A unique case of acute embolus in a renal transplant with salvage by catheter-directed thrombolysis.

INTRODUCTION: Acute renal transplant emboli can be disastrous and result in loss of the renal transplant. This case was successfully treated with thrombolysis. CASE PRESENTATION: A 66-year-old female underwent a right-sided deceased heart-beating donor renal transplant. She had excellent transplant function but presented acutely three years later with pain in the transplanted kidney, an acute rise in serum creatinine and new onset atrial fibrillation. Bedside ultrasound scan demonstrated absent transplant perfusion. Emergency angiogram confirmed acute emboli in the transplant renal artery with some kidney perfusion. Thrombolysis with alteplase and anticoagulation with heparin was commenced. Serial imaging at 24 and 36 h demonstrated significant improvement in transplant perfusion. Following a period of supportive therapy, her transplant function recovered, although not to pre-morbid baseline levels. CONCLUSION: Consider acute embolus in a renal transplant patient with acute kidney injury, transplant tenderness and cardiac arrhythmia. Early thrombolysis may salvage renal transplants and good transplant function may be regained.

The effect of piezoelectric ultrasonic instrumentation on titanium discs: a microscopy and trace elemental analysis in vitro study.

AIM: To evaluate in vitro topographical and composition changes by piezoelectric ultrasonic instrumentation with metallic and plastic tips on machined and moderately roughened titanium surfaces. METHODS: Twenty machined and moderately roughened laser-marked titanium discs were ultrasonically instrumented with metallic and plastic tips. Surface instrumentation was carried out with controlled pressure for 20 and 30 seconds at two power settings. For each time and power setting, instrumentation was repeated four times with one instrumentation per disc quadrant. Surface topography analysis was performed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Surface roughness measurements were compared between instrumented and non-instrumented surfaces. Surface element composition and rinsing solutions were evaluated using energy-dispersive spectroscopy (EDS) and trace elemental analysis using inductively coupled plasma mass spectrometry (ICPMS), respectively. RESULTS: SEM photomicrographs and CLSM 3D surface plot images of instrumented machined and moderately roughened surfaces demonstrated severe surface topographical alterations with metallic tips and mild to moderate changes for plastic tip instrumented sites. ICPMS analysis of the rinsing solutions identified titanium and other metal traces with the use of metallic tips, and mainly titanium and carbon when plastic tips were used. Surface EDS analysis showed elemental traces of the ultrasonic tips. CONCLUSION: Ultrasonic instrumentation with metallic or plastic tips created surface topographical and compositional changes. Different changes in surface topography were noted between the surfaces, as the roughness of the machined surfaces increased while the extent of roughness of the moderately roughened surfaces decreased. The clinical relevance of these changes is yet to be determined.

Validation of the Global Rating Scale for endoscopy.

The Global Rating Scale for endoscopy is a web-based tool that can be used to assess and improve the quality of an endoscopy service. It was developed by asking endoscopy health professionals what they would want from the service for themselves or their relatives if they were undergoing an endoscopic procedure. To date, the Global Rating Scale has not been validated by patients themselves. We used focus groups in order to access the views and opinions of patients who had recently had experience of endoscopy services. Six focus groups were undertaken in five different Health Board areas across Scotland; in total 26 people participated. The results indicated that from the patients' perspective the 12 items of the GRS covered all areas of the endoscopy experience. There were no specific concerns identified that were not already covered within the Global Rating Scale. We conclude that the Global Rating Scale does address quality issues that matter to patients undergoing endoscopy, and validates the use of the GRS as a quality assessment tool for endoscopy services.

Consistency as tool to support in vitro batch potency testing in GMP production.

There is great interest in the veterinary vaccine field to move away from in vivo release tests for vaccines to reduce cost and testing time, improve consistency and of course the 3Rs (reduce, refine, replace). A brief overview of Good Manufacturing Practice (GMP) and the consistency approach is discussed below and an overview of how manufacturers can use the consistency approach and GMP controls along with statistical analysis of processes at each stage of the production process (starting materials, antigen and finished product) to build in quality and reduce the need for in vivo finished product tests. A final summary and outline of some challenges we will face in moving this approach forward is covered in conclusion.

Does the trace element composition of brown trout Salmo trutta eggs remain unchanged in spawning redds?

