Biomedical subjects

R L Lester

Publications and source records attributed to R L Lester.

At least 37 records · Page 2Linked to original sources

PubMed1997

Suppressor gene analysis reveals an essential role for sphingolipids in transport of glycosylphosphatidylinositol-anchored proteins in Saccharomyces cerevisiae.

Sphingolipids are normally necessary for growth of Saccharomyces cerevisiae cells, but mutant strains that bypass the need for sphingolipids have been identified. Such bypass mutants fail to grow under stressful conditions, including low pH (pH 4.1), when they lack sphingolipids. To begin to understand why sphingolipids seem to be necessary for coping with low-pH stress, we screened a genomic library and selected a suppressor gene, CWP2 (cell wall protein 2), that when present in multiple copies partially compensates for the lack of sphingolipids and enhances survival at low pH. To explain these results, we present evidence that sphingolipids are required for a normal rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins, including Cwp2 and Gas1/Gpg1, from the endoplasmic reticulum (ER) to the Golgi apparatus. The effect of sphingolipids is specific for transport of GPI-anchored proteins because no effect on the rate of transport of carboxypeptidase Y, a non-GPI-anchored protein, was observed. Since the Gasl protein accumulated in the ER with a GPI anchor in cells lacking sphingolipids, we conclude that sphingolipids are not necessary for anchor attachment. Therefore, sphingolipids must be necessary for a step in formation of COPII vesicles or for their transport to the Golgi apparatus. Our data identify the Cwp2 protein as a vital component in protecting cells from the stress of low pH.

Biological Transport↗

PubMed1996

Sphingolipid synthesis: identification and characterization of mammalian cDNAs encoding the Lcb2 subunit of serine palmitoyltransferase.

Synthesis of the ceramide portion of sphingolipids in animals has been hypothesized to be tightly regulated thereby controlling the rate of de novo sphingolipid formation. Regulation is predicted to occur at the first and committed biosynthetic step catalyzed by serine palmitoyltransferase (SPT, EC 2.3.1.50). This hypothesis remains unproven because SPT has been refractory to purification and subsequent characterization. To begin to test this hypothesis we have used a genetic strategy to isolate LCB2 homologs from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe and a cDNA homolog from humans and mice. Identity is supported by overall amino acid sequence similarity between the predicted proteins and the known Saccharomyces cerevisiae Lcb2 protein. In addition, a motif of 56 residues from the human protein functionally substituted for the corresponding region of the S. cerevisiae Lcb2 protein. The 56 residue motif was found to be unique to Lcb2 proteins. Likewise, the base sequence encoding it is unique to the human genome. Finally, a peptide sequence in the motif is known to be part of the catalytic domain of all members of the aminolevulinate synthase superfamily of proteins of which Lcb2 is a member. These data argue that this motif is part of the catalytic domain of SPT and is a signature of Lcb2 proteins. The mammalian LCB2 cDNAs provide valuable reagents for studying the Lcb2 subunit of SPT and for studying how ceramide synthesis is regulated.

Acyltransferases↗

PubMed1996

Isolation and composition of inositolphosphorylceramide-type sphingolipids of hyphal forms of Candida albicans.

Hyphal forms of the human pathogen Candida albicans have been found to contain substantial quantities of phosphosphingolipids. These lipids were fractionated into three classes by normal-phase high-performance liquid chromatography. The first class contained equimolar amounts of phosphorus, inositol, phytosphingosines, and fatty acids; their composition and chromatographic behavior suggest that these compounds are inositolphosphorylceramides. The second class contained equimolar amounts of phosphorus, mannosylinositol, phytosphingosines, and fatty acids; their composition and chromatographic behavior indicate that these compounds are mannosylinositolphosphorylceramides. The third class of compounds contained phosphorus, mannosylinositol, inositol, phytosphingosines, and fatty acids in a molar ratio of 2:1:1:1:1; their composition and chromatographic behavior indicate that these compounds are mannosyldiinositolphosphorylceramides. Molecular species in each class differ in the composition of long chain bases and fatty acids; the most abundant long chain bases were C18 and C20 phytosphingosines, and the most abundant fatty acids were hydroxy and nonhydroxy C24-26. The array of sphingolipids in C. albicans is similar to that of Saccharomyces cerevisiae. Sphingolipids have been shown to be essential in S. cerevisiae, thus these lipids, which are not present in animals, offer a potentially unique target for antifungal chemotherapy against C. albicans.

