Densitometry Measurements Research Articles

Proteome Dynamics and Proteome Function of Cardiac 19S Proteasomes

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.

Natural history of corneal haze after collagen crosslinking for keratoconus and corneal ectasia: Scheimpflug and biomicroscopic analysis

To determine the natural history of collagen crosslinking (CXL)-associated corneal haze measured by Scheimpflug imagery and slitlamp biomicroscopy in patients with keratoconus or ectasia after laser in situ keratomileusis. Cornea and refractive surgery subpecialty practice, United States. Prospective randomized controlled clinical trial. The treatment group received ultraviolet-A/riboflavin CXL therapy. The control group received riboflavin alone without epithelial debridement. To objectively measure CXL-associated corneal haze, corneal densitometry using Scheimpflug imagery was measured and the changes in haze were analyzed over time. A similar analysis was performed using clinician-determined slitlamp haze. Correlation of CXL-associated corneal haze with postoperative outcomes was analyzed. The mean preoperative corneal densitometry was 14.9 ± 1.93 (SD) (Pentacam Scheimpflug densitometry units). Densitometry peaked at 1 month (mean 23.4 ± 4.40; P<.001), with little change at 3 months (mean 22.4 ± 4.79; P = .06) and decreased between 3 months and 6 months (19.4 ± 4.48; P<.001) and between 6 months and 12 months. By 12 months, densitometry had not completely returned to baseline in the entire cohort (mean 17.0 ± 3.82; P<.001) and the keratoconus subgroup; however, it returned to baseline in the ectasia group (16.1 ± 2.41; P = .15). The postoperative course of slitlamp haze was similar to objective densitometry measurements. Increased haze, measured by densitometry, did not correlate with postoperative clinical outcomes. The time course of corneal haze after CXL was objectively quantified; it was greatest at 1 month, plateaued at 3 months, and was significantly decreased between 3 months and 12 months. Changes in haze did not correlate with postoperative clinical outcomes.

Diffuse idiopathic skeletal hyperostosis (DISH): relation to vertebral fractures and bone density

SummaryRadiographs and spinal bone mineral density (BMD) were evaluated from 342 elderly men regarding possible effects of diffuse idiopathic skeletal hyperostosis (DISH) on vertebral fractures and densitometry measurements. Prevalent vertebral fractures were more frequent among men with DISH compared to men with no DISH even after fracture prevalence was adjusted for BMD. Paravertebral calcifications should be considered in patients with DISH when interpreting BMD measurements because both dual X-ray absorptiometry (DXA) and quantitative CT (QCT) densitometry may not be reliable.IntroductionThe purpose of this study is to evaluate the prevalence of DISH in older men and its association with vertebral fractures and with BMD determined by DXA and QCT.MethodsLateral radiographs of the spine were analyzed in a sample of 342 men aged ≥65 years participating in the MrOS Study concerning the presence and grade of DISH and vertebral fractures. Lumbar BMD was measured by both DXA (areal, grams per square centimeter) and QCT (volumetric, grams per cubic centimeter). The association between DISH, BMD, and presence of fractures was studied using χ 2 and t tests.ResultsDISH was present in 52% (178/342) of the men. Men with DISH were older (mean, 75.1 vs 73.3, p < 0.05) and more likely to have prevalent fractures (28% vs 20%, p < p = 0.09). BMD assessed with DXA (1.08 vs 1.00 g/cm2, p ≤ 0.0001), but not with QCT (0.11 vs 0.11 g/cm3, p = 0.65), was significantly higher in men with DISH compared to men without DISH. Significantly lower BMD of men with both DISH and fractures compared to men with DISH but without fractures was only detected by QCT (−25%, 0.09 vs 0.12, p < 0.05). Both DXA BMD and QCT BMD were significantly higher in severe lumbar DISH (+22% and +31%, p < 0.0001), respectively.ConclusionDISH was associated with a higher prevalence of vertebral fractures in elderly men. Lumbar ossifications related to DISH should be considered when interpreting BMD measurements to predict their fracture risk.

