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PubMed2003

Complications associated with peripherally inserted central catheter use during pregnancy.

OBJECTIVE: The purpose of this study was to evaluate the complications of peripherally inserted central catheter use in an obstetrics population at a single institution over a 5-year period. STUDY DESIGN: We conducted a descriptive retrospective review of all obstetrics patients with peripherally inserted central catheter insertion during the antepartum period at Good Samaritan Regional Medical Center, from January 1, 1997, through September 30, 2001. RESULTS: During the 5 years of the study, complete data regarding the primary admission diagnoses for peripherally inserted central catheter placement and associated complications were available for 52 cases. An associated complication rate of 50% (n = 26 cases) was noted. The complications included culture-proved line infection (n = 9/52 complications, 17%), presumed line infection (n = 6/52 complications, 12%), cellulitis (n = 4/52 complications, 8%), mechanical line failure (n = 4/52 complications, 8%), pain that required line discontinuation (n = 2/52 complications, 4%), and superficial thrombophlebitis (n = 1/52 complications, 2%). A chi(2) analysis was performed, and preterm labor may be associated with an increased risk for peripherally inserted central catheter complication (P =.012). CONCLUSION: Patients who receive a peripherally inserted central catheter during pregnancy are at significant risk for infectious complications. Alternative treatment approaches are suggested, where appropriate.

Catheterization, Peripheral↗
PubMed2003

Peripherally inserted central catheter (PICC) fracture and embolization in the pediatric population.

OBJECTIVES: To document and characterize fracture and embolization of peripherally inserted central catheters (PICCs) in the pediatric population and define predisposing features for these complications. STUDY DESIGN: A case series was assembled by examining the records of PICC insertions in a single tertiary care pediatric hospital over a 6-year period. A control group was selected by simple random sampling of eligible PICC insertions. RESULTS: Among approximately 1650 PICCs, 11 children were identified with a fractured line, requiring invasive retrieval. Patient characteristics did not reveal any specific risk factors compared with the control group. Likewise, catheter size, site, and medications infused through the line were not significant predisposing factors for fracture. However, duration of placement and a line complication (blockage of the line or leaking at the insertion site) were significantly associated with catheter fractures. In all cases, the embolized line fragment was successfully retrieved by percutaneously inserted catheters and snares. No major complications arose from these fractured catheters. CONCLUSIONS: Fracture and embolization of PICCs occur and may pose a potential risk of serious consequences. It is prudent to list PICC fracture as a rare but potentially serious complication of this device when obtaining informed consent for its insertion.

Adolescent↗
PubMed1975

Translocation of a plasmid DNA sequence which mediates ampicillin resistance: molecular nature and specificity of insertion.

A series of recombinant plasmids was generated in Escherichia coli in which the TEM beta-lactamase translocon (TnA) was inserted into the small plasmid RSF1010. RSF1010 is a 5.5 X 10(6) dalton nonconjugative plasmid which confers resistance to streptomycin and sulfonamide. The recombinant plasmids can be classified into three clearly defined phenotypic groups. Group I is ampicillin-, streptomycin- and sulfonamide-resistant. Group II is ampicillin- and sulfonamide-resistant but has lost streptomycin resistance. Group III is ampicillin-resistant but is sensitive to sulfonamide and shows a simultaneous 30-fold reduction in the minimal inhibitory concentration of streptomycin. It was possible to map the site of insertion of TnA within RSF1010 by electron microscope studies of DNA heteroduplexes formed between RSF1010 and recombinant plasmids. Insertions of TnA occur at, at least, 12 distinct sites in a region corresponding to one-third of the RSF1010 DNA molecule. Those insertions giving rise to particular phenotypes are clustered. Insertions of TnA-like insertion sequences (IS) appear to give rise to strongly polar mutations.

Ampicillin↗
PubMed1977

Genetic instability in Drosophila melanogaster: putative multiple insertion mutants at the singed bristle locus.