Alkalies↗

PubMed1994

The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis.

The first and committed step in synthesis of the ceramide moiety of sphingolipids is catalyzed by serine palmitoyltransferase (EC 2.3.1.50), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. This step is thought to be tightly regulated to control the synthesis of sphingolipids, but data supporting this hypothesis are lacking mainly because the enzyme has resisted purification and consequent characterization. Rather than attempting to purify the enzyme from normal cells, we have taken a different tack and opted to try and overproduce the enzyme to facilitate its purification. Here we demonstrate that overproduction in Saccharomyces cerevisiae requires expression of LCB1, a previously isolated yeast gene, and LCB2, the isolation and characterization of which we describe. Several lines of evidence argue that both genes encode subunits of the enzyme; however, biochemical evidence will be needed to substantiate this hypothesis. Although overproduction was modest, 2- to 4-fold, it should now be possible to devise improved overproduction vectors for yeast or other host organisms.

Acyltransferases↗

PubMed1993

A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase.

Saccharomyces cerevisiae normally requires sphingolipid biosynthesis for growth; however, mutant strains lacking sphingolipids have been isolated by suppression of a genetic defect in sphingolipid long chain base biosynthesis. To begin to understand the nature of the suppressor(s) we isolated and characterized a suppressor gene, SLC1 (sphingolipid compensation). DNA sequence analysis showed that the wild type SLC1 allele differs from the suppressor allele by a single nucleotide which changes Gln-44 in the predicted wild type protein to Leu4-4 in the predicted SLC1-1 suppressor protein. The predicted SLC1 protein sequence is homologous to the 1-acyl-sn-glycerol-3-phosphate acyltransferase of Escherichia coli encoded by the plsC gene. The homology extends to function as well since the SLC1 gene complements the growth defect in an E. coli strain mutated in plsC. These results suggest that the SLC1 protein has a fatty acyltransferase activity. SLC1 thus may be the first eucaryotic sn2-acylglyceride fatty acyltransferase gene to be cloned. SLC strains grown in the absence of long chain base make novel phosphatidylinositol derivatives (Lester, R. L., Wells, G. B., Oxford, G., and Dickson, R. C. (1993) J. Biol. Chem. 268, 845-856) having a C26 fatty acid at the sn-2 position and the same polar head groups as normal sphingolipids. We postulate that the SLC1 suppressor allele encodes a variant enzyme with an altered substrate specificity that enables it to use a C26 in place of a C16/18 fatty acid precursor to acylate the sn-2 position of inositol-containing glycerolipids.

Acyltransferases↗

PubMed1993

Mutant strains of Saccharomyces cerevisiae lacking sphingolipids synthesize novel inositol glycerophospholipids that mimic sphingolipid structures.

Mutant strains of Saccharomyces cerevisiae, termed SLC, make no detectable sphingolipids when cultured without a sphingolipid long chain base such as phytosphingosine (Dickson, R.C., Wells, G.B., Schmidt, A., and Lester, R.L. (1990) Mol. Cell. Biol. 10, 2176-2181). When grown with phytosphingosine they make sphingolipids in normal amounts. SLC strains carry a secondary suppressor mutation that bypasses the need to synthesize a long chain base. Hypothetically, the suppressor mutation could function by mutating a gene whose protein product required a sphingolipid for function, by increasing the level of one or more endogenous lipids, or by making new lipid(s). Here we demonstrate that SLC strains make novel inositol glycerophospholipids when cultured without a long chain base. The novel lipids are phosphatidylinositol (PI), mannosyl-PI, and inositol-P-(mannosyl-PI), containing 1 mol of C26 fatty acid, ordinarily found in yeast sphingolipids but not usually found in glycerophospholipids; the C26 fatty acid appears to be located at the SN-2 position of the glycerol. In addition, mono-fattyacyl versions of these three compounds were also identified. The polar head groups and hydrophobic portions of these novel lipids are strikingly similar to S. cerevisiae sphingolipids found in wild-type cells. We speculate that the novel lipids structurally mimic sphingolipids and thereby compensate for some sphingolipid function(s) necessary for growth.

Chromatography, High Pressure Liquid↗

PubMed1993

Intrinsic reinnervation--myth or reality?