Radiologic Evaluation of Putty Versus Powder Form of Demineralized Bone Matrix in Sinus Floor Elevation

The aim of this study was to evaluate differences in ossification of the 2 forms of demineralized bone matrix (DBM)-putty and powder-radiographically, using digital densitometry on panoramic radiographs in maxillary sinus floor augmentation procedures. Twelve subjects needing bilateral maxillary sinus floor augmentation for the placement of osseointegrated implants were included in this study. The left and right maxillary sinuses were augmented in the same session in each patient using the 2 preferred forms of DBM-putty and powder-during the same session. One sinus was augmented with DBM putty form and the other sinus was augmented with DBM powder form randomly. Every patient had a total of 4 panoramic radiographs taken, preoperatively and in the first, third, and sixth month postoperatively. The densitometry measurements were taken from each step of the aluminium step-wedge, from both sinuses from different points a total of 3 times, and the average of these measurements was calculated. The amount of mineralization in each graft material in every radiograph was clarified by the appearance of a difference in the equivalent aluminium thickness, and the obtained results were statistically evaluated. The results showed that there were no significant differences between two graft materials statistically. These two graft materials could be good alternatives in sinus lifting procedures because of less morbidity, lower price, and good ossification. The results indicate that 2 different types of DBM achieved good ossification in the sinus lifting procedure, and there is not a considerable distinction in these 2 forms.

Salinity and flow regime independent multiphase flow measurements

For oil production fields, there is a need for downhole measurements of the gas/water/oil multiphase flow. In extreme conditions a relatively simple, robust, and non-intrusive system will be appropriate. A measurement setup that combines multiple gamma beam (MGB) and dual modality densitometry (DMD) measurements, would be able to determine the gas volume fraction (GVF) independently of the flow pattern, and monitor changes in water salinity. MGB measurements of gamma-ray transmission along multiple sections across the oil pipe will provide information on the flow pattern. Whereas the DMD principle will give information on changes in salinity from a combination of transmission and scattering gamma-radiation measurements. In this work we present the results from MGB and DMD measurements of a multiphase flow with high-speed gamma-ray tomograph measurements as reference for the flow pattern. The MGB measurements should enable us to distinguish between stratified or wavy/slug and annular or slug flow. Flow patterns with several minor components distributed evenly over the measurement cross section, like bubble flow, will be interpreted as homogeneous flow. The DMD measurements can be used to monitor salinity changes of the water component for intervals where the GVF is low and the water cut of the liquid is high. Combined with other gauges for water cut measurements, the MGB and DMD measurement setup should improve the multiphase flow measurements, and enable increased oil/gas recovery and production water monitoring.

An Audit of Bone Densitometry Measurement in Patients with Coeliac Disease

There is a well documented association between coeliac disease and reduced bone mineral density (BMD). Osteopenia and osteoporosis are both preventable and treatable. There are clear clinical recommendations available for measuring BMD in subgroups of patients with coeliac disease. However, it is suspected that within the practice population these standards are not being achieved. This suspicion is based on routine clinical encounters with patients with coeliac disease.

HPTLC Determination of Food Emulsifiers by Iodine Staining and Densitometry

The quantitative determination by HPTLC, after iodine staining and densitometry, of three food emulsifiers phosphatidylcholine, glycerol monolaurin and sorbitan monolaurate has been fully validated. Results showed that through a temperature reduction step, the effect of iodine vapor staining could be adequately preserved for densitometry measurement. The key parameters of detection were optimized and by means of the Kubelka–Munk modeling test, the quantitative prediction of the method was verified, suggesting that this protocol would be a possible solution for the quantitation of lipids lacking a chromophore.

