Central venous catheters: incidence and predictive factors of venous thrombosis.

AIMS: Central venous catheter access in an acute setting can be a challenge given underlying disease and risk for venous thrombosis. Peripherally inserted central venous catheters (PICCs) are commonly placed but limit sites for fistula creation in patients with chronic renal failure (CKD). The aim of this study is to determine the incidence of venous thrombosis from small bore internal jugular (SBIJ) and PICC line placement. This investigation identifies populations of patients who may not be ideal candidates for a PICC and highlights the importance of peripheral vein preservation in patients with renal failure. MATERIALS AND METHODS: A venous Doppler ultrasound was performed at the time of SBIJ insertion and removal to evaluate for thrombosis in the internal jugular vein. Data was collected pre- and post-intervention to ascertain if increased vein preservation knowledge amongst the healthcare team led to less use of PICCs. Demographic factors were collected in the SBIJ and PICC groups and risk factor analysis was completed. RESULTS: 1,122 subjects had PICC placement and 23 had SBIJ placement. The incidence of thrombosis in the PICC group was 10%. One patient with an SBIJ had evidence of central vein thrombosis when the catheter was removed. Univariate and multivariate analysis demonstrated a history of transplant, and the indication of total parenteral nutrition was associated with thrombosis (p<0.001). The decrease in PICCs placed in patients with CKD 6 months before and after intervention was significant (p<0.05). CONCLUSIONS: There are subsets of patients ith high risk for thrombosis who may not be ideal candidates for a PICC.

A second mitochondrial DNA primase is essential for cell growth and kinetoplast minicircle DNA replication in Trypanosoma brucei.

The mitochondrial DNA of trypanosomes contains two types of circular DNAs, minicircles and maxicircles. Both minicircles and maxicircles replicate from specific replication origins by unidirectional theta-type intermediates. Initiation of the minicircle leading strand and also that of at least the first Okazaki fragment involve RNA priming. The Trypanosoma brucei genome encodes two mitochondrial DNA primases, PRI1 and PRI2, related to the primases of eukaryotic nucleocytoplasmic large DNA viruses. These primases are members of the archeoeukaryotic primase superfamily, and each of them contain an RNA recognition motif and a PriCT-2 motif. In Leishmania species, PRI2 proteins are approximately 61 to 66 kDa in size, whereas in Trypanosoma species, PRI2 proteins have additional long amino-terminal extensions. RNA interference (RNAi) of T. brucei PRI2 resulted in the loss of kinetoplast DNA and accumulation of covalently closed free minicircles. Recombinant PRI2 lacking this extension (PRI2ΔNT) primes poly(dA) synthesis on a poly(dT) template in an ATP-dependent manner. Mutation of two conserved aspartate residues (PRI2ΔNTCS) resulted in loss of enzymatic activity but not loss of DNA binding. We propose that PRI2 is directly involved in initiating kinetoplast minicircle replication.

Structure of discontinuities in kinetoplast DNA-associated minicircles during S phase in Crithidia fasciculata.

Kinetoplast DNA (kDNA) is a novel form of mitochondrial DNA consisting of thousands of interlocked minicircles and 20-30 maxicircles. The minicircles replicate free of the kDNA network but nicks and gaps in the newly synthesized strands remain at the time of reattachment to the kDNA network. We show here that the steady-state population of replicated, network-associated minicircles only becomes repaired to the point of having nicks with a 3'OH and 5'deoxyribonucleoside monophosphate during S phase. These nicks represent the origin/terminus of the strand and occur within the replication origins (oriA and oriB) located 180 degrees apart on the minicircle. Minicircles containing a new L strand have a single nick within either oriA or oriB but not in both origins in the same molecule. The discontinuously synthesized H strand contains single nicks within both oriA and oriB in the same molecule implying that discontinuities between the H-strand Okazaki fragments become repaired except for the fragments initiated within the two origins. Nicks in L and H strands at the origins persist throughout S phase and only become ligated as a prelude to network division. The failure to ligate these nicks until just prior to network division is not due to inappropriate termini for ligation.

Identification of new kinetoplast DNA replication proteins in trypanosomatids based on predicted S-phase expression and mitochondrial targeting.

