

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA-binding affinity

mRNP3 and mRNP4 (also called FRGY2) are two mRNA-binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3-4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3-4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3-4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3-4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins.     

Regulation of hemicholinium-3 sensitive choline uptake in Xenopus laevis oocytes by the second C2 domain of synaptotagmin

A size-fractionated torpedo electric lobe cDNA library was screened for the neuronal choline transporter by functional expression in oocytes. A clone, TLC2B, was isolated that induced a component of choline uptake that was hemicholinium-3 sensitive and inhibited by the substitution of lithium for sodium at low choline concentrations. However, [3H]choline uptake by both injected and non-injected oocytes were characterized by high affinity constants, suggesting that TLC2B could be affecting a native choline transporter. Indeed, hemicholinium-3 sensitive choline uptake could also be induced by preincubation of non-injected oocytes with a protein kinase C inhibitor, H-7. By sequence analysis and immuno-precipitation, the peptide produced by injection of TLC2B cRNA was identified as a soluble 24 kDa C-terminal fragment of the neuronal protein, synaptotagmin. Full length synaptotagmin was, however, ineffective in the functional test. The peptide encoded by TLC2B corresponds to the second protein kinase C-homologous domain of torpedo synaptotagmin, and like other soluble C2 domain peptides, was capable of calcium-dependent translocation to membranes. Its action on choline uptake in oocytes was, however, abolished by the addition of calcium in the presence of a calcium ionophore. These results suggest that the interaction of certain C2 domains, such as the C-terminal domain of synaptotagmin, with more specific targets may be anulled in the presence of calcium due to its absorption to membrane phospholipids.     

Expression of rabbit ileal N3 Na+/nucleoside cotransport activity in Xenopus laevis oocytes

OBJECTIVES: Our goal was to provide the range of cost savings associated with various catheter reuse strategies. BACKGROUND: Percutaneous transluminal coronary angioplasty catheters are commonly reused in several countries outside the United States. However, the cost-effectiveness of such reuse strategies has not been evaluated. METHODS: Three theoretical models of catheter reuse were constructed using the actual costs for treating patients with coronary angioplasty at the Cleveland Clinic. Costs were calculated based on the number of balloon catheters, the amount of contrast agent used and the rates for urgent revascularization that were observed in a prospective Canadian study on catheter reuse. RESULTS: The median cost to treat a lesion by means of coronary angioplasty using new catheters was $8,800 per patient. In reuse models, the potential to reduce cost depended on the number of balloon catheters used and the rates of urgent revascularization. The "best care" scenario offered a potential savings of $480 (5.5% of total in-hospital cost), whereas the "worst case" scenario resulted in an increased cost of $1,075 (12.2% of total in-hospital cost) compared with the single-use strategy. Cost of the "likely case" scenario was similar to that of the single-use strategy. Sensitivity analyses identified the different rates of revascularization and cost of balloon catheters required to offset potential savings in each strategy. CONCLUSIONS: Although reusing coronary angioplasty catheters may reduce total in-hospital costs, even a modest increase in complications requiring urgent revascularization may offset any potential savings. However, if an increase in complications and procedure time can be avoided, the reuse strategy has significant economic potential and, ultimately, may be extended to other percutaneous coronary interventional equipment.     

Rapid sulfation of 3,3',5'-triiodothyronine in native Xenopus laevis oocytes

Sulfation is an important metabolic pathway facilitating the degradation of thyroid hormone by the type I iodothyronine deiodinase. Different human and rat tissues contain cytoplasmic sulfotransferases that show a substrate preference for 3,3'-diiodothyronine (3,3'-T2) > T3 > rT3 > T4. During investigation of the expression of plasma membrane transporters for thyroid hormone by injection of rat liver RNA in Xenopus laevis oocytes, we found uptake and metabolism of iodothyronines by native oocytes. Groups of 10 oocytes were incubated for 20 h at 18 C in 0.1 ml medium containing 500,000 cpm (1-5 nM) [125I]T4, [125I]T3, [125I]rT3, or [125I]3,3'-T2. In addition, cytosol prepared from oocytes was tested for iodothyronine sulfotransferase activity by incubation of 1 mg cytosolic protein/ml for 30 min at 21 C with 1 microM [125I]T4, [125I]T3, [125I]rT3, or [125I]3,3'-T2 and 50 microM 3'-phosphoadenosine-5'-phosphosulfate. Incubation media, oocyte extracts, and assay mixtures were analyzed by Sephadex LH-20 chromatography for production of conjugates and iodide. After 20-h incubation, the percentage of added radioactivity present as conjugates in the media and oocytes amounted to 0.9 +/- 0.2 and 1.0 +/- 0.1 for T4, less than 0.1 and less than 0.1 for T3, 32.5 +/- 0.4 and 29.3 +/- 0.2 for rT3, and 3.8 +/- 0.3 and 2.3 +/- 0.2 for 3,3'-T2, respectively (mean +/- SEM; n = 3). The conjugate produced from rT3 was identified as rT3 sulfate, as it was hydrolyzed by acid treatment. After injection of oocytes with copy RNA coding for rat type I iodothyronine deiodinase, we found an increase in iodide production from rT3 from 2.3% (water-injected oocytes) to 46.2% accompanied by a reciprocal decrease in rT3 sulfate accumulation from 53.7% to 7.1%. After 30-min incubation with cytosol and 3'-phosphoadenosine-5'-phosphosulfate, sulfate formation amounted to 1.8% for T4, less than 0.1% for T3, 77.9% for rT3, and 2.9% for 3,3'-T2. These results show that rT3 is rapidly metabolized in native oocytes by sulfation. The substrate preference of the sulfotransferase activity in oocytes is rT3 > 3,3'-T2 > T4 > T3. The physiological significance of the high activity for rT3 sulfation in X. laevis oocytes remains to be established.