Single-beam gamma densitometry measurements of oil–water flow in horizontal and slightly inclined pipes

Gamma densitometry is a frequently used non-intrusive method for measuring component volume fractions in multiphase flow systems. The application of a single-beam gamma densitometer to investigate oil–water flow in horizontal and slightly inclined pipes is presented. The experiments are performed in a 15 m long, 56 mm diameter, inclinable stainless steel pipe using Exxsol D60 oil (viscosity 1.64 mPa s, density 790 kg/m 3) and water (viscosity 1.0 mPa s, density 996 kg/m 3) as test fluids. The test pipe inclination is changed in the range from 5° upward to 5° downward. Experimental measurements are reported at three different mixture velocities, 0.25, 0.50 and 1.00 m/s, and the inlet water cut is varied from 0 to 1. The gamma densitometer is composed of radioactive isotope of Am-241 with the emission energy of 59.5 keV, scintillation detector [NaI(Tl)] and signal processing system. The time averaged cross-sectional distributions of oil and water phases are measured by traversing the gamma densitometer along the vertical pipe diameter. Based on water volume fraction measurements, water hold-up and slip ratio are estimated. The total pressure drop over the test section is measured and frictional pressure drop is estimated based on water hold-up measurements. The measurement uncertainties associated with gamma densitometry are also discussed. The measured water hold-up and slip ratio profiles are strongly dependent on pipe inclination. In general, higher water hold-up values are observed in upwardly inclined pipes compared to the horizontal and downwardly inclined pipes. At low mixture velocities, the slip ratio decreases as the water cut increases. The decrease is more significant as the degree of inclination increases. The frictional pressure drop for upward flow is slightly higher than the horizontal flow. In general, there is a marginal difference in frictional pressure drop values for horizontal and downwardly inclined flows.

Scatter correction in digital mammography based on image deconvolution

X-ray scatter is a major cause of nonlinearity in densitometry measurements using digital mammography. Previous scatter correction techniques have primarily used a single scatter point spread function to estimate x-ray scatter. In this study, a new algorithm to correct x-ray scatter based on image convolution was implemented using a spatially variant scatter point spread function which is energy and thickness dependent. The scatter kernel was characterized in terms of its scattering fraction (SF) and scatter radial extent (k) on uniform Lucite phantoms with thickness of 0.8–8.0 cm. The algorithm operates on a pixel-by-pixel basis by grouping pixels of similar thicknesses into a series of mask images that are individually deconvolved using Fourier image analysis with a distinct kernel for each image. The algorithm was evaluated with three Lucite step phantoms and one anthropomorphic breast phantom using a full-field digital mammography system at energies of 24, 28, 31 and 49 kVp. The true primary signal was measured with a multi-hole collimator. The effect on image quality was also evaluated. For all 16 studies, the average mean percentage error in estimating the true primary signal was found to be −2.13% and the average rms percentage error was 2.60%. The image quality was seen to improve at every energy up to 25% at 49 kVp. The results indicate that a technique based on a spatially variant scatter point spread function can accurately estimate x-ray scatter.

Experimental and Theoretical Determination of the Limiting Partial Molar Volume of Indole in CCl4, Tetrahydrofuran and Acetonitrile at 293.15 K: A Comparative Study with Benzimidazole and Benzothiophene

The partial molar volumes of indole(Ind) at infinite dilution (\(V_{2}^{\infty}\)) in carbon tetrachloride (CCl4), acetonitrile (ACN) and tetrahydrofuran (THF) as solvents, were estimated from densitometry measurements at 293.15 K. The results indicate that \(V_{2}^{\infty}\mbox{(ACN)}>V_{2}^{\infty}\mbox{(CCl$_{4}$)}\) \(\approx V_{2}^{\infty}\mbox{(THF)}\). The values determined in this study are close to the values calculated from reported density for Ind in the solid state. In order to make a comparison the partial molecular volume of benzimidazole (Bim) and benzothiophene (BT) in solvents with appropriate solubility were measured too, and the results have revealed that \(V_{2}^{\infty}\mbox{(BT)}>V_{2}^{\infty}\mbox{(Ind)}\) in CCl4 and \(V_{2}^{\infty}\mbox{(Ind)}>V_{2}^{\infty}\mbox{(Bim)}\) in THF. In this work the role of solvent reorganization around to solute cavity, and specific and nonspecific interactions on the volumetric behavior of these molecules in solution are discussed using the Terasawa-Itsuki-Arakawa model, the Lee-Graziano model, molar volumes of solutes calculated at the DFT-B3LYP/cc-pVTZ and aug-cc-pVTZ level in the gas phase and considering solvent presence with the Onsager’s reaction field, and the van der Waals volume. This analysis suggests that the molecular volumes of solutes are overestimated by the quantum methods employed in this work and that the volumetric contribution from the van der Waals components to the limiting partial molecular volumes of solutes is important, with the exception of Ind in CCl4 where the solvent reorganization is the dominant factor.

