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Biomedical subjects

A Werner

Publications and source records attributed to A Werner.

At least 163 records · Page 9Linked to original sources
PubMed1993

Expression of Na/Pi cotransport from opossum kidney cells in Xenopus laevis oocytes.

Xenopus laevis oocytes have been used for the expression of Na/Pi-cotransport activity by injections of poly(A)+ RNA (mRNA) isolated from an established renal cell line (OK cells). 3-5 days after mRNA injection, Na-dependent phosphate (Pi) uptake by oocytes was increased in a dose-dependent manner; there was no increase in Na-independent Pi uptake. Sucrose density-gradient fractionation indicated that the mRNA species encoding this activity is 2.4-2.8 kb in length. In Northern blots, using a cDNA probe related to human kidney-cortex Na/Pi-cotransport activity (NaPi-3), hybridization with a mRNA-species of 2.4-2.6 kb was obtained. Kinetic characterization ([Pi], [Na]) showed that expressed transport activity has properties similar to apical Na/Pi cotransport in OK cells.

Animals↗
PubMed1993

Expression cloning of a human renal cDNA that induces high affinity transport of L-cystine shared with dibasic amino acids in Xenopus oocytes.

A renal cDNA clone (rBAT) that induces system bo,+-like amino acid transport activity in Xenopus oocytes has recently been isolated (Bertran, J., Werner, A., Moore, M. L., Strange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacín, and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5601-5605). Here we show the isolation of a cDNA clone by screening a human kidney cortex cDNA library for expression of sodium-independent transport of L-[3H]arginine in Xenopus oocytes. The cRNA of this clone induces in oocytes, in addition to the uptake of L-arginine, that of L-[35S]cystine and L-[3H]leucine. Expressed uptake of these amino acids is mutually cis-inhibitable by the other 2 amino acids. Expressed uptake of L-cystine is saturable and shows an apparent Km in the micromolar range. All these characteristics resemble induction of system bo,+ related to rBAT in the oocytes. Human rBAT mRNA (approximately 2.5 kilobases) is found in kidney, small intestine (i.e., jejunum), pancreas, and liver. Human kidney poly(A)+ RNA (mRNA) induces sodium-independent uptake of L-cystine, L-arginine, and L-leucine in Xenopus oocytes. Hybrid depletion with an antisense oligonucleotide of the isolated clone greatly prevents (80-97%) human kidney mRNA-dependent induction of the uptake of these amino acids (i.e., L-cystine, L-arginine, and L-leucine). The isolated clone (2304 base pairs in length) contains a poly(A) tail and encodes a predicted 78.8-kDa protein which is 85 and 80% identical to the rabbit and rat rBAT, respectively. This predicted protein corresponds to a membrane glycoprotein, and contains six potential N-glycosylation sites which might be functional in the oocyte: [35S] methionine labeling of oocytes shows a specific band of 94 kDa in crude membranes of these human cRNA-injected oocytes; treatment of these oocytes with tunicamycin shifts the cRNA-specific translation product to approximately 72 kDa. We conclude that we have isolated a functional cDNA corresponding to human rBAT. The isolation of this human cDNA would lead to the study of the possible involvement of rBAT in human hyperaminoacidurias.

Amino Acid Sequence↗
PubMed1993

Expression cloning of human and rat renal cortex Na/Pi cotransport.

We have isolated two cDNA clones, NaPi-2 and NaPi-3, by screening rat kidney cortex and human kidney cortex cDNA libraries, respectively, for expression of sodium-dependent phosphate transport in Xenopus laevis oocytes. Substrate specificity and a detailed kinetic analysis (Na, Pi, H+ concentrations) suggested that expressed uptake activities relate to proximal tubular brush border membrane Na/Pi cotransport. NaPi-2 cDNA contains 2464 bp encoding a protein of 637 aa; NaPi-3 cDNA contains 2573 bp encoding a protein of 639 aa. NaPi-2- and NaPi-3-deduced protein sequences show high homology to each other but are different from the protein sequence deduced from the previously cloned NaPi-1 cDNA (from rabbit proximal tubules). Hydropathy profile predictions suggest at least eight membrane-spanning regions in NaPi-2/3-related proteins. In vitro translation results in proteins of the expected size and suggests glycosylation. Northern blot analysis shows corresponding mRNA species (approximately 2.7 kb) in kidney cortex of various species but no hybridization with RNAs isolated from a variety of other tissues (including intestinal segments); a hybridization signal (approximately 4.8 kb) was observed only in the lung (human). We conclude that we have structurally identified two closely related proteins most likely involved in human and rat renal brush border Na/Pi cotransport.

