Ceruloplasmin receptors in liver cell suspensions are limited to the endothelium

The interaction of ceruloplasmin (CP) with isolated liver cell suspensions was studied using 125I-labeled and latex minibead-derivatized CP. Fractionation of liver cell suspensions was done using metrizamide gradient centrifugation. In crude liver cell suspensions only endothelial cells, but not hepatocytes and Kupffer cells bound the minibead probe. The binding was specific and inhibited by excess native CP. These results were confirmed using 125I-CP combined with cell fractionation technique. Kinetic data, obtained from the latter system, indicated a dissociation constant (Kd) of 1 X 10(-7) M and the number of receptors to be 5.7 X 10(5) per endothelial cell. The exclusive binding of CP to liver endothelium suggests that this cell may mediate the hepatocytes uptake of CP and is, therefore, a crucial element of the tissue-blood barrier.     

Calcium-induced compaction and its inhibition in embryonal carcinoma cell aggregates

H6 embryonal carcinoma cells form aggregates of cells in culture medium which contains 2 mM calcium. These aggregates are described as uncompacted, indicating that the individual cells of the aggregate are spherical and are in limited contact with each other. In contrast, compaction of the aggregate, induced by increasing the calcium concentration, results in a tight mass of cells flattened against one another and connected by intercellular junctions. At least 85-97% of the aggregates undergo compaction in 7 mM calcium and are subsequently decompacted if removed to 2 mM calcium. Since calcium ionophore A23187 does not induce compaction, extracellular rather than intracellular calcium seems to be the limiting factor. We have demonstrated that this calcium-induced morphogenetic change is sensitive to inhibition by agents which also prevent the calcium-dependent compaction of the 8-cell mouse embryo. The cytoskeletal-binding drugs tetracaine HCl, colcemid, vinblastine, colchicine, and cytochalasin B each inhibit compaction of H6 aggregates. Interference at surface molecule sites by exposure to the lectins wheat germ agglutinin or concanavalin A or by interruption of glycosylation with exposure to tunicamycin, or by reaction with anti-H6 Fab or anti-F9, also prevent compaction. Since the mouse embryo and embryonal carcinoma cells share certain processes which are involved in initiating and maintaining compaction, these processes and their subsequent roles in differentiation may be examined using embryonal carcinoma cell aggregates.     

Human Y-chromatin : I. Dispersion and condensation

The fluorescent Y-body was observed to manifest striking variations in morphology in interphase lymphocyte and fibroblast nuclei. These variations appeared to be largely independent of the specific fixation or hypotonic treatment. The Y-chromatin body ranged from a highly condensed mass to a constellation of smaller discrete particles. Dispersion of Y-chromatin was not seen in mitotically inactive cells from buccal mucosa and hair root sheaths. Comparison of uncultured and cultured lymphocytes confirmed a higher proportion of dispersed Y-bodies in cultured cells, particularly those with a relatively large mean nuclear diameter. In serially cultured lymphocytes, the frequency of Y-body dispersion showed a direct relationship to the time in culture. Mean nuclear diameter also increased directly with time in culture. In cultured fibroblasts dispersion of the Y-body increased during the logarithmic phase of growth and declined during the post-logarithmic phase.     

Analysis of Chemopredictive Assay for Targeting Cancer Stem Cells in Glioblastoma Patients