A series of eleven independent mutants at the X chromosome singed bristle (sn) locus of Drosophila melanogaster is described. All mutants descend from flies caught in the wild and bred in the laboratory. On the basis of their inordinately high spontaneous mutation frequency, ten of the mutants are classified as putative insertion mutants. Reversions to wild type occur at frequencies of 10(-4)-10(-3). Some reversions appear to be losses of the inserted element, others appear (by analogy with prokaryotes) to be changes in the orientation of the inserted elements. Consistent with the insertion hypothesis, some sn mutants generate what are interpreted to be deletions at the sn locus. In their mutational properties, the sn mutants are analogous to insertion sequence (IS) elements and bacteriophage Mu of Escherichia coli, but the precise nature of the insertion remains unknown.

Alleles↗
PubMed1982

Expression of herpes simplex virus glycoprotein C from a DNA fragment inserted into the thymidine kinase gene of this virus.

Previous reports have described mutants of herpes simplex virus type 1 that fail to produce or accumulate one of the major glycoproteins, glycoprotein C (gC). This defect is not lethal in cell culture, has been associated with the syncytial plaque morphology of some mutants, and may result from mutations that map to a region on the genome noncontiguous with the structural gene for gC. To investigate the conditions required for, and consequences of, gC expression in a specific genetic background, we have inserted a wild-type allele of the gC gene into the thymidine kinase gene (tk) of a gC- fusion-inducing viral mutant, strain MP. This was accomplished by identifying cloned viral DNA fragments homologous to gC mRNA, inserting the appropriate fragments into the viral tk cloned in pBR322, and then cotransfecting cells with the recombinant plasmids and DNA from strain MP, for selection of insertional TK- mutants. All TK- mutants containing insertions of appropriate sequences (in either orientation) into tk were found to express gC while maintaining the syncytial plaque morphology of strain MP. Elimination of the insertion from one of the TK- mutants was accompanied by loss of ability to produce gC. Our results permit more precise mapping of the DNA sequence encoding gC, to a subfragment of Sal I fragment R (map coordinates 0.620-0.640) and indicate also that promoter sequences for the gC gene may be located in this fragment. Moreover, we can conclude that the previously described regulatory mutation of strain MP does not prevent expression of gC from the DNA inserted into its gene tk and that the syncytial phenotype of MP cannot be due solely to absence of gC.

Chromosome Mapping↗
PubMed1987

Efficient Tn10 transposition into a DNA insertion hot spot in vivo requires the 5-methyl groups of symmetrically disposed thymines within the hot-spot consensus sequence.

Transposon Tn10 inserts preferentially at particular insertion "hot spots" that share a symmetrical 6-base-pair consensus sequence: 5' GCTNAGC 3'. The protein that recognizes this sequence is not known but is likely to be the Tn10-encoded transposase protein. We present evidence that the 5-methyl groups of the two thymines in this sequence are essential for efficient transposon insertion; in their absence the sequence is still recognized, but at lower efficiency. We have reached this conclusion by examination of a specific hot spot whose sequence is 5' GCCAGGC 3'. The innermost cytosines of this sequence happen to be substrates for methylation at their 5 positions by the bacterial dcm-encoded methylase. We find that Tn10 transposes into this site 15 times more frequently in a Dcm+ host than in a Dcm- host; in the Dcm- host, insertions still occur, but at a low frequency. Thus, at this site, the absence of pyrimidine 5-methyl groups at the third positions of the consensus sequence is sufficient to convert a strong insertion hot spot into a weaker but still recognizable hot spot. This observation supports the general proposition, suggested previously by comparisons among consensus sequences, that the presence or absence of these 5-methyl groups is one major feature that can make the difference between a strong and a weak Tn10 insertion hot spot.

Base Sequence↗
PubMed1990

Insertion in the mRNA of a metachromatic leukodystrophy patient with sphingolipid activator protein-1 deficiency.