Decreasing trend and fluctuations in the mean ring density of Norway spruce through the twentieth century

• Studies on wood density variations are necessary for estimating the forest carbon pool. Further, they can help predict the technological properties of wooden end-products. While there have been frequent reports on the relationships between wood density, cambial age, and ring width, there is little information about the historical trend in wood density for the last century, particularly in the context of global climate change. • In this study, different sources of variations in mean ring density (site, tree, ring age, ring width, and calendar date) were studied using an original sampling design. A total of 105 Norway spruce (Picea abies Karst.) trees were sampled in north-eastern France, from thirteen sites with trees of different ages growing at the same site and in similar conditions. X-ray densitometry measurements were performed on samples taken at breast height. The chronology of the mean ring density over the 20th century was estimated by using a statistical modelling approach based on linear mixed-effects models after accounting for the effect on the mean ring density introduced by different ring widths, cambial ages, sites, and trees. • The mean ring density of Norway spruce was seen to decrease by about 18 kg m −3 relative to the year 1900. The chronology showed no evolution between 1900 and 1950, a steep decline from 1950 to 1980 (reaching a maximum decrease of 30 kg m −3 ), followed by an increase from 1980 to 2000. • The observed decrease was consistent with the results of previous works and supports the hypothesis that this could indicate a global trend and that this trend is independent of the wood structure. Moreover, high inter-annual density variations were found. In future studies, the influence of climate on the wood density and within-ring properties must be clarified to identify the anatomic causes for wood density variations.

Swimming Training Increases the Post-Yield Energy of Bone in Young Male Rats

The purpose of this study is to investigate the effects of non-weight-bearing exercise on growing bone. Male Wistar rats (7 week-old) were assigned to one baseline control group, one control group and two swimming training groups, which were trained with 2 and 4% body-weight mass added, respectively. After an 8-week training period, three groups showed significant development compared to the baseline control group. Among the three 15-week-old groups, swimming-trained rats were lower in body weight (BW), densitometry and size-related measurements. In femoral biomechanical testing, swimming training groups were significantly lower in yield moment and ultimate moment, which may be due to a significantly lower long bone cross-sectional moment of inertia. However, the two swimming groups were higher in post-yield energy absorption and displacement. Further, in estimated tissue-level biomaterial properties, no differences were shown in yield stress, strain or toughness among the three groups. Using BW as a covariate, results of ANCOVA showed no differences in size-related parameters among the three groups, and some parameters were even higher in the two swimming groups. Regarding Pearson's correlation, size-related parameters correlated well to BW and whole bone strength but not to tissue post-yield behaviors. In conclusion, when compared to age-matched control group, swimming rats showed lower bone strength and lower yield energy absolutely at the structural level, but similar yield stress and yield toughness at the tissue level. Moreover, swimming training benefited growing bone in post-yield behaviors. Further studies should investigate the parameters that contribute to this exercise-induced post-yield behavior.

O-GlcNAc Protein Modification in Cancer Cells Increases in Response to Glucose Deprivation through Glycogen Degradation

When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.

Beam hardening artifacts in micro-computed tomography scanning can be reduced by X-ray beam filtration and the resulting images can be used to accurately measure BMD