Trypanosomatid parasites contain an unusual form of mitochondrial DNA (kinetoplast DNA [kDNA]) consisting of a catenated network of several thousand minicircles and a smaller number of maxicircles. Many of the proteins involved in the replication and division of kDNA are likely to have no counterparts in other organisms and would not be identified by similarity to known replication proteins in other organisms. A new kDNA replication protein conserved in kinetoplastids has been identified based on the presence of posttranscriptional regulatory sequences associated with S-phase gene expression and predicted mitochondrial targeting. The Leishmania major protein P105 (LmP105) and Trypanosoma brucei protein P93 (TbP93) localize to antipodal sites flanking the kDNA disk, where several other replication proteins and nascent minicircles have been localized. Like some of these kDNA replication proteins, the LmP105 protein is only present at the antipodal sites during S phase. RNA interference (RNAi) of TbP93 expression resulted in a cessation of cell growth and the loss of kDNA. Nicked/gapped forms of minicircles, the products of minicircle replication, were preferentially lost from the population of free minicircles during RNAi, suggesting involvement of TbP93 in minicircle replication. This approach should allow the identification of other novel proteins involved in the duplication of kDNA.

Posterior reversible encephalopathy syndrome: a case study.

A young woman 4 days postpartum was admitted after experiencing two seizures. Her mentation waxed and waned until, after several hours, staff were unable to arouse her with voice or touch. A computed tomography scan demonstrated considerable white-matter edema. The patient's condition declined to a coma. She remained comatose despite therapeutic interventions to control increased intracranial pressure. To her family, her condition was a source of anguish. To the physicians and nurses, she was a puzzle. The final diagnosis was posterior reversible encephalopathy syndrome, which was related to a preeclamptic condition and its associated hypertension. The collaboration of obstetricians and neurologists with vigilant care by neuroscience nurses resulted in a positive outcome for this challenging patient.

Increased Inlet Blood Flow Velocity Predicts Low Wall Shear Stress in the Cephalic Arch of Patients with Brachiocephalic Fistula Access.

BACKGROUND: An autogenous arteriovenous fistula is the optimal vascular access for hemodialysis. In the case of brachiocephalic fistula, cephalic arch stenosis commonly develops leading to access failure. We have hypothesized that a contribution to fistula failure is low wall shear stress resulting from post-fistula creation hemodynamic changes that occur in the cephalic arch. METHODS: Twenty-two subjects with advanced renal failure had brachiocephalic fistulae placed. The following procedures were performed at mapping (pre-operative) and at fistula maturation (8-32 weeks post-operative): venogram, Doppler to measure venous blood flow velocity, and whole blood viscosity. Geometric and computational modeling was performed to determine wall shear stress and other geometric parameters. The relationship between hemodynamic parameters and clinical findings was examined using univariate analysis and linear regression. RESULTS: The percent low wall shear stress was linearly related to the increase in blood flow velocity (p < 0.01). This relationship was more significant in non-diabetic patients (p < 0.01) than diabetic patients. The change in global measures of arch curvature and asymmetry also evolve with time to maturation (p < 0.05). CONCLUSIONS: The curvature and hemodynamic changes during fistula maturation increase the percentage of low wall shear stress regions within the cephalic arch. Low wall shear stress may contribute to subsequent neointimal hyperplasia and resultant cephalic arch stenosis. If this hypothesis remains tenable with further studies, ways of protecting the arch through control of blood flow velocity may need to be developed.

Health literacy, cervical cancer risk factors, and distress in low-income African-American women seeking colposcopy.

OBJECTIVES: To describe the relationship between health literacy, distress, and cervical cancer risk factors in women at high risk for developing cervical cancer. DESIGN: Cross-sectional, prospective cohort design. SETTING: Two university-based gynecological oncology colposcopy clinics and 3 Planned Parenthood community dinics. PATIENTS/PARTICIPANTS: One hundred-thirty English-speaking African-American women > or = 18 years referred for colposcopy following abnormal Pap testing. MAIN OUTCOME MEASURES: Avoidance and Intrusion subscales of the Impact of Events Scale (IES), Rapid Estimate of Adult Literacy in Medicine (REALM), and demographics. RESULTS: Forty-five percent of women had a low level of health literacy (< 9th grade). Low health literacy was related to fewer risk factors (P < .01) and higher levels of distress on the Impact of Events avoidance subscale (P < .05) after controlling for covariates. Forty-three percent of women with low literacy had excessive levels of distress as compared to 25% in women with high literacy (P < .05). CONCLUSIONS: A low level of health literacy is associated with increased levels of distress among women at high risk for developing cervical cancer. To the extent that distress serves as a barrier to treatment, culturally informed, effective interventions are needed.