The lysosomal catabolism of sulfatide requires arylsulfatase A and a specific sphingolipid activator protein, SAP-1. While most patients with metachromatic leukodystrophy have mutations in the gene for arylsulfatase A, some patients have deficient SAP-1, as determined by immunological techniques. We now describe the molecular findings in a patient who died at 22 years of age with SAP-1 deficiency. The DNA polymerase chain reaction was used to amplify regions of cDNA which were subcloned in M13 phage DNA and sequenced by the dideoxy chain-termination method. The patient was found to have a 33-base-pair insertion between nucleotides 777 and 778 (numbered from the A of the ATG initiation codon). No other changes were found in the coding sequence of the cDNA from this patient. At the site of the insertion some normal people have an additional 9 base pairs, which correspond to the last 9 nucleotides at the 3' end of the insertion. The cDNAs from the second-cousin parents were amplified and sequenced, and in both two alleles were identified, one with the 33-base-pair insertion and one with no insertion. Two brothers were found to have only the normal alleles and a sister was found to have the 33-base-pair insertion and a normal allele. The findings confirm studies performed on leukocyte extracts demonstrating normal antigen levels in the two brothers and a lower level in the sister. The presence of 11 additional amino acids in the coding region of mature SAP-1 in this patient causes significant changes in the hydropathy profile compatible with the previous findings at the protein level.

Amino Acid Sequence↗
PubMed1997

Insertional editing of nascent mitochondrial RNAs in Physarum.

Maturation of Physarum mitochondrial RNA involves the highly specific insertion of nonencoded nucleotides at multiple locations. To investigate the mechanism(s) by which this occurs, we previously developed an isolated mitochondrial system in which run-on transcripts are accurately and efficiently edited by nucleotide insertion. Here we show that under limiting concentrations of exogenous nucleotides the mitochondrial RNA polymerases stall, generating a population of nascent RNAs that can be extended upon addition of limiting nucleotide. Several of these RNA species have been characterized and were found to be fully edited, indicating that nascent RNA is a substrate for nucleotide insertion in isolated Physarum mitochondria. Remarkably, these RNAs are edited at positions located within 14-22 nucleotides of the polymerase active site, suggesting that insertional editing may be physically or functionally associated with transcription. The absence of unedited RNA in these experiments indicates that large tracts of RNA downstream of editing sites are not required for nucleotide addition, and argues that insertional editing in Physarum occurs with a 5' to 3' polarity. These data also provide strong evidence that insertional editing in Physarum is mechanistically distinct from editing in kinetoplastid systems.

Animals↗
PubMed1999

Circular permutation and receptor insertion within green fluorescent proteins.

Many areas of biology and biotechnology have been revolutionized by the ability to label proteins genetically by fusion to the Aequorea green fluorescent protein (GFP). In previous fusions, the GFP has been treated as an indivisible entity, usually appended to the amino or carboxyl terminus of the host protein, occasionally inserted within the host sequence. The tightly interwoven, three-dimensional structure and intricate posttranslational self-modification required for chromophore formation would suggest that major rearrangements or insertions within GFP would prevent fluorescence. However, we now show that several rearrangements of GFPs, in which the amino and carboxyl portions are interchanged and rejoined with a short spacer connecting the original termini, still become fluorescent. These circular permutations have altered pKa values and orientations of the chromophore with respect to a fusion partner. Furthermore, certain locations within GFP tolerate insertion of entire proteins, and conformational changes in the insert can have profound effects on the fluorescence. For example, insertions of calmodulin or a zinc finger domain in place of Tyr-145 of a yellow mutant (enhanced yellow fluorescent protein) of GFP result in indicator proteins whose fluorescence can be enhanced severalfold upon metal binding. The calmodulin graft into enhanced yellow fluorescent protein can monitor cytosolic Ca(2+) in single mammalian cells. The tolerance of GFPs for circular permutations and insertions shows the folding process is surprisingly robust and offers a new strategy for creating genetically encodable, physiological indicators.

Amino Acid Sequence↗
PubMed1996

Signal anchor sequence insertion into the outer mitochondrial membrane. Comparison with porin and the matrix protein targeting pathway.

We have addressed the question of overlap between the pathways for protein insertion into the outer mitochondrial membrane and import to the matrix compartment, using competition studies in vitro. A synthetic peptide corresponding to the matrix-targeting signal of pre-ornithine carbamyl transferase competed for outer membrane insertion of porin but did not compete for membrane insertion of outer membrane signal anchor-containing proteins. Conversely, however, a synthetic peptide corresponding to the signal anchor sequence of Tom70 competed for import of all proteins examined. Both peptides competed for a step beyond receptor binding. Import of all precursors examined was inhibited by antibodies raised against the import receptor Tom20. Following binding to the surface of the organelle, outer membrane integration of porin was sensitive to depletion of nucleoside triphosphates by apyrase, whereas signal anchor protein insertion was not. The results demonstrate that outer membrane signal anchor insertion overlaps with a general insertion pathway. However, it exhibits both properties and steps that differ from the pathway followed by porin and matrix-targeted protein.