Introduction: The prognosis of glioblastoma (GBM) treated with standard-of-care maximal surgical resection and concurrent adjuvant temozolomide (TMZ)/radiotherapy remains very poor (less than 15 months). GBMs have been found to contain a small population of cancer stem cells (CSCs) that contribute to tumor propagation, maintenance, and treatment resistance. The highly invasive nature of high-grade gliomas and their inherent resistance to therapy lead to very high rates of recurrence. For these reasons, not all patients with similar diagnoses respond to the same chemotherapy, schedule, or dose. Administration of ineffective anticancer therapy is not only costly but more importantly burdens the patient with unnecessary toxicity and selects for the development of resistant cancer cell clones. We have developed a drug response assay (ChemoID) that identifies the most effective chemotherapy against CSCs and bulk of tumor cells from of a panel of potential treatments, offering great promise for individualized cancer management. Providing the treating physician with drug response information on a panel of approved drugs will aid in personalized therapy selections of the most effective chemotherapy for individual patients, thereby improving outcomes. A prospective study was conducted evaluating the use of the ChemoID drug response assay in GBM patients treated with standard of care. Methods: Forty-one GBM patients (mean age 54 years, 59% male), all eligible for a surgical biopsy, were enrolled in an Institutional Review Board–approved protocol, and fresh tissue samples were collected for drug sensitivity testing. Patients were all treated with standard-of-care TMZ plus radiation with or without maximal surgery, depending on the status of the disease. Patients were prospectively monitored for tumor response, time to recurrence, progression-free survival (PFS), and overall survival (OS). Odds ratio (OR) associations of 12-month recurrence, PFS, and OS outcomes were estimated for CSC, bulk tumor, and combined assay responses for the standard-of-care TMZ treatment; sensitivities/specificities, areas under the curve (AUCs), and risk reclassification components were examined. Results: Median follow-up was 8 months (range 3-49 months). For every 5% increase in in vitro CSC cell kill by TMZ, 12-month patient response (nonrecurrence of cancer) increased two-fold, OR = 2.2 (P = .016). Similar but somewhat less supported associations with the bulk tumor test were seen, OR = 2.75 (P = .07) for each 5% bulk tumor cell kill by TMZ. Combining CSC and bulk tumor assay results in a single model yielded a statistically supported CSC association, OR = 2.36 (P = .036), but a much attenuated remaining bulk tumor association, OR = 1.46 (P = .472). AUCs and [sensitivity/specificity] at optimal outpoints (>40% CSC cell kill and >55% bulk tumor cell kill) were AUC = 0.989 [sensitivity = 100/specificity = 97], 0.972 [100/89], and 0.989 [100/97] for the CSC only, bulk tumor only, and combined models, respectively. Risk categorization of patients was improved by 11% when using the CSC test in conjunction with the bulk test (risk reclassification nonevent net reclassification improvement [NRI] and overall NRI = 0.111, P = .030). Median recurrence time was 20 months for patients with a positive (>40% cell kill) CSC test versus only 3 months for those with a negative CSC test, whereas median recurrence time was 13 months versus 4 months for patients with a positive (>55% cell kill) bulk test versus negative. Similar favorable results for the CSC test were observed for PFS and OS outcomes. Panel results across 14 potential other treatments indicated that 34/41 (83%) potentially more optimal alternative therapies may have been chosen using CSC results, whereas 27/41 (66%) alternative therapies may have been chosen using bulk tumor results. Conclusions: The ChemoID CSC drug response assay has the potential to increase the accuracy of bulk tumor assays to help guide individualized chemotherapy choices. GBM cancer recurrence may occur quickly if the CSC test has a low in vitro cell kill rate even if the bulk tumor test cell kill rate is high.     

A simple method for detecting steroid aggregation and estimating solubility in aqueous solutions

Steroids are generally sparingly soluble in water. Thus, for in vitro studies of steroid metabolism or enzymology it is common practice to solubilize steroids by the addition of a small amount (2–10%, v/v) of an organic cosolvent. Methanol, ethanol, and 1,2-propanediol, singly or in combination, have been widely used (1). Effects of organic solvents on the kinetic parameters, K_m and V_max, of steroid-metabolizing enzymes with various substrates have been demonstrated (2,3), and the results are consistent with the conclusion that organic solvent influences on catalytic activity reflect, in part, effects on the aggregation state and solubility of steroid substrates.Light-scattering measurements have been applied extensively in studies of macromolecular structure (4) and micelle formation by a large variety of amphiphilic substances [reviewed in Ref. (5)]. Jones and Gordon (6) used a commercial instrument, designed specifically for light-scattering measurements, to characterize micelle formation in aqueous solutions by Δ5-3-ketosteroids containing various substituents at the 17β position. They showed that turbidity versus concentration plots were of the form seen in studies of micelle formation (5) and that steroids can exist in solution in monomeric or micellar forms, their aggregation state being a function of the polarity of the steroid solute and the composition of the solvent.To estimate solubility quantitatively ³H- or ¹⁴C-labeled steroids have been used in conjunction with centrifugation (3), dialysis (7), or filtration (8). These techniques allow for accurate estimates of solubility, but one may encounter problems due to nonspecific absorption on membranes or the unavailability of the labeled steroid of interest.We have observed that steroid aggregation and solubility can be estimated easily and with high sensitivity with a commercially available fluorometer. In this report the method is described and examples demonstrating the reproducibility and sensitivity of the technique are presented.     

