Functional early endosomes are required for maturation of major histocompatibility complex class II molecules in human B lymphoblastoid cells

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place.</p

The application of biochemical methods involving enzyme assays in the study of certain pathological conditions, studies with isocitrate dehydrogenase and b-glucuronidase

The thesis consists of two separate studies on two enzymes which have potentially useful applications in the biochemical study of various human diseases. In Part 1, the activity of the enzyme, is citrate dehydrogenase, in serum of patients with diseases involving the liver or biliary tract, was studied and compared with established biochemical tests of liver function, in order to assess its application to the differential diagnosis of jaundice. Particular attention was paid to results on patients with obstructive jaundice as previous authors have reached differing conclusions of the ultimate usefulness of this enzyme, due to the equivocal results shown in these cases. Very high values were found in severe, acute breakdown of liver cells. Those coincided with large increases in activity of two other enzymes used in the diagnosis of acute hepatic damage - serum giutamic-oxalacetic transaminase and serum glutamic-pyruvic transaminase. The serum isocitrate dehydrogenase was extremely sensitive in detecting liver cell damage, even at a subclinical level, and was comparable and perhaps slightly superior to the serum transaminases in this respect, while all three enzymes were very much more sensitive than other conventional tests of liver function. In chronic liver damage, variable values for serum isocitrate dehydrogenase were found, with only small to moderate increases which could not be related to the clinical state of the patient. Higher values were found in biliary cirrhosis than in portal cirrhosis. The enzyme results were significantly correlated with the transaminase results, but not with the other liver function tests. In obstructive jaundice, the enzyme was elevated in the serum of over 50% of the cases, and particularly high values were associated with secondary involvement of the liver in malignant oases, and acute inflammation of the biliary tract in benign obstructive jaundice. The enzyme could not be used to differentiate malignant from non-malignant cases, and was not related to the severity or duration of the biliary retention. In these cases, the enzyme was not closely related to serum transaminase values. It was concluded that the enzyme did not offer great advantages in the differential diagnosis of jaundice due to the abnormal values which may be found in obstructive jaundice. However, it was an easily estimated enzyme, very sensitive for detecting and assessing acute damage to liver cells, and could be a useful adjunct or alternative to the serum transaminasess Part R involved the study of the enzyme, p-glucuronidase, in various biological material taken from patients with carcinoma of the cervix uteri. Biopsy specimens of cervical carcinoma wore obtained and fractionated by homogenisation and differential centrifugation into three cytoplasmic fractions - mitochondria, miorosomes and soluble supernatant. p-Glucuronidase activity was measured in each fraction and the results compared with specimens eV non-malignant corvix treated in the ammo way. The activity of the carcinoma specimens was found to be considerably higsher than non-malignant specimens in the majority or caeca. Xneroasee in p-glucurenldase activity were mainly in the soluble supernatant fraction and not in particle enzyme even though tho enoyme had previously boon doocribod no located mainly within cytoplasmic particles. There was no correlation with the enzyme activity and dearie of malignancy of the cervical lesion. Sample of cervical carcinoma were taken before and aft treatment oi the lesion by irradiation from radium implant at and their 0.glucuronidase activity compared. There was not a uniform change in enzyme activity following radiation duo to larao unexplained increases and decreases being observed. The excretion of p-gluecuronidase in urine of patient with cervical carcinoma receiving radiotherapy was followed over the Pu11 course of treatment. Pronounced increase in excretion of the enzyme following radium treatment wove obaorvod in the majority of patients studied and the pattern of 0.61ucuronidaso excretion could be related to the enzyme activity of the tissue being irradiated. There was also increase in p-glucuronidase activity of the serum in these patients, and the results indicated that radiation of the lesion caused mobilisation or the enzyme into the patient circulation, which was then cleared by the kidneys into the urine

Space Suit Portable Life Support System Test Bed (PLSS 1.0) Development and Testing