Apyrase↗
PubMed1996

Insertion of the 70-kDa peroxisomal membrane protein into peroxisomal membranes in vivo and in vitro.

Biosynthesis and intracellular transport of 70-kda peroxisomal membrane protein (pmp70) has been studied in rat hepatoma, h-4-ii-e cells. Pulse-chase analysis showed that a newly synthesized 35S-PMP70 first appeared in the cytosolic fraction and was then transported into the peroxisomal fraction. The half-life of 35S-PMP70 in the cytosolic fraction was approximately 3 min. Integration of 35S-PMP70 into membranes occurred in the peroxisomal fraction and was completed within 30 min. No proteolytic processing of 35S-PMP70 was observed. An in vitro import system was reconstituted to characterize the insertion mechanism of PMP70 into peroxisomes. Peroxisomes isolated from rat liver were incubated at 26 degrees C with [35S]methionine-labeled in vitro translation products of PMP70 mRNA in the presence of the cytosolic fraction. The peroxisomes were reisolated and insertion of 35S-PMP70 into the membrane was analyzed using a Na2CO3 procedure. The binding and insertion of 35S-PMP70 were dependent on temperature and incubation time and was specific for peroxisomes. Pretreatment of peroxisomes with trypsin and chymotrypsin almost abolished the binding and insertion of 35S-PMP70. The translation products contained several truncated 35S-PMP70s. The NH2 terminally truncated 35S-PMP70s, with a molecular mass greater than 50 kDa, bound to and inserted into peroxisomal membranes, whereas truncated 35S-PMP70s smaller than 45 kDa did not. These results suggest that PMP70 is post-translationally transported to peroxisomes without processing and inserted into peroxisomal membranes by a specific mechanism in which a proteinaceous receptor and a certain internal sequence of PMP70 are involved.

ATP-Binding Cassette Transporters↗
PubMed1997

Alteration of a mitochondrial outer membrane signal anchor sequence that permits its insertion into the inner membrane. Contribution of hydrophobic residues.

Tom70p is targeted and inserted into the mitochondrial outer membrane in the Nin-Ccyto orientation, via an NH2-terminal signal anchor sequence. The signal anchor is comprised of two domains: an NH2-terminal hydrophilic region which is positively charged (amino acids 1-10) followed by the predicted transmembrane segment (amino acids 11-29). Substitution of the NH2-terminal domain with a matrix-targeting signal caused the signal anchor to adopt the reverse orientation in the outer membrane (Ncyto-Cin) or, if presented to mitoplasts, to arrest protein translocation at the inner membrane without insertion. Physically separating the transmembrane segment from the matrix-targeting signal by moving it downstream within the protein resulted in a failure to arrest in either membrane, and consequently the protein was imported to the matrix. However, if the mean hydrophobicity of the Tom70p transmembrane segment was increased in these constructs, the protein inserted into the inner membrane with an Nin-Cout orientation. Therefore we have determined conditions that allow the Tom70p transmembrane domain to insert in either membrane, pass through both membranes, or arrest without insertion in the inner membrane. These results identify the mean hydrophobicity of potential transmembrane domains within bitopic proteins as an important determinant for insertion into the mitochondrial inner membrane.

Amino Acid Sequence↗
PubMed1997

Identification of membrane insertion sequences of the rabbit gastric cholecystokinin-A receptor by in vitro translation.