Evaluating the accuracy of geometrical distortion correction of magnetic resonance images for use in intracranial brain tumor radiotherapy

AimDetermine the 1) effectiveness of correction for gradient-non-linearity and susceptibility effects on both QUASAR GRID^3D and CIRS phantoms; and 2) the magnitude and location of regions of residual distortion before and after correction.BackgroundUsing magnetic resonance imaging (MRI) as a primary dataset for radiotherapy planning requires correction for geometrical distortion and non-uniform intensity.Materials and MethodsPhantom Study: MRI, computed tomography (CT) and cone beam CT images of QUASAR GRID^3D and CIRS head phantoms were acquired. Patient Study: Ten patients were MRI-scanned for stereotactic radiosurgery treatment. Correction algorithm: Two magnitude and one phase difference image were acquired to create a field map. A MATLAB program was used to calculate geometrical distortion in the frequency encoding direction, and 3D interpolation was applied to resize it to match 3D T1-weighted magnetization-prepared rapid gradient-echo (MPRAGE) images. MPRAGE images were warped according to the interpolated field map in the frequency encoding direction. The corrected and uncorrected MRI images were fused, deformable registered, and a difference distortion map generated.ResultsMaximum deviation improvements: GRID^3D, 0.27 mm y-direction, 0.07 mm z-direction, 0.23 mm x-direction. CIRS, 0.34 mm, 0.1 mm and 0.09 mm at 20-, 40- and 60-mm diameters from the isocenter. Patient data show corrections from 0.2 to 1.2 mm, based on location. The most-distorted areas are around air cavities, e.g. sinuses.ConclusionsThe phantom data show the validity of our fast distortion correction algorithm. Patient-specific data are acquired in <2 min and analyzed and available for planning in less than a minute.     

Relaxation kinetics of E. coli ribosomes.

Relaxation kinetics measurements on two types of ribosome preparations were parformed by the pressure-jump and temperature-Jump techniques, using light scattered at 90° as detector. For freshly prepared tibosomes isolated as 70S tight coupled from 26 000 RPM sucrose gradint sedimentation in 10 mM Mg²⁺, surprisingly large reaction amplitudes were found in 10 mM Mg²⁺ wilh both techniques, leading to an overall formation constant for 70S couples approximately three orders of magnitude smaller than that reported fot tight couples. For pelleted, two-tunes salt-washed ribosomes, amplitude titration versus Mg²⁺ in the pressure-jump apparatus showed an amplitude maximum near 10 mM Mg²⁺ with a relaxation time near 20 ms, and a second amplitude maximum near 2.5 mM Mg²⁺ with a relaxation time near 25 s. Both types of preparation on reanalysis on sucrose gradients at 5 mM Mg²⁺ showed approximately 15% of subunits, with a distinct zone in the 50S region. 70S light couples recovered from a sucrose density gradient separation at 5 mM Mg²⁺ on pelleted two-times salt-washed ribosomes behaved in the same way as the original sample in pressure-jump experiments at 10 mM Mg²⁺. These findings have been interpreted as follows (I) the processes observed at 10 mM Mg²⁺ are due entirety to the relatively small loose couple content of the samples, even in the case of material isolated as 70S tight couples, (2) the processes observed at 2.5 mM Mg²⁺ are due almost entirely to the preponderant tight couple population of the material, and (3) samples isolated as 70S tight couples from sucrose gradients at 5 mM Mg²⁺ spontaneously revert within hours into micro-heterogeneous material containing about 15% loose couples, for both types of ribosomes.