A mitochondrial DNA primase is essential for cell growth and kinetoplast DNA replication in Trypanosoma brucei.

Kinetoplast DNA in African trypanosomes contains a novel form of mitochondrial DNA consisting of thousands of minicircles and dozens of maxicircles topologically interlocked to form a two-dimensional sheet. The replication of this unusual form of mitochondrial DNA has been studied for more than 30 years, and although a large number of kinetoplast replication genes and proteins have been identified, in vitro replication of these DNAs has not been possible since a kinetoplast DNA primase has not been available. We describe here a Trypanosoma brucei DNA primase gene, PRI1, that encodes a 70-kDa protein that localizes to the kinetoplast and is essential for both cell growth and kinetoplast DNA replication. The expression of PRI1 mRNA is cyclic and reaches maximum levels at a time corresponding to duplication of the kinetoplast DNA. A 3'-hydroxyl-terminated oligoriboadenylate is synthesized on a poly(dT) template by a recombinant form of the PRI1 protein and is subsequently elongated by DNA polymerase and added dATP. Poly(dA) synthesis is dependent on both PRI1 protein and ATP and is inhibited by RNase H treatment of the product of PRI1 synthesis.

Survival following resuscitative thoracotomy for combined left ventricle and left atrium ruptures secondary to blunt trauma.

Improvements in pre-hospital care and the development of integrated Trauma Systems have streamlined access for the severely injured to sophisticated, specialist Trauma Centre reception and resuscitation. We describe the initial care of a survivor of combined ruptures of the left ventricle and left atrium secondary to blunt injury. This case emphasises the contribution of such a Trauma System in achieving a favourable outcome for a severely injured trauma patient with injuries previously considered non-survivable.

Cell cycle-dependent localization and properties of a second mitochondrial DNA ligase in Crithidia fasciculata.

The mitochondrial DNA in kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kbeta) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kalpha). LIG kalpha localizes to the kinetoplast primarily in cells that have completed mitosis and contain either a dividing kinetoplast or two newly divided kinetoplasts. Essentially all dividing or newly divided kinetoplasts show localization of LIG kalpha. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kalpha to the kDNA but at a much lower frequency. The mRNA level of LIG kalpha varies during the cell cycle out of phase with that of LIG kbeta. LIG kalpha transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG kbeta transcript levels are maximal during S phase. The LIG kalpha protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG kalpha transcript and the instability of the LIG kalpha protein suggest a possible role of the ligase in regulating minicircle replication.

Mitochondrial DNA ligases of Trypanosoma brucei.

The mitochondrial DNA of Trypanosoma brucei, termed kinetoplast DNA or kDNA, consists of thousands of minicircles and a small number of maxicircles catenated into a single network organized as a nucleoprotein disk at the base of the flagellum. Minicircles are replicated free of the network but still contain nicks and gaps after rejoining to the network. Covalent closure of remaining discontinuities in newly replicated minicircles after their rejoining to the network is delayed until all minicircles have been replicated. The DNA ligase involved in this terminal step in minicircle replication has not been identified. A search of kinetoplastid genome databases has identified two putative DNA ligase genes in tandem. These genes (LIG k alpha and LIG k beta) are highly diverged from mitochondrial and nuclear DNA ligase genes of higher eukaryotes. Expression of epitope-tagged versions of these genes shows that both LIG k alpha and LIG k beta are mitochondrial DNA ligases. Epitope-tagged LIG k alpha localizes throughout the kDNA, whereas LIG k beta shows an antipodal localization close to, but not overlapping, that of topoisomerase II, suggesting that these proteins may be contained in distinct structures or protein complexes. Knockdown of the LIG k alpha mRNA by RNA interference led to a cessation of the release of minicircles from the network and resulted in a reduction in size of the kDNA networks and rapid loss of the kDNA from the cell. Closely related pairs of mitochondrial DNA ligase genes were also identified in Leishmania major and Crithidia fasciculata.