To determine which amino acid sequences account for transmembrane folding of G7 receptors, the membrane domain of the rabbit cholecystokinin-A (CCK-A) G-protein-coupled receptor has been investigated by in vitro transcription/translation of two types of fusion vectors containing sequences that include putative transmembrane segments. First, the seven putative transmembrane domains of the CCK-A receptor were inserted individually into pGEM vectors beginning with the cDNA encoding the first 101 (HK-M0) or 139 (HK-M1) amino acids of the alpha subunit of the gastric H, K-ATPase. These were separated by the cDNA for the inserted transmembrane domains from the cDNA encoding the last 177 amino acids of the beta subunit of the H,K-ATPase containing five N-linked glycosylation consensus sequences (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). Transcription/translation of these fusion vectors in rabbit reticulocyte lysate +/- dog pancreatic microsomes followed by SDS-polyacrylamide gel electrophoresis defined the presence of signal anchor sequences in HK-M0 by glycosylation and stop transfer sequences in HK-M1 by inhibition of glycosylation. Six out of the seven putative transmembrane domains had membrane insertion signals, but no membrane insertion activity was found for the H3 segment in these vectors. To test the effect of specific upstream and downstream sequences on membrane insertion, vectors were also made starting with the cDNA encoding the N terminus of the CCK-A receptor separated from the last 177 amino acids of the H,K-ATPase beta subunit by cDNA encoding CCK-A receptor sequences of different lengths. In addition to transcription/translation, endoglycosidase H treatment was used to verify glycosylation when multiple bands were found in the presence of microsomes. The four positive charges in the loop between H1 and H2 were required for the correct orientation of the first transmembrane domain. The H3 segment acted as a stop transfer sequence only when the whole N terminus and H3 were followed by the positive charges in the cytoplasmic loop between H3 and H4. The activity of H6 as a signal anchor sequence depended on preceding positive charges. These translation data using two types of fusion vectors establish a seven-transmembrane folding model using only in vitro translation for the CCK-A receptor beginning with two signal anchor sequences and then alternating stop transfer and signal anchor insertions. Positive charges between H1 and H2, H3 and H4, and H5 and H6 function as cytoplasmic anchors in the membrane folding of this receptor.

Amino Acid Sequence↗
PubMed1999

Topological and functional analysis of the human reduced folate carrier by hemagglutinin epitope insertion.

The membrane topology of the human reduced folate carrier protein (591 amino acids) was assessed by single insertions of the hemagglutinin epitope into nine sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary cells expressing each of these constructs were probed with anti-hemagglutinin epitope monoclonal antibodies to assess whether the insertion was exposed to the external environment or to the cytoplasm. The results are consistent with the 12-transmembrane topology predicted for this protein. The hemagglutinin epitope insertion mutants were also tested for their effects on the function of the reduced folate carrier. For these studies, each of the constructs had a carboxyl-terminal fusion of the enhanced green fluorescent protein to monitor and quantitate expression. Insertions into the external loop between transmembrane regions 7 and 8 (Pro-297), the cytoplasmic loop between transmembrane regions 6 and 7 (Ser-225), and near the cytoplasmic amino and carboxyl termini (Pro-20 and Gly-492, respectively) had minor effects on methotrexate binding and uptake. The insertion into the cytoplasmic loop between transmembrane regions 10 and 11 (Gln-385) greatly reduced both binding and uptake of methotrexate, whereas the insertion into the external loop between transmembrane regions 11 and 12 (Pro-427) selectively interfered with uptake but not binding.

Amino Acid Sequence↗
PubMed2000

Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane beta-sheet from a prepore intermediate.

Perfringolysin O (PFO), a member of the cholesterol-dependent cytolysin family of pore-forming toxins, forms large oligomeric complexes comprising up to 50 monomers. In the present study, a disulfide bridge was introduced between cysteine-substituted serine 190 of transmembrane hairpin 1 (TMH1) and cysteine-substituted glycine 57 of domain 2 of PFO. The resulting disulfide-trapped mutant (PFO(C190-C57)) was devoid of hemolytic activity and could not insert either of its transmembrane beta-hairpins (TMHs) into the membrane unless the disulfide was reduced. Both the size of the oligomer formed on the membrane and its rate of formation were unaffected by the oxidation state of the Cys(190)-Cys(57) disulfide bond; thus, the disulfide-trapped PFO was assembled into a prepore complex on the membrane. The conversion of this prepore to the pore complex was achieved by reducing the C190-C57 disulfide bond. PFO(C190-C57) that was allowed to form the prepore prior to the reduction of the disulfide exhibited a dramatic increase in the rate of PFO-dependent hemolysis and the membrane insertion of its TMHs when compared with toxin that had the disulfide reduced prior mixing the toxin with membranes. Therefore, the rate-limiting step in pore formation is prepore assembly, not TMH insertion. These data demonstrate that the prepore is a legitimate intermediate during the insertion of the large transmembrane beta-sheet of the PFO oligomer. Finally, the PFO TMHs do not appear to insert independently, but instead their insertion is coupled.