The temporal stability of trace element concentrations in fertilized, artificially incubated anadromous brown trout Salmo trutta eggs and newly hatched fry was investigated. The anadromous status of the parental fish was confirmed using strontium isotopic analysis of otoliths. Whilst manganese concentrations in eggs varied over time, concentrations of aluminium, potassium, magnesium, strontium, barium and calcium were all unchanged 1 week and 6 weeks post-fertilization as well as in recently hatched larvae. The results clearly suggest that the distinctive trace element signature present in the eggs and newly hatched larvae of anadromous S. trutta (typically characterized by high strontium, low barium) is stable over time. Therefore analysis of the trace element composition of eggs is concluded to be a cost-effective and reliable method for determining the spatial and temporal extent of upstream spawning migration by anadromous salmonids. The temporal variability of at least one element in this study suggests the stability of untested multi-element signatures cannot automatically be assumed.

A bioavailable strontium (87Sr/86Sr) isoscape for Aotearoa New Zealand: Implications for food forensics and biosecurity.

As people, animals and materials are transported across increasingly large distances in a globalized world, threats to our biosecurity and food security are rising. Aotearoa New Zealand is an island nation with many endemic species, a strong local agricultural industry, and a need to protect these from pest threats, as well as the economy from fraudulent commodities. Mitigation of such threats is much more effective if their origins and pathways for entry are understood. We propose that this may be addressed in Aotearoa using strontium isotope analysis of both pests and products. Bioavailable radiogenic isotopes of strontium are ubiquitous markers of provenance that are increasingly used to trace the origin of animals and plants as well as products, but currently a baseline map across Aotearoa is lacking, preventing use of this technique. Here, we have improved an existing methodology to develop a regional bioavailable strontium isoscape using the best available geospatial datasets for Aotearoa. The isoscape explains 53% of the variation (R2 = 0.53 and RMSE = 0.00098) across the region, for which the primary drivers are the underlying geology, soil pH, and aerosol deposition (dust and sea salt). We tested the potential of this model to determine the origin of cow milk produced across Aotearoa. Predictions for cow milk (n = 33) highlighted all potential origin locations that share similar 87Sr/86Sr values, with the closest predictions averaging 7.05 km away from their true place of origin. These results demonstrate that this bioavailable strontium isoscape is effective for tracing locally produced agricultural products in Aotearoa. Accordingly, it could be used to certify the origin of Aotearoa's products, while also helping to determine if new pest detections were of locally breeding populations or not, or to raise awareness of imported illegal agricultural products.

Severe statin-induced rhabdomyolysis mimicking Guillain-Barré syndrome in four patients with diabetes mellitus treated with fusidic acid.

BACKGROUND: An interaction between fusidic acid and HMG coenzyme A reductase inhibitors (statins), resulting in rhabdomyolysis, has been described. Pain and mild weakness are common presenting symptoms. CASE REPORT: We report four patients with Type 2 diabetes prescribed long-term statin treatment who, following treatment with fusidic acid, presented atypically with painless, severe flaccid paralysis suggestive of Guillain-Barré syndrome. This, together with nerve conduction studies consistent with Guillain-Barré syndrome, resulted in the delayed recognition of rhabdomyolysis in these cases. CONCLUSIONS: The addition of fusidic acid can precipitate rhabdomyolysis in patients with diabetes already taking a statin. This can present with rapidly progressive weakness resembling Guillain-Barré syndrome. We recommend that creatine kinase is checked in patients with diabetes on statin therapy who present with profound weakness and routinely in those commenced on prolonged courses of fusidic acid.

Ssh1p determines the translocation and dislocation capacities of the yeast endoplasmic reticulum.

Sec61p is required both for protein translocation and dislocation across the membrane of the endoplasmic reticulum (ER). However, the cellular role of the Sec61p homolog Ssh1p has not been clearly defined. We show that deltassh1 mutant cells have strong defects in both SRP-dependent and -independent translocation. Moreover, these cells were also found to be induced for the unfolded protein response and to be defective in dislocation of a misfolded ER protein. In addition, deltassh1 mutant cells rapidly became respiratory deficient. The other defects discussed above were suppressed in the respiratory-deficient state or under conditions where the rate of polypeptide translation was artificially reduced. These data identify Ssh1p as a component of a second, functionally distinct translocon in the yeast ER membrane.

Protein translocation across membranes: common themes in divergent organisms.