Amino Acid Sequence↗
PubMed1993

Two mRNA transcripts (rBAT-1 and rBAT-2) are involved in system b0,(+)-related amino acid transport.

Previously, we isolated a cDNA clone (rBAT-1) of 2.2 kilobase pairs (kb) from a rabbit kidney cortex cDNA library, encoding a protein involved in sodium-independent transport of L-dibasic amino acids, L-cystine, and some neutral amino acids via a system related to b0,(+)-like activity (Bertran, J., Werner, A., Moore, M. L., Stange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5601-5605). In Northern blot hybridization using an rBAT-1 cDNA probe, 2.2- and 3.9-kb mRNA species were observed. Here we describe the isolation of a 3.9-kb cDNA clone (rBAT-2) by expression cloning using Xenopus laevis oocytes corresponding to the 3.9-kb mRNA species. On the basis of sequence analysis, in vitro translation (major protein of approximately 78 kDa), and functional analysis (expression of transport function), we conclude that rBAT-1- and rBAT-2-related proteins are identical: 677 amino acids in length, with most likely only one transmembrane-spanning domain. There are seven differences in the nucleotide composition within a common overlap of 2189 nucleotides, resulting in 2 amino acid replacements. In comparison with rBAT-1, rBAT-2 has 26 additional nucleotides at the 5'-end, an identical location of the first polyadenylation signal, and approximately 1.7 kb of 3'-untranslated sequence (rich in AT(U) motifs) prior to a poly(A) tail of 63 adenines. We conclude that rBAT-1 and rBAT-2 encode the same protein and that the major difference seems to be related to the use of different polyadenylation signals.

Amino Acid Sequence↗
PubMed1993

Impairment of retinal increment thresholds in Huntington's disease.

We have investigated detection thresholds for a foveal blue test light using a Maxwellian view system in 61 normal subjects, 19 patients with Huntington's chorea, 14 patients with Tourette's syndrome, and 20 patients with schizophrenia. Ten measurements were made: The blue test light (1 degree diameter, 500 msec duration) was presented either superimposed on a yellow adaptation field (5 degree diameter) or 500 msec after switching off this field (transient tritanopia effect). In both cases five different background intensities were presented. The only abnormality found was in patients with Huntington's chorea. During adaptation these patients' thresholds are significantly higher than normal (p < 0.005). No change was found in the transient tritanopia effect. Huntington's disease causes degeneration of several different transmitter systems in the brain. Increment threshold testing allows for noninvasive investigation of patients and confirms the involvement of the retina in the degenerative process in Huntington's chorea.

Adaptation, Ocular↗
PubMed1993

Localization of NaPi-1, a Na/Pi cotransporter, in rabbit kidney proximal tubules. II. Localization by immunohistochemistry.

Polyclonal antibodies have been raised against a C-terminal peptide of NaPi-1, a recently cloned Na-Pi cotransport system of rabbit kidney cortex with a predicted (unglycosylated) molecular mass of 52 kDa. By Western blot analysis using brush-border membranes isolated from rabbit kidney cortex, two proteins with apparent molecular masses of 64 kDa and 35 kDa were specifically recognized (peptide protectable) by the antiserum obtained. The 64-kDa protein was found to migrate in parallel with the luminal membrane during separation by free-flow electrophoresis of brush-border and basolateral membranes. In immunofluorescence studies using cryostat sections of rabbit kidney, specific binding of antibodies was observed in proximal tubules (including S1, S2 and S3 segments) of superficial and deep nephrons. Anti-(NaPi-1)-antibody-mediated fluorescence was restricted to the brush border of proximal tubular cells. No specific immunoreaction was observed in other tubular segments. The results suggest that the native NaPi-1-related protein (Na-Pi cotransport system) has an apparent molecular mass of 64 kDa and is uniformly expressed in the apical membrane of proximal tubules of all nephron generations in the rabbit kidney. Immunohistochemical localization of the Na-Pi cotransport system NaPi-1 confirms the segmental localization within the nephron of NaPi-1-related mRNA as revealed by the reverse transcriptase/polymerase chain reaction (see preceding paper).

Amino Acid Sequence↗
PubMed1993

Effect of low-phosphate diet on sodium/phosphate cotransport mRNA and protein content and on oocyte expression of phosphate transport.

