Effects of Ionic Strength, Salt, and pH on Aggregation of Boehmite Nanocrystals: Tumbler Small-Angle Neutron and X-ray Scattering and Imaging Analysis.

The US government currently spends significant resources managing the legacies of the Cold War, including 300 million liters of highly radioactive wastes stored in hundreds of tanks at the Hanford (WA) and Savannah River (SC) sites. The materials in these tanks consist of highly radioactive slurries and sludges at very high pH and salt concentrations. The solid particles primarily consist of aluminum hydroxides and oxyhydroxides (gibbsite and boehmite), although many other materials are present. These form complex aggregates that dramatically affect the rheology of the solutions and, therefore, efforts to recover and treat these wastes. In this paper, we have used a combination of transmission and cryo-transmission electron microscopy, dynamic light scattering, and X-ray and neutron small and ultrasmall-angle scattering to study the aggregation of synthetic nanoboehmite particles at pH 9 (approximately the point of zero charge) and 12, and sodium nitrate and calcium nitrate concentrations up to 1 m. Although the initial particles form individual rhombohedral platelets, once placed in solution they quickly form well-bonded stacks, primary aggregates, up to ∼1500 Å long. These are more prevalent at pH = 12. Addition of calcium nitrate or sodium nitrate has a similar effect as lowering pH, but approximately 100 times less calcium than sodium is needed to observe this effect. These aggregates have fractal dimension between 2.5 and 2.6 that are relatively unaffected by salt concentration for calcium nitrate at high pH. Larger aggregates (>∼4000 Å) are also formed, but their size distributions are discrete rather than continuous. The fractal dimensions of these aggregates are strongly pH-dependent, but only become dependent on solute at high concentrations.

Successful surgical treatment of Q fever endocarditis with mitral valve repair.

We report a case of successful surgical treatment of Q fever endocarditis with mitral valve repair in a 66-year old retired British soldier. Valve replacement is invariably undertaken in Q fever endocarditis due to the degree of valvular damage and concerns about eradicating the organism, Coxiella burnetii. Our unique case allowed valve repair since pre-existing myxomatous degeneration and subsequent posterior mitral valve leaflet prolapse resulted in significant excess valve tissue, allowing quadrangular resection of the damaged and perforated P2 portion of this leaflet. Follow-up at four years (including three years of antibiotic treatment) has confirmed excellent valve repair, with no echocardiographic, clinical or microbiological evidence of recurrence. We are only the second group to describe valve repair in a patient with chronic Q fever endocarditis. Valve repair is preferable to valve replacement for Q fever endocarditis, if technically possible.

Traumatic avulsion of the suprascapular artery.

Trauma to the subclavian artery and its branches is rare, and usually the result of penetrating injuries. Blunt trauma presents its own peculiar management difficulties, particularly when causing haemorrhage into the thoracic cavity. Cardiothoracic surgeons may be asked to deal with such cases, so an understanding of the anatomy and options for surgical access are essential. We present a case of blunt avulsion of the suprascapular artery resulting in massive haemothorax, a previously unreported injury.

Metabolic labeling and immunoprecipitation of yeast proteins.

The proteins of Saccharomyces cervsiae can be metabolically labeled, as described here, with (35)methionine and (35)cysteine or a hydrolysate of E. coli labeled with (35)O4(2-). After the labeling, protocols are provided for the mechanical disruption of the yeast cells or conversion to spheroplasts, with subsequent lysis before immunoprecipitation of the proteins.

The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast.

BACKGROUND: In eukaryotic cells, clathrin-coated vesicles transport specific cargo from the plasma membrane and trans-Golgi network to the endosomal system. Removal of the clathrin coat in vitro requires the uncoating ATPase Hsc70 and its DnaJ cofactor auxilin. To date, a requirement for auxilin and Hsc70 in clathrin function in vivo has not been demonstrated. RESULTS: The Saccharomyces cerevisiae SWA2 gene, previously identified in a synthetic lethal screen with arf1, was cloned and found to encode a protein with a carboxy-terminal DnaJ domain which is homologous to that of auxilin. Like auxilin, Swa2p has a clathrin-binding domain and is able to stimulate the ATPase activity of Hsc70. The swa2-1 allele recovered from the original screen carries a point mutation in its tetratricopeptide repeat (TPR) domain, a motif not found in auxilin but known in other proteins to mediate interaction with heat-shock proteins. Swa2p fractionates in the cytosol and appears to be heavily phosphorylated. Disruption of SWA2 causes slow growth and several phenotypes that are very similar to those exhibited by clathrin mutants. Furthermore, the swa2Delta mutant exhibits a significant increase in membrane- associated or -assembled clathrin relative to a wild-type strain. CONCLUSIONS: These results indicate that Swa2p is a clathrin-binding protein required for normal clathrin function in vivo. They suggest that Swa2p is the yeast ortholog of auxilin and has a role in disassembling clathrin, not only in uncoating clathrin-coated vesicles but perhaps in preventing unproductive clathrin assembly in vivo.