Specific signal sequences are required for the translocation of proteins into and across both the endoplasmic reticulum of eukaryotes and the plasma membrane of prokaryotes. The similar properties of these signals, together with their ability to function when transferred between systems, suggested that the mechanisms of translocation in the two cases may be fundamentally similar. Indeed, recent findings have revealed striking similarities between essential components of the prokaryotic and eukaryotic translocation systems, suggesting that both are derived from a common ancestor.

A novel subfamily of Hsp70s in the endoplasmic reticulum.

The endoplasmic reticulum contains a number of proteins involved in the processing of secretory polypeptides. These include BiP, which is an Hsp70-family member highly conserved throughout evolution. BiP is known to be intimately involved in several aspects of protein biogenesis, but our understanding of these events has been complicated by the recent description of a novel Hsp70-related protein in yeast, Lhauthorp, whose functions overlap with those of BiP. Current indications are that this protein is distributed widely among eukaryotes and that it represents a distinct subfamily of the Hsp70 class of molecular chaperones.

Radioautographic localization of sodium pump sites in rabbit intestine.

Direct demonstration of the cellular location of sodium pumping constitutes a key problem in the solution of intestinal sodium absorption. Utilizing silicone-impregnated epoxy sections of freeze-dried, osmium-fixed tissue, ouabain-(3)H and inulin-(3)H light microscope radioautographs have been produced which show that: lateral but not brush border membranes of rabbit small intestine bind ouabain-(3)H (high specific activity) with an affinity so great that a subsequent washing in ouabain-free medium has little effect on binding; lateral membrane binding is not apparent with low specific activity ouabain-(3)H, and inulin-(3)H and ouabain-(3)H (low specific activity) in the cores of the villi do not equilibrate with the intercellular spaces. Preliminary tracer measurements of ouabain-(3)H and inulin-(14)C spaces also agree with these findings As ouabain is a specific inhibitor of active sodium transport, these observations provide direct support for the view that lateral membrane pumping of sodium into the intercellular spaces causes, through osmotic forces on water, a flow of fluid out of these spaces into the interstitium.

High-resolution radioautography of phlorizin-3H in rings of hamster intestine.

Quantitative light microscope radioautographs of galactose-(3)H and phlorizin-(3)H were prepared from freeze-dried plastic-embedded hamster small intestine incubated in vitro. The usual uphill epithelial cell accumulation of galactose accompanied by a somewhat smaller lamina propria accumulation was observed in control tissue incubated 3 min in 1 mM galactose-(3)H. The addition of 5 x 10(-4)M phlorizin to the medium blocked uphill accumulation, but did not prevent galactose equilibration with the epithelial cells. The galactose content of the lamina propria was considerably less than the galactose content of the epithelial cell. Varying the phlorizin-(3)H content of the medium from 0.6 to 60 microM revealed a brush border binding of phlorizin which followed a Langmuir adsorption isotherm with a half-saturation constant of 13 microM and a maximum binding of 84 micromoles of phlorizin/liter of microvilli or 2.6 x 10(6) sites/epithelial cell. The phlorizin content of the epithelial cell compartment, excluding microvilli, never exceeded 10% that of the medium after 20 min of incubation. These findings directly support the view that phlorizin is a nontransported inhibitor which binds glucose-galactose carriers at the surface of epithelial cell microvilli.

Ouabain binding to renal tubules of the rabbit.

It is well known that ouabain, a specific inhibitor of Na-K ATPase-dependent transport, interferes with renal tubular salt reabsorption. In this study, we employed radiochemical methods to measure the kinetics of [3H]ouabain binding to slices of rabbit renal medulla and high resolution quantitative autoradiography to determine the location and number of cellular binding sites. The kinetics obeyed a simple bimolecular reaction with an association constant of 2.86 +/- 0.63 SD x 10(3) M-1 min-1 and a dissociation constant of 1.46 x 10(-3) min-1, yielding an equilibrium binding constant of 0.51 x 10(-6) M. Binding was highly dependent upon temperature. At a concentration of 10(-6) M, the rate of accumulation between 25 degrees C and 35 degrees C exhibited a Q10 of 1.8. At 0 degree C the rate of ouabain dissociation was negligible. The specificity of binding was demonstrated with increasing potassium concentrations. At a concentration of 1 microM, 6 mM, and 50 mM K+ produced a 2.5- and 7-fold decrease, respectively, in the rate of ouabain accumulation observed at zero K+. Binding was completely inhibited by 1 mM strophanthin K. The major site of ouabain binding was the thick ascending limb; little or no binding was observed in thin limbs and collecting ducts. Moreover, binding was confined to the basolateral membranes. From autoradiographic grain density measurements, it was estimated that each cell contains over 4 x 10(6) ouabain binding sites or Na-K ATPase molecules. These results taken together with physiological and biochemical observations suggest that Na-K ATPase plays a key role in salt reabsorption by this segment.