Recently, we have isolated a complementary DNA most likely related to rabbit kidney cortex brush border membrane sodium/phosphate (Na/Pi) cotransport activity [NaPi-1 (1)]. To further elucidate the cellular mechanisms involved in dietary 'adaptation' of renal Na/Pi cotransport, we have exposed young rabbits for 2 weeks to either a low phosphate (Pi) diet (LPD) or a high Pi diet (HPD). Initial linear uptake of Na/Pi cotransport in isolated brush border membrane vesicles was increased in rabbits on a LPD compared with those on a HPD. Injection of equal amounts of total mRNA isolated from kidney cortex of LPD or HPD rabbits into Xenopus laevis oocytes resulted in a higher stimulation of Na-dependent oocyte Pi uptake in LPD than HPD preparations. No difference in the content of 'specific' mRNA (NaPi-1 cDNA probe, Northern blots) and of the content of the 'specific' brush border membrane protein (NaPi-1 antipeptide antibody, Western blots) between LPD and HPD preparations was observed. We conclude that 'chronic' dietary Pi deprivation leads to a protein synthesis-dependent alteration of Na/Pi cotransport activity which does not involve a change in the total amount of a protein related to the recently cloned NaPi-1 protein.

Animals↗
PubMed1993

Endocarditis caused by a Leptotrichia buccalis-like bacterium in a patient with a prosthetic aortic valve.

The isolation of a Leptotrichia buccalis-like bacterium from the blood of a patient with endocarditis associated with a prosthetic aortic valve is reported. The organism was isolated from different blood cultures. It was similar but not completely identical to Leptotrichia buccalis. The microbiological identification was confirmed independently by three laboratories, including two reference institutions. The case and the characteristics of the microorganism are reported.

Aged↗
PubMed1993

[In vitro fertilization. Prognostic factors].

Multiple factors influence the outcome of in vitro fertilisation and embryo transfer (IVF-ET). In our prospective study different factors have been subject of examination concerning their effect on the outcome of in vitro fertilisation and embryo transfer. 1237 couples undergoing 1675 consecutive treatment cycles between 1.1.1990-31.12.1991 were included in this study. Prior to treatment, couples were divided into "good" and "poor" prognosis groups. Cycles were prospectively labelled as carrying a potentially "poor prognosis", if one or more of the following factors were noted: 1) female age > 35; 2) an existence of male factor; 3) couples with more than 3 previous unsuccessful treatment cycles. Couples with none of these factors were assigned to the "good" prognosis group. The pregnancy rate per cycle in the "poor" prognosis group was 5.96%, compared with 17.92% per cycle in the "good" prognosis group (p < 0.001). The most important factors determining pregnancy rates were female age and male factor, and we observed that the rate of pregnancy declined after the third treatment cycle. An explanation may be seen in lower fertilisation rates after the age of 35 and cases of poor semen quality. Both will result in poor embryo quality.

Adult↗
PubMed1993

Detection of plasma viremia in human immunodeficiency virus-infected individuals at all clinical stages.

Free virus (virus not present within cells) was detected in the plasma of all human immunodeficiency virus (HIV)-infected individuals studied. Plasma samples from asymptomatic individuals and individuals with HIV disease were tested. The levels of virus varied, but high virus titers correlated directly with HIV-related symptoms and low CD4+ lymphocyte counts. Effective detection of infectious virus depended on the use of an enzyme-linked immunosorbent assay for p24 core antigen and culture conditions in which plasma was added to mitogen-stimulated lymphocytes within 3 h of venipuncture. When there were delays in the time to culturing of plasma, neutralizing antibodies and perhaps other factors present in the plasma were found to reduce the efficiency of virus recovery. Plasma stored at -70 degrees C for several months maintained a stable level of free virus. These results suggest that measurement of HIV present in plasma under optimal conditions could be an efficient way of monitoring the clinical state of an individual and the effects of antiviral therapy.

Antiviral Agents↗
PubMed1993

Distinguishing features of an infectious molecular clone of the highly divergent and noncytopathic human immunodeficiency virus type 2 UC1 strain.