Sequence elements in both the intergenic space and the 3' untranslated region of the Crithidia fasciculata KAP3 gene are required for cell cycle regulation of KAP3 mRNA.

mRNA levels of several Crithidia fasciculata genes involved in DNA metabolism have previously been found to cycle as cells progress through the cell cycle. Octamer consensus sequences in the 5' untranslated regions (5' UTRs) of these transcripts were shown to be required for cycling of these mRNAs. The KAP3 gene encodes a kinetoplast histone H1-like DNA binding protein, and its mRNA levels cycle in parallel with those of the kinetoplast DNA topoisomerase (TOP2), dihydrofolate reductase-thymidylate synthase (DHFR-TS), and the large subunit of the nuclear single-stranded DNA binding protein (RPA1). KAP3 mRNA contains two octamer consensus sequences in its 3' UTR but none in its 5' UTR. Mutation of these octamer sequences was not sufficient to prevent cycling of a sequence-tagged KAP3 mRNA expressed from a plasmid. Mutation of an octamer sequence contained on the precursor transcript but not on the mRNA, in addition to mutation of the two octamer sequences in the 3' UTR, was necessary to abolish cycling of the mRNA. The requirement for a sequence not present on the mature mRNA indicates that regulation of the mRNA levels by the octamer sequences occurs at or prior to splicing of the transcript. Incompletely processed RNAs containing octamer sequences were also found to accumulate during the cell cycle when the mRNA levels were lowest. These RNA species hybridize to both the KAP3 coding sequence and that of the downstream drug resistance gene, indicating a lack of processing within the intergenic region separating these genes. We propose a cell cycle-dependent interference in transcript processing mediated by octamer consensus sequences as a mechanism contributing to the cycling of such transcripts.

Mitochondrial DNA ligase in Crithidia fasciculata.

Kinetoplast DNA (kDNA), the form of mitochondrial DNA in trypanosomatids, consists of thousands of interlocked circular DNAs organized into a compact disk structure. A type II DNA topoisomerase, a DNA polymerase beta, and a structure-specific endonuclease have been localized to antipodal sites flanking the kDNA disk along with nascent DNA minicircles. We have cloned a gene (LIG k) encoding a mitochondrial DNA ligase in the trypanosomatid Crithidia fasciculata, and we show that an epitope-tagged form of the ligase colocalizes with the other replication proteins at the antipodal sites and also at the two faces of the kDNA disk. DNA LIG k becomes adenylated in reactions with ATP, and the adenylate moiety is removed by incubation with pyrophosphate or nicked DNA. The ligase interacts physically with the beta polymerase and is proposed to be involved in the repair of gaps in the newly synthesized minicircles. In yeast and mammals, a single gene encodes both nuclear and mitochondrial forms of DNA ligase. The LIG K protein sequence has low similarity to mitochondrial DNA ligases in other eukaryotes and is distinct from the C. fasciculata nuclear DNA ligase (LIG I).

Presence of multiple mRNA cycling sequence element-binding proteins in Crithidia fasciculata.

A consensus sequence present in the 5'- or 3'-untranslated regions of several Crithidia fasciculata messenger RNAs encoding proteins involved in DNA metabolism has been shown to be necessary for the periodic accumulation of these mRNAs during the cell cycle. A protein complex termed cycling sequence-binding protein (CSBP) has two subunits, CSBPA and CSBPB, and binds the consensus sequence with high specificity. The binding activity of CSBP was shown to vary during the cell cycle in parallel with the levels of putative target mRNAs. Although disruption of the CSBPA gene resulted in loss of both CSBPA and CSBPB, the putative target message levels still continued to vary during the cell cycle. The presence of an additional and distinct binding activity was revealed in these CSBPA null mutant cells. This activity, termed CSBP II, was also expressed in wild-type Crithidia cells. CSBP II has higher binding specificity for the cycling sequence element than the earlier described CSBP complex. Three polypeptides associated with purified CSBP II show specific binding to the cycling sequence. These proteins may represent a family of sequence-specific RNA-binding proteins involved in post-transcriptional regulation.