Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.     

Laboratory studies in cross-species lung transplantation

The lack of sufficient suitable human donor lungs for the many patients requiring pulmonary transplantation as life-saving therapy for end-stage lung diseases has generated extensive interest in cross-species lung transplantation. Ethical concerns and those of animal rights advocates have prompted studies of nonprimate species as potential solid organ donors for humans. This paper provides an overview of some of the laboratory studies of cross-species pulmonary transplantation performed over the past 20 years and focuses, in particular, on more recent work (from our laboratory and others) in the area of porcine-to-primate pulmonary xenotransplantation.     

Effects of Plasmodium berghei infection on arteether metabolism and disposition

Arteether (AE) is primarily deethylated to dihydroqinghaosu (DQHS) in rats and humans. Conversion of AE to DQHS was impaired in microsomes from rats infected with Plasmodium berghei. The Km for AE was 175.1 +/- 49.1 and 124.4 +/- 115.1 mumol/l, and Vmax was 2.24 +/- 0.45 and 1.22 +/- 0.67 nmol AE formed/mg protein/min in control and infected microsomes (p < 0.05), respectively. Calculated intrinsic clearance (CLint = initial Vmax/Km) for AE was only 4% lower in infected microsomes. Apparent pharmacokinetic parameter estimates for AE using the isolated perfused rat liver demonstrated no differences (p > 0.05) in volume of distribution, clearance, and half-life between normal and infected animals. Malaria infection resulted in decreased biliary excretion of free AE and DQHS. The majority of AE is eliminated via biliary excretion of conjugated DQHS, which is approximately 500-fold higher than free DQHS and 75-fold higher than free AE on a molar basis.     

Age and ethnicity affect the risk and outcome of focal segmental glomerulosclerosis

In patients with proteinuria, African-American (AA) ethnicity is reported to be a risk factor for focal segmental glomerulosclereosis (FSGS) and its progression to end-stage renal disease (ESRD). We reviewed our single-center experience to determine the probability of FSGS and its progression to ESRD based on ethnicity and age at presentation in children with proteinuria with or without nephrotic syndrome. Proteinuria without systemic disease or acute glomerulonephritis was the presenting feature in 17% (236/1,403) of children in the renal patient database of Texas Children's Hospital, Baylor College of Medicine. Histopathological diagnoses were established in 107 of 236 patients (45%). FSGS was identified in 65 patients, accounting for 28% of all patients with proteinuria and 61% of patients who underwent renal biopsy. FSGS was more prevalent in AA (45%) than in non-AA patients (22%) (P=0.001), and AA patients with FSGS were older at presentation (12.7+/-4.4 years) than non-AA patients (5.6+/-4.6 years) (P<0.001). Among patients who underwent renal biopsy, increasing age at presentation increased the probability of having FSGS in AA but not non-AA patients (P=0.04). Five-year actuarial renal survival of FSGS was worse in AA (8%) than in non-AA patients (31%) (P=0.01). These data suggest an increased risk and worse outcome of FSGS in AA compared with non-AA children.     

Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, β1, β2, β3, and phospholemman. The α1, α2, β1, and β2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.     

Interactions between immunoglobulin-like and catalytic modules in Clostridium thermocellum cellulosomal cellobiohydrolase CbhA

Cellobiohydrolase CbhA from Clostridium thermocellum cellulosome is a multi-modular protein composed starting from the N-terminus of a carbohydrate-binding module (CBM) of family 4, an immunoglobulin(Ig)-like module, a catalytic module of family 9 glycoside hydrolases (GH9), X1(1) and X1(2) modules, a CBM of family 3 and a dockerin module. Deletion of the Ig-like module from the Ig-GH9 construct results in complete inactivation of the GH9 module. The crystal structure of the Ig-GH9 module pair reveals the existence of an extensive module interface composed of over 40 amino acid residues of both modules and maintained through a large number of hydrophilic and hydrophobic interactions. To investigate the importance of these interactions between the two modules, we compared the secondary and tertiary structures and thermostabilities of the individual Ig-like and GH9 modules and the Ig-GH9 module pair using both circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). Thr230, Asp262 and Asp264 of the Ig-like module are located in the module interface of the Ig-GH9 module pair and are suggested to be important in 'communication' between the modules. These residues were mutated to alanyl residues. The structure, stability and catalytic properties of the native Ig-GH9 and its D264A and T230A/D262A mutants were compared. The results indicate that despite being able to fold relatively independently, the Ig-like and GH9 modules interact and these interactions affect the final fold and stability of each module. Mutations of one or two amino acid residues lead to destabilization and change of the mechanism of thermal unfolding of the polypeptides. The enzymatic properties of native Ig-GH9, D264A and T230A/D262A mutants are similar. The results indicate that inactivation of the GH9 module occurs as a result of multiple structural disturbances finally affecting the topology of the catalytic center.     