Organization of the yeast Golgi complex into at least four functionally distinct compartments.

Pro-alpha-factor (pro-alphaf) is posttranslationally modified in the yeast Golgi complex by the addition of alpha1,6-, alpha1,2-, and alpha1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the alpha1,6- and alpha1,3-mannosylation and Kex2p-dependent processing of pro-alphaf are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that alpha1,2-mannosylation of pro-alphaf is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-alpha1,6-D-mannanase (endoM) were used to quantitate the amount of each pro-alphaf intermediate during transport through the Golgi complex. We found that alpha1,6-, alpha1,2-, and alpha1,3-mannose were sequentially added to pro-alphaf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. In addition, most of the Golgi-modified pro-alphaf that accumulated in brefeldin A-treated cells received only alpha1,6-mannosylation as did approximately 50% of pro-alphaf transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an alpha1,6-mannosyltransferase but lacks alpha1,2-mannosyltransferase activity in vivo. We propose that the alpha1,6-, alpha1,2-, and alpha1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial, trans, and trans-Golgi network of the yeast Golgi complex, respectively.

Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function.

ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Delta synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-alpha-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Delta cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

Techniques in conduit harvesting.

Coronary artery bypass grafting is a commonly performed operation for the treatment of ischaemic heart disease. The success of it is largely dependent upon the quality and type of graft used. This article describes the application of and rationale behind techniques commonly used in conduit harvesting.

The high osmolarity glycerol response (HOG) MAP kinase pathway controls localization of a yeast golgi glycosyltransferase.

The yeast alpha-1,3-mannosyltransferase (Mnn1p) is localized to the Golgi by independent transmembrane and lumenal domain signals. The lumenal domain is localized to the Golgi complex when expressed as a soluble form (Mnn1-s) by exchange of its transmembrane domain for a cleavable signal sequence (Graham, T. R., and V. A. Krasnov. 1995. Mol. Biol. Cell. 6:809-824). Mutants that failed to retain the lumenal domain in the Golgi complex, called lumenal domain retention (ldr) mutants, were isolated by screening mutagenized yeast colonies for those that secreted Mnn1-s. Two genes were identified by this screen, HOG1, a gene encoding a mitogen-activated protein kinase (MAPK) that functions in the high osmolarity glycerol (HOG) pathway, and LDR1. We have found that basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions. Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a concomitant increase of Mnn1p in later Golgi compartments.

An arf1Delta synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar protein transport in Saccharomyces cerevisiae.

ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Delta allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Delta). Most of the swa mutants exhibit phenotypes comparable to arf1Delta mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y.

COPI in ER/Golgi and intra-Golgi transport: do yeast COPI mutants point the way?

Coat complexes facilitate the formation of transport vesicles which are essential for proper trafficking of protein and lipids through the secretory pathway. Since its initial identification in the mid-1980s, the COPI coat complex has been credited with mediating multiple distinct transport events and intracellular processes in the exocytic pathway. Not surprisingly, the diversity of these functions has led to significant debate concerning the primary function of COPI. Specifically, within the ER/Golgi and intra-Golgi systems, does COPI mediate anterograde protein transport, retrograde protein transport, or both? This review will focus on the in vivo roles of COPI, primarily examining data from studies of yeast COPI mutants but also including evidence from mammalian systems as appropriate. Some of the current controversies surrounding whether COPI acts directly or indirectly in anterograde and retrograde transport will also be addressed. Because recruitment of COPI to membranes requires the small GTP-binding protein ARF, we will also discuss ARF and proteins that regulate ARF function, and how these proteins might modulate both COPI-driven events and overall membrane composition. Finally, we will point out some of the links still missing from our understanding of COPI-driven events and discuss possible future directions for studies of COPI function.

von Willebrand factor and urinary albumin excretion are possible indicators of endothelial dysfunction in cardiopulmonary bypass.

