Electron microscopic observations on the large subunit of the rat liver ribosome.

Active large subunits obtained by urea treatment of rat liver ribosomes, 59S, were compared with large subunits in intact ribosomes and with the 50S subunits obtained by EDTA treatment. For electron microscopy the specimens were negatively stained or shadow cast. The negatively stained 59S subunits had a slightly ovoidal form; their average dimensions, 244 +/- 17 x 207 +/- 18 A, were very close to the dimensions of the large subunits in intact ribosomes, and lay between the theoretical dimensions for anhydrous and fully hydrated particles that were calculated from the physical properties of the subunits in solution. The shadow-cast preparations showed particles of similar shape. The 50S subunits, which had lost their 5S RNA, were shadow cast at the same time. They appeared to be more spread out than the 59S subunits and had threadlike extensions. In the positively stained regions of uranyl oxalate-stained preparations the 50S particles varied greatly in shape and size, with average dimensions of 330 +/- 21 x 276 +/- 33 A, and showed threadlike extensions like those of the shadow-cast particles. For 50S particles in solution the frictional drag of these extensions probably accounts for their low sedimentation coefficient.

Fine control of endothelial VEGFR-2 activation: caveolae as fluid shear stress shelters for membrane receptors.

Recent experimental evidence points to the possibility that cell surface-associated caveolae may participate in mechanotransduction. The particular shape of caveolae suggests that these structures serve to prevent exposure of putative mechanosensors residing within these membrane invaginations to shear stresses at magnitudes associated with initiation of cell signaling. Accordingly, we numerically analyzed the fluid flow in and around caveolae using the equation of motion for flow of plasma at low Reynolds numbers and assuming no slip-condition on the membrane. The plasma velocity inside a typical caveola and the shear stress acting on its membrane are markedly reduced compared to the outside membrane. Computation of the diffusion field in the vicinity of a caveola under flow, however, revealed a rapid equilibration of agonist concentration in the fluid inside a caveola with the outside plasma. Western blots and immunocytochemistry support the role of caveolae as shear stress shelters for putative membrane-bound mechanoreceptors such as flk-1. Our results, therefore, suggest that caveolae serve to reduce the fluid shear stress acting on receptors in their interior, while allowing rapid diffusion of ligands into the interior. This mechanism may permit differential control of flow and ligand activation of flk-1 receptor in the presence of ligands.

In-air micro-particle induced X-ray emission analysis of asbestos and metals in lung tissue.

Inhalation of asbestos increases the risk of lung cancer and pulmonary fibrosis. It is difficult to directly assess the distribution and content of inhaled particles in lung tissue sections. The purpose of this study is to employ an in-air micro particle induced X-ray emission (in-air micro-PIXE) system for assessment of the spatial distribution and content of asbestos and other metals in lung tissue. A proton ion-microbeam from this system was applied to irradiate lung tissue of patients with or without asbestosis, tumor tissue from both groups, and asbestos fibers (in vitro). The content of each element composing asbestos and those of other metals were calculated and their distribution was assessed from the characteristic X-ray pattern for each element obtained after irradiation. This in-air micro-PIXE system could identify the location of asbestos bodies composed of Si, Mg, and Fe in lung tissue sections. Macrophage and lymphocytes accumulated in that area. This new system also revealed deposits of titanium, nickel, and cobalt in the lung tissues, in addition to asbestos bodies. The Si and Fe content were higher in lungs with asbestosis than in lungs without asbestosis or in tumor tissue. Analysis of asbestos fibers composed of chrysotile, crocidolite, and amosite showed that the ratios of Si, Fe, and Mg corresponded with those for the chemical structures. In-air micro-PIXE analysis is useful for assessing the distribution and quantities of asbestos bodies and also other metals in lung tissue comparing to immune-related cell localizations, and is also useful for analysis of standard asbestos fibers.

Observation of co-segregation of titanium and boron at the interface between recrystallized and unrecrystallized grains in cold-rolled interstitial-free steel sheets.

It has been reported that the addition of ppm levels of B strongly retarded the growth of recrystallized grain into unrecrystallized grains in the process of cold-rolling and annealing of Ti-added interstitial-free (IF) ferritic steels. This phenomenon was explained by solute drag effect based on the assumption that, during annealing, B atoms segregate at the interface between recrystallized and unrecrystallized grains where they interact with Ti atoms. To verify this, atom probe tomography analysis of the interface was performed in Ti-added IF steels with and without B addition. Needle tips containing the interface identified from electron backscattering diffraction analysis, were produced by focused ion beam milling with the lift-out method. To increase the experiment reliability, the misorientation angle of the aimed interface was compared with that estimated by field ion microscopy analysis. Considerable amount of Ti segregation was observed at the interface in the steel without B addition, which increased with increasing amount of B segregation in the steel with B addition. The results suggest that the retardation of the interface migration was caused by solute drag effect based on the simultaneous co-segregation of Ti and B due to their attractive interaction.