Bacterial Toxins↗
PubMed2001

Cholesterol is an important factor affecting the membrane insertion of beta-amyloid peptide (A beta 1-40), which may potentially inhibit the fibril formation.

beta-Amyloid peptide (A beta), a normal constituent of neuronal and non-neuronal cells, has been proven to be the major component of extracellular plaque of Alzheimer's disease. Interactions between A beta and neuronal membranes have been postulated to play an important role in the neuropathology of Alzheimer's disease. Here we show that A beta is able to insert into lipid bilayer. The membrane insertion ability of A beta is critically controlled by the ratio of cholesterol to phospholipids. In a low concentration of cholesterol A beta prefers to stay in membrane surface region mainly in a beta-sheet structure. In contrast, as the ratio of cholesterol to phospholipids rises above 30 mol%, A beta can insert spontaneously into lipid bilayer by its C terminus. During membrane insertion A beta generates about 60% alpha-helix and removes almost all beta-sheet structure. Fibril formation experiments show that such membrane insertion can reduce fibril formation. Our findings reveal a possible pathway by which A beta prevents itself from aggregation and fibril formation by membrane insertion.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2003

Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions.

The study of mutant enzymes can reveal important details about the fundamental mechanism and regulation of RNA polymerase, the central enzyme of gene expression. However, such studies are complicated by the multisubunit structure of RNA polymerase and by its indispensability for cell growth. Previously, mutant RNA polymerases have been produced by in vitro assembly from isolated subunits or by in vivo assembly upon overexpression of a single mutant subunit. Both approaches can fail if the mutant subunit is toxic or incorrectly folded. Here we describe an alternative strategy, co-overexpression and in vivo assembly of RNA polymerase subunits, and apply this method to characterize the role of sequence insertions present in the Escherichia coli enzyme. We find that co-overexpression of its subunits allows assembly of an RNA polymerase lacking a 188-amino acid insertion in the beta' subunit. Based on experiments with this and other mutant E. coli enzymes with precisely excised sequence insertions, we report that the beta' sequence insertion and, to a lesser extent, an N-terminal beta sequence insertion confer characteristic stability to the open initiation complex, frequency of abortive initiation, and pausing during transcript elongation relative to RNA polymerases, such as that from Bacillus subtilis, that lack the sequence insertions.

Bacillus subtilis↗
PubMed2003

Conditional lethal mutations separate the M13 procoat and Pf3 coat functions of YidC: different YIDC structural requirements for membrane protein insertion.

Conditional lethal YidC mutants have been isolated to decipher the role of YidC in the assembly of Sec-dependent and Sec-independent membrane proteins. We now show that the membrane insertion of the Sec-independent M13 procoat-lep protein is inhibited in a short time in a temperature-sensitive mutant when shifted to the nonpermissive temperature. This provides an additional line of evidence that YidC plays a direct role in the insertion of the Sec-independent M13 procoat protein. However, in the temperature-sensitive mutant, the insertion of the Sec-independent Pf3 phage coat protein and the Sec-dependent leader peptidase were not strongly inhibited at the restricted temperatures. Conversely, using a cold-sensitive YidC strain, we find that the membrane insertion of the Sec-independent Pf3 coat protein is blocked, and the Sec-dependent leader peptidase is inhibited at the nonpermissive temperature, whereas the insertion of the M13 procoat protein is nearly normal. These data show that the YidC function for procoat and its function for Pf3 coat and possibly leader peptidase are genetically separable and suggest that the YidC structural requirements are different for the Sec-independent M13 procoat and Pf3 coat phage proteins that insert by different mechanisms.

Adenosine Triphosphatases↗