A clinical and electromyographic study of major nerve transections at the wrist in 22 patients has shown that electrical recovery nearly always occurs in the intrinsic muscles, despite the absence of clinically detectable function. There appears to be a level of electrical reinnervation above which clinically detectable intrinsic muscle power is usually present. This level would appear to be 50% of the contralateral maximum evoked muscle action potential; above this the "myth" of clinical recovery becomes a reality.

Adolescent↗

PubMed1993

Sphingolipids with inositolphosphate-containing head groups.

InsPCers have been characterized in many plants, fungi, and protozoans but not in animals. There are no well-documented reports of the absence of InsPCers in organisms of these categories and one might possibly consider these lipids to be ubiquitous in plants, fungi, and protozoans. The polar headgroups of these lipids display quite heterogeneous structures depending on the source, including attachment to proteins as possible membrane anchors. The ceramides are with some exceptions composed of phytosphingosine and a very long-chain, usually hydroxylated, fatty acid. The vital nature of such sphingolipids in the plasma membrane is indicated in S. cerevisiae. Clearly, much remains to be discovered about the structure, metabolism, and function of the InsPCers.

Amino Acid Sequence↗

PubMed1992

Characterization of a novel, potent, and specific inhibitor of serine palmitoyltransferase.

We have examined the mechanism of action of two natural products identified as broad spectrum antifungal agents (VanMiddlesworth, F., Dufresne, C., Wincott, F. E., Mosley, R. T., and Wilson, K. E. (1992) Tetrahedron Lett., in press; VanMiddlesworth, F., Giacobbe, R. A., Lopez, M. Garrity, G., Bland, J. A., Bartizal, K., Fromtling, R. A., Polishook, J., Zweerink, M. M., Edison, A. M., Rozdilsky, W., Wilson, K. E., and Monaghan, R. L. (1992) J. Antibiot. (Tokyo) 45, 861-867), designated sphingofungin B (2S-amino-3R,4R,5S,14-tetrahydroxyeicos-6-enoic acid) and sphingofungin C (2S-amino-5S-acetoxy-3R,4R,14-trihydroxyeicos-6-enoic acid), and find they are potent specific inhibitors of serine palmitoyltransferase, which catalyze the committed step of sphingolipid biosynthesis. We used Saccharomyces cerevisiae as a model to investigate the mechanism of the antifungal activity of these compounds. Macromolecular synthesis was not immediately affected by either sphingofungin B or C, synthesis continued for 60-90 min following the addition of drug to growing cultures. Significant loss of viability with sphingofungins required growing cultures and began only after several hours, with greater than 99.9% of drug-treated cells non-viable after 24 h. No lysis or other gross changes in cell morphology were observed in drug-treated cells. The structural similarity of sphingofungin B and C to sphingosine and phytosphingosine prompted us to investigate their effects on sphingolipid synthesis. Nanomolar levels of the drugs inhibited the incorporation of [3H]inositol into sphingolipid before incorporation into the sphingolipid precursor, phosphatidylinositol was affected, suggesting specific inhibition of sphingolipid synthesis. This hypothesis was confirmed by experiments in which the growth inhibitory activity of both drugs was completely ablated by the addition of phytosphingosine, dihydrosphingosine, or ketodihydrosphingosine to the culture medium. Reversal of antifungal activity by ketodihydrosphingosine suggested that serine palmitoyltransferase could be the actual target of these compounds. Direct evidence for this hypothesis was the observation of inhibition of serine palmitoyltransferase activity in crude membrane preparations at nanomolar concentrations of each drug. The potent inhibition of serine palmitoyltransferase coupled with the apparent lack of effect of these compounds on other cellular functions suggests that sphingofungin B and C will prove to be important new tools for studying the role of sphingolipids in yeast and perhaps in other organisms.

Acyltransferases↗

PubMed1992

Two different types of lipid moieties are present in glycophosphoinositol-anchored membrane proteins of Saccharomyces cerevisiae.

Numerous glycoproteins of Saccharomyces cerevisiae are anchored in the lipid bilayer by a glycophosphatidylinositol (GPI) anchor. Mild alkaline hydrolysis reveals that the lipid components of these anchors are heterogeneous in that both base-sensitive and base-resistant lipid moieties can be found on most proteins. The relative abundance of base-resistant lipid moieties is different for different proteins. Strong alkaline or acid hydrolysis of the mild base-resistant lipid component liberates C18-phytosphingosine indicating the presence of a ceramide. Two lines of evidence suggest that proteins are first attached to a base-sensitive GPI anchor, the lipid moiety of which subsequently gets exchanged for a base-resistant ceramide: (i) an early glycolipid intermediate of GPI biosynthesis only contains base-sensitive lipid moieties; (ii) after a pulse with [3H]myo-inositol the relative abundance of base-sensitive GPI anchors decreases significantly during chase. This decrease does not take place if GPI-anchored proteins are retained in the ER.