Bone mineral density (BMD) measurements are critical in many research studies investigating skeletal integrity. For pre-clinical research, micro-computed tomography (μCT) has become an essential tool in these studies. However, the ability to measure the BMD directly from μCT images can be biased by artifacts, such as beam hardening, in the image. This three-part study was designed to understand how the image acquisition process can affect the resulting BMD measurements and to verify that the BMD measurements are accurate. In the first part of this study, the effect of beam hardening-induced cupping artifacts on BMD measurements was examined. In the second part of this study, the number of bones in the X-ray path and the sampling process during scanning was examined. In the third part of this study, μCT-based BMD measurements were compared with ash weights to verify the accuracy of the measurements. The results indicate that beam hardening artifacts of up to 32.6% can occur in sample sizes of interest in studies investigating mineralized tissue and affect mineral density measurements. Beam filtration can be used to minimize these artifacts. The results also indicate that, for murine femora, the scan setup can impact densitometry measurements for both cortical and trabecular bone and morphologic measurements of trabecular bone. Last, when a scan setup that minimized all of these artifacts was used, the μCT-based measurements correlated well with ash weight measurements ( R 2 = 0.983 when air was excluded), indicating that μCT can be an accurate tool for murine bone densitometry.

Repeatability and validity of lens densitometry measured with Scheimpflug imaging

To assess the repeatability and validity of lens densitometry derived from the Pentacam Scheimpflug imaging system. Eye Clinic, Queensland University of Technology, Brisbane, Australia. This prospective cross-sectional study evaluated 1 eye of subjects with or without cataract. Scheimpflug measurements and slitlamp and retroillumination photographs were taken through a dilated pupil. Lenses were graded with the Lens Opacities Classification System III. Intraobserver and interobserver reliability of 3 observers performing 3 repeated Scheimpflug lens densitometry measurements each was assessed. Three lens densitometry metrics were evaluated: linear, for which a line was drawn through the visual axis and a mean lens densitometry value given; peak, which is the point at which lens densitometry is greatest on the densitogram; 3-dimensional (3D), in which a fixed, circular 3.0 mm area of the lens is selected and a mean lens densitometry value given. Bland and Altman analysis of repeatability for multiple measures was applied; results were reported as the repeatability coefficient and relative repeatability (RR). Twenty eyes were evaluated. Repeatability was high. Overall, interobserver repeatability was marginally lower than intraobserver repeatability. The peak was the least reliable metric (RR 37.31%) and 3D, the most reliable (RR 5.88%). Intraobserver and interobserver lens densitometry values in the cataract group were slightly less repeatable than in the noncataract group. The intraobserver and interobserver repeatability of Scheimpflug lens densitometry was high in eyes with cataract and eyes without cataract, which supports the use of automated lens density scoring using the Scheimpflug system evaluated in the study.

PET/CT for the Characterization of Adrenal Masses in Patients with Cancer: Qualitative Versus Quantitative Accuracy in 150 Consecutive Patients

The objective of our study was to evaluate a large cohort of patients with PET/CT to determine whether qualitative (visual) assessment, quantitative standardized uptake value (SUV), or standardized uptake ratio (SUR) techniques should be used when attempting to characterize adrenal masses in patients with cancer. The study group was composed of 150 consecutive patients (78 men, 72 women; mean age, 60 years; range, 24-88 years) with documented adrenal lesions. All patients were known to have an underlying primary malignancy and were referred for PET/CT to evaluate the underlying primary and metastatic tumor burden. Definitive lesion characterization was determined by evaluating all histologic adrenal specimens and all relevant prior and follow-up CT scans, including unenhanced, contrast-enhanced, and delayed contrast-enhanced washout studies. Of the 139 benign lesions, 109 were considered benign by CT densitometry measurements and 135 by qualitative PET data. Qualitative PET characterized 28 of 30 benign lesions that were considered indeterminate by unenhanced CT. All 26 malignant lesions were characterized by PET: All showed qualitative and quantitative signal intensity greater than the liver. By combining unenhanced and qualitative CT data with the retrospective PET data, the analysis yielded a sensitivity of 100% for the detection of malignancy, a specificity of 99%, a positive predictive value (PPV) of 93%, a negative predictive value (NPV) of 100%, and an accuracy of 99% (Table 1). Conversely, for the detection of benignity, the sensitivity, specificity, PPV, NPV, and accuracy were 99%, 100%, 100%, 93%, and 99%, respectively. PET/CT is a highly accurate method for differentiating benign from malignant adrenal masses particularly when using qualitative, rather than quantitative, PET data. The routine use of quantitative mean or maximal SUV or SUR data may be unnecessary. Occasional benign lesions do show mild to moderate increased FDG uptake compared with that of the liver and may mimic some malignant lesions. Without evidence that these lesions are benign by unenhanced CT densitometry or adrenal mass stability or growth from previous CT scans, we recommend that these lesions be characterized using contrast-enhanced washout tests and that if those tests are inconclusive, using percutaneous biopsy if early lesion characterization is mandatory.