Designing smarter touch-based interfaces for educational contexts

In next-generation classrooms and educational environments, interactive technologies such as surface computing, natural gesture interfaces, and mobile devices will enable new means of motivating and engaging students in active learning. Our foundational studies provide a corpus of over 10,000 touch interactions and nearly 7,000 gestures collected from nearly 70 adults and children aged 7 years and above, which can help us understand the characteristics of children’s interactions in these modalities and how they differ from adults. Based on these data, we identify key design and implementation challenges of supporting children’s touch and gesture interactions, and we suggest ways to address them. For example, we find children have more trouble successfully acquiring onscreen targets and having their gestures recognized than do adults, especially the youngest age group (7–10 years old). The contributions of this work provide a foundation that will enable touch-based interactive educational apps that increase student success.     

Structural study of titanium oxide films synthesized by ion beam-assisted deposition

The application of titanium dioxide (TiO(2)) films as surgical implant coatings for antibiotic attachment depends crucially on their available surface area and thus their surface morphology and crystallinity. Here, we report our fabrication of high Wenzel ratio TiO(2) films targeted to increase the film surface area using the ion beam-assisted deposition (IBAD) technique at high-deposition temperatures (approximately 610 degrees C). The modulation of the films' surface morphology was accomplished by varying the chemical identity of the concurrent ion beams bombarded on the films during the e-beam evaporation process. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were employed to investigate the surface morphology of the as-deposited films. X-ray diffractometry (XRD) revealed that these nanocrystalline films primarily consist of anatase phase TiO(2). Wenzel ratio, the ratio of the actual surface area to the projected area, of IBAD films prepared with argon, oxygen, and nitrogen ion beams was measured to be 1.52, 1.31 and 1.49, respectively. The effect of the differences in chemical reactivity and ion size of these three type ion beams are discussed to explain the present results.     

Evolution of the cranial computed tomography scan in child abuse

Computed tomography (CT) scans obtained at the time of clinical presentation have occasionally been reported to be normal in children with history and findings of significant abusive head injury. We have retrospectively observed abnormalities in "normal" scans of some similar children. We have also seen abnormalities develop on serial scanning. To determine how frequently these situations occur, we reviewed charts of 34 children with a final diagnosis of child abuse who also had cranial CT scans performed. Their CT scans were retrospectively reviewed by a pediatric radiologist. Eleven (11/34) CT scans had initially been interpreted as normal. Four (4/11) of these had been reinterpreted during the hospitalization as abnormal, affecting medical (1) and legal (3) outcome. Repeat scanning in three of the remaining seven resulted in surgical drainage of a subdural effusion (1) and affected legal outcome (2). Four of the seven initial scans felt normal throughout the hospitalizations were judged abnormal on retrospective review. This evaluation was confirmed in the two rescanned. Initial CT interpretation most often failed to appreciate changes in parenchymal density and small amounts of falcine or cortical subdural blood. Subsequent scans also showed evolving effusions and infarcts. Changes were noted in 1 1/2 to 5 days. The CT scan frequently shows subtle changes in the immediate posttrauma period. If the child does not recover promptly, subsequent scans frequently result in significant changes in clinical and legal management.     

Heat conduction by liquid helium II in capillary tubes. IV. Mutual friction under pressure

We report measurements of supercritical heat conduction in liquid helium II at pressures above the vapor pressure. The measurements were made in glass capillary tubes of diameter 50.5 and 366.8 m in the temperature range from 1.2 to 2.1 K. As in our previous results at the vapor pressure, a clear distinction can be made between two regimes in which the average separation of vortex lines is either comparable with or larger than the size of the flow channel. In the wider tube, at high relative velocities v between the superfluid and normal component, where the vortex spacing is small, a modified form of Gorter-Mellink mutual friction F _sn is observed which varies as F_sn=A_snv(v–v₀)². The coefficient A increases with pressure, and its variation with T/T(p) is in fairly good agreement with Schwarz ' microscopic theory of superfluid turbulence. However, the absolute magnitude of A is in general larger than in Schwarz ' theory and also larger than most other experimental values. In the narrower tube the measurements are all in the regime where the vortex spacing is comparable with the tube size, and they do not agree with the predictions of the Gorter-Mellink law. At present, the data cannot be analyzed quantitatively in the terms of the vortex line density, because B, the vortex line scattering coefficient, is not known at high pressures.Preparation of this paper was supported by a U.S. National Science Foundation grant DMR 7901073 and by a UK Science Research Council Senior Fellowship (DFB).SRC Senior Fellow.