Chromatography, Affinity↗

PubMed1992

Phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and the phosphoinositol sphingolipids are found in the plasma membrane and stimulate the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

Several plasma membrane phospholipids have been studied for their ability to modulate the activity of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae. We show here that phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2), and/or the phosphatidylinositol and PIP kinases are localized primarily in the plasma membrane. Previous in vivo studies with S. cerevisiae have shown that large, rapid, and reversible changes occur in the levels of PIP and PIP2 congruent with changes in cellular ATP levels. We demonstrate here that isolated plasma membranes exhibit the same changes in PIP and PIP2 content when they are supplied with or washed free of ATP. Using a mixed micellar assay we systematically studied the efficacy of the plasma membrane lipids in sustaining the activity of the plasma membrane H(+)-ATPase. We demonstrate for the first time that a number of plasma membrane glycerophospholipids effectively stimulate the ATPase, including PIP, PIP2, and cardiolipin. Phosphoinositol-containing sphingolipids, major components of the plasma membrane, are also shown to stimulate the ATPase at significantly lower levels than the glycerophospholipids and must also be considered as important effectors in vivo.

Cell Fractionation↗

PubMed1992

Phenotypes of sphingolipid-dependent strains of Saccharomyces cerevisiae.

To study sphingolipid function(s) in Saccharomyces cerevisiae, we have investigated the effects of environmental stress on mutant (SLC) strains (R. C. Dickson, G. B. Wells, A. Schmidt, and R. L. Lester, Mol. Cell. Biol. 10:2176-2181, 1990) that either contain or lack sphingolipids, depending on whether they are cultured with a sphingolipid long-chain base. Strains lacking sphingolipid were unable to grow at low pH, at 37 degrees C, or with high salt concentrations in the medium; these environmental stresses are known to inhibit the growth of some S. cerevisiae strains with a defective plasma membrane H(+)-ATPase. We found that sphingolipids were essential for proton extrusion at low pH and furthermore found that cells lacking sphingolipid no longer exhibited net proton extrusion at normal pH after a 1-min exposure to pH 3. Cells lacking sphingolipid appeared to rapidly become almost completely permeable to protons at low pH. The deleterious effects of low pH could be partially prevented by 1 M sorbitol in the suspension of cells lacking sphingolipid. Proton extrusion at normal pH (pH 6) was significantly inhibited at 39 degrees C only in cells lacking sphingolipid. Thus, the product of an SLC suppressor gene permits life without sphingolipids only in a limited range of environments. Outside this range, sphingolipids appear to be essential for maintaining proton permeability barriers and/or for proton extrusion.

Fungal Proteins↗

PubMed1992

Sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae: genetics, physiology, and a method for their selection.

A selection method for sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae was devised after observing that strains that require a long-chain base for growth become denser when starved for this substance. Genetic analysis of over 60 such strains indicated only two complementation classes, lcb1 and lcb2. Mutant strains from each class grew equally well with 3-ketodihydrosphingosine, erythrodihydrosphingosine or threodihydrosphingosine, or phytosphingosine. Since these metabolites represent the first, second, and last components, respectively, of the long-chain-base biosynthetic pathway, it is likely that the LCB1 and LCB2 genes are involved in the first step of long-chain-base synthesis. The results of long-chain-base starvation in the Lcb- strains suggest that one or more sphingolipids have a vital role in S. cerevisiae. Immediate sequelae of long-chain-base starvation were loss of viability, exacerbated in the presence of alpha-cyclodextrin, and loss of phosphoinositol sphingolipid synthesis but not phosphatidylinositol synthesis. Loss of viability with long-chain-base starvation could be prevented by also blocking either protein or nucleic acid synthesis. Without a long-chain-base, cell division, dry mass accumulation, and protein synthesis continued at a diminished rate and were further inhibited by the detergent Tergitol. The cell density increase induced by long-chain-base starvation is thus explained as a differential loss of cell division and mass accumulation. Long-chain-base starvation in Lcb- S. cerevisiae and inositol starvation of Inos- S. cerevisiae share common features: an increase in cell density and a loss of cell viability overcome by blocking macromolecular synthesis.