A multi-year effort has been carried out at NASA-JSC to develop an advanced extra-vehicular activity Portable Life Support System (PLSS) design intended to further the current state of the art by increasing operational flexibility, reducing consumables, and increasing robustness. Previous efforts have focused on modeling and analyzing the advanced PLSS architecture, as well as developing key enabling technologies. Like the current International Space Station Extra-vehicular Mobility Unit PLSS, the advanced PLSS comprises three subsystems required to sustain the crew during extra-vehicular activity including the Thermal, Ventilation, and Oxygen Subsystems. This multi-year effort has culminated in the construction and operation of PLSS 1.0, a test bed that simulates full functionality of the advanced PLSS design. PLSS 1.0 integrates commercial off the shelf hardware with prototype technology development components, including the primary and secondary oxygen regulators, Ventilation Subsystem fan, Rapid Cycle Amine swingbed carbon dioxide and water vapor removal device, and Spacesuit Water Membrane Evaporator heat rejection device. The overall PLSS 1.0 test objective was to demonstrate the capability of the Advanced PLSS to provide key life support functions including suit pressure regulation, carbon dioxide and water vapor removal, thermal control and contingency purge operations. Supplying oxygen was not one of the specific life support functions because the PLSS 1.0 test was not oxygen rated. Nitrogen was used for the working gas. Additional test objectives were to confirm PLSS technology development components performance within an integrated test bed, identify unexpected system level interactions, and map the PLSS 1.0 performance with respect to key variables such as crewmember metabolic rate and suit pressure. Successful PLSS 1.0 testing completed 168 test points over 44 days of testing and produced a large database of test results that characterize system level and component performance. With the exception of several minor anomalies, the PLSS 1.0 test rig performed as expected; furthermore, many system responses trended in accordance with pre-test predictions

Advanced Spacesuit Portable Life Support System Oxygen Regulator Development and Testing

The advanced spacesuit portable life support system (PLSS) oxygen regulators represent an evolutionary approach to regulator development. Several technology development prototypes have been produced that borrow much of the mechanical regulator design from the well proven Shuttle/ISS Extravehicular Mobility Unit (EMU) Secondary Oxygen Regulator, but incorporate a motor-settable pressure set-point feature that facilitates significantly greater operational flexibility. For example, this technology would enable EVA to begin at a higher suit pressure, which would reduce pre-breathe time, and then slowly step down to a lower pressure to increase suit mobility for the duration of the EVA. Comprehensive testing of the prototypes was performed on the component level as well as part of the PLSS 1.0 system level testing. Results from these tests characterize individual prototype performance and demonstrate successful operation during multiple nominal and contingency EVA mode

TLR ligand-induced podosome disassembly in dendritic cells is ADAM17 dependent

Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin–rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling–induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor α–converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this “sheddase” in regulating an actin-based structure

Structural and functional basis for p38-MK2 activated Rsk signalling in Toll-Like receptor-stimulated dendritic cells

Rsk kinases play important roles in several cellular processes such as proliferation, metabolism, and migration. Until recently, Rsk activation was thought to be exclusively initiated by Erk1/2, but in dendritic cells (DC) Rsk is also activated by p38 mitogen-activated protein (MAP) kinase via its downstream substrates, MK2/3. How and why this noncanonical configuration of the MAP kinase pathway is adopted by these key immune cells are not known. We demonstrate that the Erk1/2-activated C-terminal kinase domain of Rsk is dispensable for p38-MK2/3 activation and show that compared with fibroblasts, a greater fraction of p38 and MK2/3 is located in the cytosol of DC prior to stimulation, suggesting a partial explanation for the operation of the noncanonical pathway of Rsk activation in these cells. p38/MK2/3-activated Rsk phosphorylated downstream targets and is physiologically important because in plasmacytoid DC (pDC) stimulated with Toll-like receptor 7 (TLR7) agonists, Erk1/2 activation is very weak relative to p38. As a result, Rsk activation is entirely p38 dependent. We show that this unusual configuration of MAP kinase signaling contributes substantially to production of type I interferons, a hallmark of pDC activation

Intracellular binding sites for nonsteroidal antioestrogens

This thesis is concerned with the properties of intracellular binding sites for nonsteroidal antioestrogens

Assessing and monitoring intratumor heterogeneity in glioblastoma: how far has multimodal imaging come?

Glioblastoma demonstrates imaging features of intratumor heterogeneity that result from underlying heterogeneous biological properties. This stems from variations in cellular behavior that result from genetic mutations that either drive, or are driven by, heterogeneous microenvironment conditions. Among all imaging methods available, only T1-weighted contrast-enhancing and T2-weighted fluid-attenuated inversion recovery are used in standard clinical glioblastoma assessment and monitoring. Advanced imaging modalities are still considered emerging techniques as appropriate end points and robust methodologies are missing from clinical trials. Discovering how these images specifically relate to the underlying tumor biology may aid in improving quality of clinical trials and understanding the factors involved in regional responses to treatment, including variable drug uptake and effect of radiotherapy. Upon validation and standardization of emerging MR techniques, providing information based on the underlying tumor biology, these images may allow for clinical decision-making that is tailored to an individual's response to treatment.Stephen Price is funded by a Clinician Scientist Award from the National Institute for Health Research.This is the author accepted manuscript. The final version is available from Future Medicine via http://dx.doi.org/10.2217/cns.15.2