A full-length infectious molecular clone was derived from the noncytopathic human immunodeficiency virus type 2 UC1 strain (HIV-2UC1) that was originally recoverd from an individual from the Ivory Coast. Like the parental isolate, the molecularly cloned virus (HIV-2UC1mc or UC1 mc) demonstrates a reduced ability to induce syncytium formation, to kill cells, and to down-modulate the cell surface CD4 receptor in infected cells. Phylogenetic analysis of the DNA sequence of UC1mc revealed that it is the first full-length infectious molecular clone in the second HIV-2 subgroup previously identified by partial sequence analysis of the HIV-2D205 and HIV-2GH-2 strains. These highly divergent HIV-2 strains appear to be genetically equidistant from other HIV-2 and simian immunodeficiency virus SIVmac/sm strains. UC1mc is unlike any other HIV-2 or SIVmac/sm strain in that it lacks a cysteine residue at the proposed signal peptide cleavage site in Env. However, site-directed mutagenesis experiments indicate that this missing cysteine is not alone important in the noncytopathic phenotype of UC1mc. Like other HIV-2 and SIV strains, the UC1mc Env transmembrane protein (gp43) is mutated to a truncated form (gp34) after passage in certain T-cell lines. The UC1 molecular clone should be helpful in determining the genetic sequences associated with HIV-2 cytopathicity.

Amino Acid Sequence↗
PubMed1993

Human immunodeficiency virus type 1 envelope gp120 is cleaved after incubation with recombinant soluble CD4.

Human immunodeficiency virus type 1 (HIV-1) infects human CD4+ cells by a high-affinity interaction between its envelope glycoprotein gp120 and the CD4 molecule on the cell surface. Subsequent virus entry into the cells involves other steps, one of which could be cleavage of the gp120 followed by virus-cell fusion. The envelope gp120 is highly variable among different HIV-1 isolates, but conserved amino acid sequence motifs that contain potential proteolytic cleavage sites can be found. Following incubation with a soluble form of CD4, we demonstrate that gp120 of highly purified HIV-1 preparations is, without addition of exogenous proteinase, cleaved most likely in the V3 loop, yielding two proteins of 50 and 70 kDa. The extent of gp120 proteolysis is HIV-1 strain dependent and correlates with the recombinant soluble CD4 sensitivity to neutralization of the particular strain. The origin of the proteolytic activity in the virus preparations remains unclear. The results support the hypothesis that cleavage of gp120 is required for HIV infection of cells.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Influence of shearing action of food on contact stress and subsequent wear of stress-bearing composites.

The influence of sliding action of the antagonist on occlusal three-body wear of composites and an amalgam was investigated in vitro by gradual change in the distance between the opposing substrates. When the distance was decreased from 10 microns to approximately 3 microns, wear increased significantly by a factor of two to three and was exclusively of erosive nature. At a slurry-film thickness of approximately 1 micron, direct contacts between the antagonist and protruding composite filler particles started to occur. This consequently slowed the erosive wear. Ultimately, direct contact phenomena predominated, decreasing the wear rate of the various materials to different degrees. Loss of material due to subsurface fatigue could not be demonstrated with a contact pressure of 45 MPa at which the experiments were performed. From this study, it can be concluded that minor alterations of the food-film thickness at the contact areas result in considerable changes in wear rates and wear-rate ranking of composite materials, which may partly explain inconsistencies among clinical trials.

Composite Resins↗
PubMed1993

National survey of neonatal transfusion practices: II. Blood component therapy.

Neonatal transfusion practices during 1989 of 452 institutions involved in transfusing infants were surveyed by questionnaire. Most respondents (77%) transfused fresh frozen plasma appropriately (ie, primarily to treat coagulation disorders). However, 11% stated that their most frequent use of fresh frozen plasma was solely to treat hypovolemia, a practice generally not recommended. Seventy-eight percent of respondents transfused platelets to treat bleeding infants with blood platelet counts of less than 50 x 10(9)/L; 84% gave platelets to sick, premature neonates with counts of less than 50 x 10(9)/L whether or not bleeding was evident. Only 35% of respondents transfused granulocytes for neonatal sepsis; most institutions used buffy coats isolated from units of blood--a product readily available, but of questionable efficacy when compared with leukapheresis granulocytes. Ninety-three percent of respondents provided blood components with low risk of transmitting cytomegalovirus: components from seronegative donors were used by 84%, leukocyte-reduced products by 6%, and a combination by 10%. Thirteen percent of respondents gave gamma-irradiated blood components to all and 46% gave them to some neonates to prevent graft vs host disease. Forty-one percent did not routinely irradiate. Ten percent of respondents used leukocyte reduction instead of gamma irradiation to prevent graft vs host disease, a practice currently not advocated. Thus, national transfusion practices for neonates are variable, controversial, and, occasionally, other than those usually recommended. Additional research and educational efforts are needed to ensure optimal transfusion therapy.