[3H]ouabain autoradiography of frog retina.

The kinetics and distribution of ouabain binding in retinas of Rana pipiens were examined quantitatively by scintillation counting and freeze-dry autoradiography. The time-course of binding at several concentrations was consistent with a bimolecular reaction. Estimated equilibrium binding levels gave a Michaelis-Menton relationship with a Km = 8.3 x 10(-8) M and a maximum binding level (Bmax) = 4.4 x 10(-8) mol/g protein. The distribution of binding sites measured autoradiographically varied considerably between layers. The photoreceptor, inner plexiform, and optic nerve fiber layers exhibited the heaviest binding. Within the photoreceptor layer, binding was nonuniform. Binding in the outer segment decreased distally, averaging approximately 4% of that in the proximal receptor layers (Bmax = 4.6 x 10(-6) M). The origin of the outer segment activity is uncertain at light microscope resolution, as it may be a result of inner segment calyceal processes. Binding within the proximal receptor layers was also nonuniform. Several peaks were observed, with those at the inner segment and synaptic layers being especially noticeable. Assuming an absence of glial cell binding in the proximal receptor layers, we calculated there to be 13 x 10(6) ouabain or Na+,K+ pump sites per rod receptor. Limited measurements suggest a Bmax of approximately 8 x 10(-6) M for the inner plexiform layer.

High-resolution radioautography of galactose-3H accumulation in rings of hamster intestine.

Radioautography of water-soluble substances has posed a major technical problem for the past decade. Utilizing silicone-impregnated plastic sections of frozen-dried tissue, a quantitative method was developed for studying distribution of (3)H-labeled galactose, mannitol, and phlorizin. The content of a 2-micro band may be measured with an accuracy of +/-20% by light microscopy; radioautographs may also be prepared for the electron microscope. Results with intestinal tissue incubated 1-10 min in vitro and, then, frozen rapidly indicate that the first step in galactose absorption is uphill transport into the brush border of the columnar epithelium. Correction of galactose content for the mannitol space in the brush border suggests that the sugar pump is located at the surface of the microvilli. Further evidence for the surface locus of the glucose-galactose pump was obtained with phlorizin (next paper, reference 40). The galactose content of columnar cell cytoplasm always equalled that of microvilli and no transcellular diffusion gradient could be detected; during the first minutes of incubation, however, a gradient did exist between nucleoplasm and cytoplasm. Downhill exit of galactose from columnar cells may have proceeded either directly across basal membranes to adjacent lamina propria or indirectly via open intercellular spaces. Lastly, even in the absence of muscularis, the connective tissue of the lamina propria constituted enough of a diffusion barrier so that it served as a secondary accumulating compartment for galactose under present in vitro conditions.

Teleost chloride cell. II. Autoradiographic localization of gill Na,K-ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments.

The specific binding and inhibitory action of (3H)ouabain were employed to localize transport Na,K-ATPase in the euryhaline teleost gill, a NaCl-transporting osmoregulatory tissue in which both enzyme activity and transepithelial transport vary with environmental salinity. In killifish fully adapted to 10%, 100%, or 200% seawater, the gills were internally perfused and externally irrigated in situ. After suitable internal or external exposure to (3H)ouabain, individual gill arches were excised for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative high-resolution autoradiography. Internal exposure to 50 muM ouabain resulted in essentially complete enzyme inhibition, and binding paralleled the increases in enzyme activity at higher salinities; in contrast, external exposure gave minimal and erratic results consistent with leakage of external ouabain into interstitial fluid. (3H)Ouabain autoradiographs demonstrated that, irrespective of exposure or salinity, most of the gill binding was associated with chloride cell. These cells increased in size and number with salinity and, at the subcellular level, the distribution pattern for bound ouabain was always identical to that for the amplified basal-lateral (tubular system) membrane. The combined physiologicmorphologic results constitute final direct proof that chloride cells are the primary site of gill Na,K-ATPase. More important, they provide convincing evidence for unexpected increases in basal-lateral enzyme at higher salinities and thus raise a fundamental objection to the long-postulated role of the Na pump in secretory NaCl transport.