Diagnostic Potential of 3D-Data–Based Reconstruction Software: An Analysis of the Rare Disease Pattern of Cherubism

Cherubism is an autosomal-dominant syndrome characterized by bilateral maxillomandibular bony degeneration, fibrous connective tissue hyperplasia, and displacement of permanent tooth germs. Reossification of the cystic lumen occurs spontaneously, but dislocated teeth must be realigned orthodontically. Advancements in virtual 3D reconstruction of anatomic structures based on computed tomography (CT) or cone beam CT data have provided for more predictable individual treatment planning. We evaluated two software programs for making densitometry and volume measurements of cystic areas in the mandibles, and for 3D visualization of permanent tooth germs within the cystic lumen, in two siblings with cherubism.

Adapted liquid crystal display backlighting unit for densitometry of stained polyacrylamide electrophoresis gels

Appropriate monochromatic illumination that is spatially uniform will ensure densitometry of stained polyacrylamide electrophoresis gels with high sensitivity and repeatability. The approach of using an array of LEDs as an illuminator has practical limitations. Here we describe an alternative method based on adapted liquid crystal display backlighting. By changing the fluorescent light source and using a region where light intensity is uniform, we demonstrate the ability to achieve highly sensitive and repeatable densitometry measurements of polyacrylamide electrophoresis gels with Coomassie-stained proteins.

On the nucleolar and cytoplasmic RNA density during "cell dedifferentiation" represented by blastic transformation of human mature T lymphocytes - a cytochemical study.

The present study was undertaken to provide information on the nucleolar and cytoplasmic density in specimens stained for RNA during "cell dedifferentiation" represented by blastic transformation of mature T lymphocytes. Nucleolar and cytoplasmic RNA's were visualized using a simple cytochemical method followed by computer assisted densitometry and size measurements of digitised images. An increased nucleolar and cytoplasmic RNA density accompanying the blastic transformation was significant after 48 hours of cultivation with phytohemaglutinin (PHA) when stimulated cells were characterized the largest nucleolar size reflecting S or G2 phase of the cell cycle. On the other hand, significantly larger ratio of the nucleolar to cytoplasmic density was noted only after a shorter cultivation when stimulated cells were presumably in the G1 phase. Thus the increased nucleolar and cytoplasmic RNA density together represented an accompanying phenomenon of the cell proliferation and cycling state. From the methodical point of view, the nucleolar and cytoplasmic RNA densitometry appeared as a simple as well as useful additional method to study "dedifferentiation" or various cell states at the single cell level. In addition, it was also interesting that the increase of the nucleolar diameter in stimulated cells was much larger than that of the nucleolar density. Such difference suggested that the RNA content in nucleoli was related mainly to their size.

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Potassium channels are tetrameric proteins that mediate K(+)-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (approximately 40 kDa) on SDS-PAGE. Addition of Ba(2+) (K(1/2) approximately 50 microm) prior to loading results in an additional tetramer band (approximately 160 kDa). Mutation A109C, at a residue located near the expected Ba(2+)-binding site, decreased tetramer stabilization by Ba(2+) (K(1/2) approximately 300 microm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba(2+). Neither mutation affected Ba(2+) block of channel activity (using (86)Rb(+) flux assay). In contrast to Ba(2+), Mg(2+) had no effect on tetramer stability (even though Mg(2+) was a potent blocker). Many studies have shown Cd(2+) block of K(+) channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd(2+) on KirBac1.1 with cysteine substitutions in M2. Cd(2+) block potency followed an alpha-helical pattern consistent with the crystal structure. Significantly, Cd(2+) strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba(2+), consistent with both ions simultaneously occupying the channel: Ba(2+) at the selectivity filter entrance and Cd(2+) coordinated by I138C side chains in the inner cavity.