OBJECTIVE: Experimental evidence suggests that cardiopulmonary bypass (CPB) associated inflammatory response leads to endothelial injury and increased permeability, but this has been difficult to show clinically. We have investigated the use of von Willebrand factor (vWF), and urinary albumin excretion, as measured by the urinary albumin creatinine ratio (ACR), to demonstrate this. METHODS: A total of 23 patients undergoing elective coronary artery bypass grafting were studied. Complement fragment C3a, leukotrienne B4 (LTB4), interleukin 6 (IL6), neutrophil elastase, vWF and ACR were measured on anaesthetic induction (baseline), 20 min after starting CPB, 5 min after cross-clamp removal, 5 min, 2, 6 and 24 h after termination of CPB. Anaesthetic, CPB and myocardial protection techniques were standardised. ANOVA was performed by using the distribution free Friedman test for each measured parameter. When significance differences were found (P < 0.05), post hoc analysis with Wilcoxon signed rank test was used for comparison of each time point with the base line level and differences were only accepted as significant following the Bonferroni correction (P < 0.008). Summary measures of peak versus peak and area under the cure were also analysed for ACR with vWF. RESULTS: Peak vs. baseline levels for C3a were 4.9 vs. 2.1 microg/ml (P < 0.0001), LTB4 was 800 vs. 20 pg/ml (P < 0.0001), neutrophil elastase was 250 vs. 115 ng/ml (P < 0.001), IL6 was 620 vs. 1.4 pg/ml (P < 0.0001), vWF was 2.2 vs. 1.3 IU/ml (P < 0.0001) and ACR was 17.6 vs. 2.0 mg/mmol (P < 0.0001). C3a, LTB4 and ACR peaked during the operation. Neutrophil elastase peaked at 2 h following CPB. IL6 and vWF peaked at 6 h following CPB. The correlation coefficient between vWF and ACR following peak versus peak analysis was 0.48 (P = 0.035), and area under the curve analysis was 0.6 (P < 0.01). CONCLUSION: These results demonstrate that endothelial permeability and injury, as measured by urinary albumin excretion and vWF, respectively, are related and the use of these easily detectable and sensitive biochemical markers warrants further investigation.

ARF is required for maintenance of yeast Golgi and endosome structure and function.

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Delta, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4-64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4-64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.

Platelets and cardiopulmonary bypass.

Exposure of blood to an extracorporeal circulation, such as CPB, causes a variety of physiological responses. Haematological derangements are just one of many potential dangers to the patient who undergoes CPB. The paradox of CPB-related problems with the haematological system is that there are some factors tipping the balance towards a bleeding tendency, and others that favour a prothrombotic state. Both of these issues must be dealt with independently to create the safest environment for surgery. It has been demonstrated that platelets play a key role in both haemostatic dysfunction and thrombotic complications of CPB. Much has been achieved, both clinically and in the laboratory, in the understanding of the precise role platelets play in these events, but the exact mechanisms involved have yet to be completely identified. As research progresses, our understanding will increase, but until then clinical practice must be dictated by the current evidence available.

Prevention of remote organ injury in cardiopulmonary bypass: the impact of flow generation technique.

The purpose of this study was to investigate the effects of 3 different types of flow generation for cardiopulmonary bypass on gastrointestinal permeability and on neutrophil expression of CD11b, a surface marker of neutrophil activation. Fourteen patients undergoing elective coronary revascularization were selected randomly to receive 1 of the 3 flow generation techniques (roller, pulsatile, or centrifugal). Intestinal permeability was assessed by the fraction of an oral dose of 51chromium-ethylenediaminetetraacetate (51Cr-EDTA) recovered in the urine over 24 h. Neutrophil activation was determined by expression of CD11b markers at 6 time points. Overall, the 14 patients showed significant increases in intestinal permeability. It was not possible to demonstrate statistically significant differences among the flow generation groups; however, when compared to both roller pump groups, the centrifugal pump group showed a 3.2% reduction in intestinal permeability. There was no change in the expression of CD11b receptors throughout the time points, nor was there a relationship of CD11b markers to the flow generation technique.

Novel approach to investigate a source of microbial contamination of central venous catheters.

The potential route of contamination by skin microorganisms onto the distal tip of central venous catheters during insertion was investigated. Thirty patients undergoing cardiac surgery who required a central venous catheter (CVC) as part of their clinical management were studied. Following catheter placement, the device insertion equipment and the skin at the insertion site were sampled for microorganisms. The distal tips of the CVCs were also sampled in situ within 90 min post insertion. Bacteria were isolated from 20 of 30 (66%) CVC skin insertion sites, from 15 of 30 (50%) guidewires, and from five of 30 (16%) catheter distal tips in situ. These findings suggest that despite rigorous skin disinfection and strict aseptic technique, viable microorganisms are impacted during insertion onto the distal tip of the CVC, which may act as a subsequent nidus of infection.

Traumatic disruption of the thoracic aorta: a rational approach to imaging.

An imaging strategy is crucial in patients who have sustained a traumatic disruption of the thoracic aorta. Of those who reach hospital alive, 70-90 per cent will survive if diagnosed early and treated appropriately. The clinician has many imaging techniques to choose from, but they vary considerably in their degree of accuracy and performance time. Consequently their appropriateness is dependent on the type of injury suspected, the haemodynamic stability of the patient and the availability and experience of the radiologists. This article describes the types and presentation of traumatic thoracic aortic disruption so that the advantages and disadvantages of the various imaging modalities can be explained. It concludes by presenting an imaging strategy for use when this condition is suspected.