Cell Division↗

PubMed1992

Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity.

We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium of wild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both lcb1 and lcb2 mutant strains exhibited negligible SPT activity when tested in vitro. Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPH-dependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an lcb1 mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcb- phenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCB1 and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.

Acyltransferases↗

PubMed1991

The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane.

To investigate the vital function(s) of the phosphoinositol-containing sphingolipids of Saccharomyces cerevisiae, we measured their intracellular distribution and found these lipids to be highly localized in the plasma membrane. Sphingolipids were assayed in organelles which had been uniformly labeled with [3H]inositol or 32P and by chemical measurements of alkali-stable lipid P, of long chain bases, and of very long chain fatty acids. We have developed an improved method for the preparation of plasma membranes which is based on the procedure of Duran et al. (Proc. Natl. Acad. Sci. USA 72:3952-3955, 1975). On the basis of marker enzyme and DNA assays carried out with a number of preparations, the plasma membranes contained less than 10% vacuolar membranes (alpha-mannosidase) and nuclei (DNA); the contamination by the endoplasmic reticulum (NADPH-cytochrome c reductase) varied from 0 to 20%. The plasma membrane preparations showed a 13-fold increase in the specific activity of vanadate-sensitive ATPase, compared with that in the homogenate, with a yield ranging from 50 to 80%. A comparison of the distribution of the ATPase with that of sphingolipids assayed by a variety of methods showed that 80 to 100% of the sphingolipids are localized in the plasma membrane; the sphingolipids constitute about 30% of the total phospholipid content of the plasma membrane. Minor amounts of sphingolipids that were found in isolated mitochondria and nuclei can be attributed to the presence of small amounts of plasma membrane in these fractions. These results suggest that one or more essential functions of these lipids is in the plasma membrane. Furthermore, sphingolipids may be useful chemical markers of the plasma membrane of S. cerevisiae.

Adenosine Triphosphatases↗

PubMed1991

Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis of the long-chain base component of sphingolipids.

The existence of auxotrophic mutants of Saccharomyces cerevisiae having an absolute requirement for the long-chain base (lcb) component of sphingolipids suggests that sphingolipids are crucial for viability and growth. One mutant, termed the lcb1-1 mutant, lacks the activity of serine palmitoyltransferase, the first enzyme in the pathway for long-chain base synthesis. Here, we present evidence that LCB1 has been molecularly cloned. The size of the LCB1 transcript, the direction of transcription, and transcription initiation sites were determined. In addition, the coding region and its 5' and 3' flanking regions were sequenced. Analysis of the DNA sequence revealed a single open reading frame of 1,674 nucleotides, encoding a predicted peptide of 558 amino acids. The hydropathy profile of the predicted peptide suggests a hydrophobic, globular, membrane-associated protein with two potential transmembrane helices. Comparison of the predicted amino acid sequence to known protein sequences revealed homology to 5-aminolevulinic acid synthase and to 2-amino-3-ketobutyrate coenzyme A ligase. These homologies, the similarity of the chemical reactions catalyzed by the three enzymes, and the finding that LCB1 restores serine palmitoyltransferase activity to an lcb1-defective strain indicate that serine palmitoyltransferase or a subunit of the enzyme is the most likely product of LCB1. Homology of the LCB1 predicted protein to the Escherichia coli biotin synthetase was also observed, but the biological significance of this observation is not clear. A role for sphingolipids in sporulation is implicated by our finding that diploids homozygous for lcb1 failed to sporulate.

Acyltransferases↗

PubMed1990

Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids.

Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.

Blotting, Southern↗

PubMed1990

Heparin versus saline flushing solutions in a small community hospital.

Since no nursing policy on flushing procedures existed in their small community hospital, the authors thought it would be useful to clarify recent reports of the equivalency of saline to heparin flushes in their patient group before establishment of such a policy. They conducted a 2 month double-blinded, cross-over study of catheter failures when using either saline or heparin 10 units/mL flushing solutions. All medical and surgical floor patients were included in the study. Floor nurses were used as observers and characterized catheter failures as either loss of patency or phlebitis. Heparin was shown to cause more phlebitis than saline (p less than .025), but no difference was found between the two flushing solutions in loss of patency. Data revealed a statistically significant advantage to using saline flushes when both loss of patency and phlebitis were combined (p less than .05). They recommended adoption of a flushing procedure which did not contain heparin.

Catheters, Indwelling↗