The Hsp70 homologue Lhs1p is involved in a novel function of the yeast endoplasmic reticulum, refolding and stabilization of heat-denatured protein aggregates.

Heat stress is an obvious hazard, and mechanisms to recover from thermal damage, largely unknown as of yet, have evolved in all organisms. We have recently shown that a marker protein in the ER of Saccharomyces cerevisiae, denatured by exposure of cells to 50 degrees C after preconditioning at 37 degrees C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24 degrees C. Here we show that refolding of the marker enzyme Hsp150Delta-beta-lactamase, inactivated and aggregated by the 50 degrees C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of Lhs1p, Hsp150Delta-beta-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Delta- beta-lactamase, whereas no association with native marker protein molecules could be detected. Similar findings were obtained for a natural glycoprotein of S. cerevisiae, pro-carboxypeptidase Y (pro-CPY). Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150Delta-beta-lactamase or pro-CPY, suggesting that the machinery repairing heat-damaged proteins may have specific features as compared to chaperones assisting de novo folding. After preconditioning and 50 degrees C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24 degrees C, but only 10% were able to form colonies, as compared to wild-type cells. We suggest that Lhs1p is involved in a novel function operating in the yeast ER, refolding and stabilization against proteolysis of heatdenatured protein. Lhs1p may be part of a fundamental heat-resistant survival machinery needed for recovery of yeast cells from severe heat stress.

Orbital forcing of the marine isotope stage 9 interglacial.

Milankovitch orbital forcing theory has been used to assign time scales to many paleoclimate records. However, the validity of this theory remains uncertain, and independent sea-level chronologies used to test its applicability have been restricted largely to the past approximately 135,000 years. Here, we report U-series ages for coral reefs formed on Henderson Island during sea-level high-stands occurring at approximately 630,000 and approximately 330,000 years ago. These data are consistent with the hypothesis that interglacial climates are forced by Northern Hemisphere summer solar insolation centered at 65 degrees N latitude, as predicted by Milankovitch theory.

The yeast CDC9 gene encodes both a nuclear and a mitochondrial form of DNA ligase I.

BACKGROUND: The yeast CDC9 gene encodes a DNA ligase I activity required during nuclear DNA replication to ligate the Okazaki fragments formed when the lagging DNA strand is synthesised. The only other DNA ligase predicted from the yeast genome sequence, DNL4/LIG4, is specifically involved in a non-homologous DNA end-joining reaction. What then is the source of the DNA ligase activity required for replication of the yeast mitochondrial genome? RESULTS: We report that CDC9 encodes two distinct polypeptides expressed from consecutive in-frame AUG codons. Translational initiation at these two sites gives rise to polypeptides differing by a 23 residue amino-terminal extension, which corresponds to a functional mitochondrial pre-sequence sufficient to direct import into yeast mitochondria. Initiation at the first AUG codon results in a 755 amino-acid polypeptide that is imported into mitochondria, whereupon the pre-sequence is proteolytically removed to yield the mature mitochondrial form of Cdc9p. Initiation at the second AUG codon produces a 732 amino-acid polypeptide, which is localised to the nucleus. Cells expressing only the nuclear isoform were found to be specifically defective in the maintenance of the mitochondrial genome. CONCLUSIONS: CDC9 encodes two distinct forms of DNA ligase I. The first is targeted to the mitochondrion and is required for propagation and maintenance of mitochondrial DNA, the second localises to the nucleus and is sufficient for the essential cell-division function associated with this gene.

Quantitative radioautography of sugar transport in intestinal biopsies from normal humans and a patient with glucose-galactose malabsorption.

Both galactose accumulation and phlorizin binding by columnar epithelial cells have been investigated in vitro with a recently developed technique for high-resolution, plastic-section radioautography which is particularly suited to small quantities of biopsy tissue. Grain density analysis of the radioautographs provides definitive support for the view that the cellular mechanisms underlying glucose-galactose absorption in laboratory animals are fully applicable to the small intestine of man. Even the number of sugar carriers at the microvillar membrane appears similar and the major quantitative difference, lower affinity for phlorizin in man, correlates with the finding that phlorizin is also a less potent inhibitor of uphill, galactose transport at the microvilli. In addition, radioautographs of biopsies taken 2 yr apart from a patient with glucose-galactose malabsorption provide evidence that the cellular defect in this inborn error of transport is a persistent reduction in the number of functioning sugar carriers at the microvillar membrane.