Blood↗
PubMed1993

National survey of neonatal transfusion practices: I. Red blood cell therapy.

Neonatal blood component transfusion practices during 1989 were surveyed via a questionnaire developed by the Pediatric Hemotherapy Committee of the American Association of Blood Banks. Of 1790 questionnaires mailed, 452 were selected to form the database for this analysis because they were from institutions in which neonates were transfused. Nearly all institutions contained intensive care units directed by neonatologists and were involved in the management of high-risk infants. Results from institutions serving as the primary pediatric teaching hospital of a medical school were compared with those with no medical school affiliation. Thirty-six percent of primary pediatric teaching hospitals and 52% of hospitals with no medical school affiliation performed pretransfusion testing in excess of that required, resulting in additional blood loss in neonates. Sixty-six percent of primary pediatric teaching hospitals used fresh frozen plasma to adjust the hematocrit of red blood cell concentrates prior to transfusion (a practice increasing donor exposure), compared with only 29% of hospitals with no medical school affiliation. The usual indication for small-volume red blood cell transfusions in severely ill neonates was to maintain a desired hematocrit level, whereas for stable infants, red blood cell transfusions were given to treat symptomatic anemia, rather than to maintain a predetermined hematocrit. As found in 1985, neonatal transfusion practices in 1989 were variable. However, improvements have occurred since 1985 to suggest that further research and educational efforts may serve to promote even better neonatal transfusion therapy.

Blood Component Transfusion↗
PubMed1992

Poly(A)+ RNA from rabbit intestinal mucosa induces b0,+ and y+ amino acid transport activities in Xenopus laevis oocytes.

Injection of poly(A)+ RNA (mRNA) isolated from rabbit intestinal mucosa into Xenopus laevis oocytes results in an increase in sodium-independent uptake of L-[3H]leucine, L-[35S]cystine, and L-[3H]arginine. This uptake activity is related to an mRNA species corresponding to the recently isolated rabbit kidney cortex cDNA clone rBAT (related to b0,+ amino acid transporter; Bertran, J., Werner, A., Stange, G., Markovich, D., Moore, M. L., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 281, 717-723) and to a protein involved in amino acid transport via system y+. This conclusion is based on the following observations: 1) mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, induces sodium-independent uptake of L-leucine, L-cystine, and L-arginine. 2) In Northern blot analysis, mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, hybridizes to an rBAT cDNA probe, with signals of 2.2-2.3 kilobases and 3.7-3.9 kilobases. 3) mRNA isolated from mucosa of jejunum induces sodium-independent uptake of L-leucine and L-cysteine which shows an inhibition pattern corresponding to system b0,+; the inhibition pattern of mRNA-induced uptake of L-arginine is compatible with the contribution of system b0,+ and y+. 4) Hybrid depletion with an rBAT antisense oligonucleotide greatly prevents the mRNA-dependent induction of uptake of L-cystine (greater than 90%) and of L-leucine (approximately 75%); it reduces to about 50% the induction of L-arginine uptake. 5) After separation of mRNA on a sucrose density gradient, the fractions resulting in expression of b0,+ transport activity were also those hybridizing with rBAT cDNA; induction of transport activity from these fractions was also sensitive to hybrid depletion. 6) The mRNA-induced component of L-arginine uptake which is resistant to rBAT hybrid depletion is inhibited by L-homoserine, only in the presence of sodium; thus, it is related to a system y(+)-like activity.

Amino Acids↗
PubMed1992

Expression cloning of a cDNA from rabbit kidney cortex that induces a single transport system for cystine and dibasic and neutral amino acids.

We have isolated a cDNA clone by screening a rabbit kidney cortex cDNA library for expression of sodium-independent transport of L-arginine and L-alanine in Xenopus laevis oocytes. Expressed uptake relates to a single component of sodium-independent transport for dibasic and neutral amino acids. This transport activity resembles the functionally defined system b0,+ and carries cystine and dibasic amino acids with high affinity. The rBAT (b0,+ amino acid transporter-related) mRNA is found mainly in kidney and intestinal mucosa. It encodes a predicted 77.8-kDa protein with only one putative transmembrane domain and seven potential N-glycosylation sites. This protein could either be a constitutive element or a specific activator of system b0,+.

Amino Acid Sequence↗