Differential expression of a ligand induced binding site (LIBS) by GPIIb-IIIa ligand recognition peptides and parenteral antagonists.

The glycoprotein (GP) IIb-IIIa complex is an attractive anti-platelet target for the prevention of thrombotic events associated with coronary artery disease. Although GPIIb-IIIa antagonists inhibit GPIIb-IIIa binding to its ligands, the interactions have not been fully clarified, particularly with respect to their ability to induce structural changes in the complex that lead to exposure of neoantigenic epitopes or ligand-induced binding sites (LIBS). In this study we used the anti-LIBS monoclonal antibody (mAb) D3 to further define the activation states of purified active and inactive GPIIb-IIIa. We also compared the data obtained in the purified system to that observed with intact human platelets. Active GPIIb-IIIa expressed significantly greater high-affinity D3 LIBS sites compared to the inactive form. In addition, the ligand recognition peptides RGDS and H12 caused increased expression of the D3 epitope, with RGDS eliciting a much more potent response. The response of the purified GPIIb-IIIa to these peptides paralleled that observed with human platelets. To explore whether the platelet antagonists abciximab, eptifibatide and tirofiban induced expression of the D3 LIBS site, a modified competitive ELISA was developed. Our data indicate that the use of purified GPIIb-IIIa with this ELISA system provides a reproducible approach for exploring the interactions between GPIIb-IIIa and its antagonists. Whereas abciximab caused no detectable increase in the expression of the D3 epitope on purified GPIIb-IIIa, eptifibatide, tirofiban, RGDS, and H12 induced differential expression of the high-affinity LIBS. Studies with intact platelets suggested that abciximab blocked the binding of the D3 and LIBS6 mAbs, and that the pre bound anti-LIBS D3 sterically hindered abciximab binding.

Effect of HLA phenotype and gender on expression of various forms of class I HLA in plasma.

To understand the complexity of plasma HLA antigens, the distribution of different molecular weight forms of class I HLA in plasma was investigated in 44 HLA-phenotyped and unrelated individuals. Plasma class I HLA were immunoprecipitated by using the W6/32 anti-HLA monoclonal antibody, separated by SDS-polyacrylamide gel electrophoresis and characterized by immunoblotting with the HC-10 monoclonal antibody. Four different forms of HLA heavy chains (HLA-HC) with relative molecular masses of 44, 39, 36, and 34 kd were detected. Plasma samples from all individuals contained 44 and 36 kd HLA-HC, but varied as to the presence of 39 and 34 kd HLA-HC. Eighteen percent of the individuals did not have any detectable class I HLA with 39-kd heavy chains in their plasma and 61% did not have plasma class I HLA with 34-kd heavy chains. Thus, four different distribution patterns were identified for plasma class I HLA among all individuals included in our study. The distribution patterns in four different individuals were evaluated quarterly and remained unchanged during 1 year follow-up. A significant association of absence of 39-kd plasma class I HLA-HC with female gender (p less than 0.05) and HLA-B7 phenotype (p less than 0.00015) was also found. Further pedigree analyses of four families of HLA-B7-positive and 39-kd HLA-HC-negative probands indicated that genetic factor(s) other than those associated with HLA-B7 allele and female gender is involved in regulating the expression of the plasma class I HLA with 39-kd heavy chains.

Development of micromachining technology in ion microbeam system at TIARA, JAEA.

An ion-beam-lithography technique has been progressed in the microbeam systems at Japan Atomic Energy Agency (JAEA) Takasaki. In order to obtain a high-precision measure for microbeam size estimation with a high precision, we applied this technique combined with the electroplating process to make a Ni relief pattern as a resolution standard used in secondary electron imaging. As a result, the smallest beam size could be recorded. The scattering of ions in the materials influenced the spatial resolution and this is also discussed.

Biochemical characterization of 39-kDa class I histocompatibility antigen in plasma. A secretable membrane protein derived from transmembrane domain deletion.

Three human class I major histocompatibility antigens (HLA) with molecular masses of 44, 39, and 36 kDa were identified in plasma by immunoprecipitation and immunoblotting. Further biochemical characterization showed that these antigens in plasma could be fractionated by Sephacryl S-300 column chromatography into two different pools. The 44-kDa intact HLA heavy chains are detected only in pool I and have an apparent molecular weight of 200,000 as determined by calibrated gel filtration column chromatography. The 39- and 36-kDa HLA heavy chains are present only in pool II and have an apparent molecular weight of 50,000. HLA in pool I can be extracted by Triton X-114 detergent, but 39- and 36-kDa plasma HLA in pool II are water soluble and not extractable by Triton X-114. Amino acid sequences of NH2 termini for 44- and 39-kDa plasma HLA are identical to that of cellular HLA. In contrast, the NH2-terminal amino acid sequence for 36-kDa plasma HLA has not been reported previously for any other proteins. Since the loss of both transmembrane domain and cytoplasmic tail at the carboxyl terminus of HLA will generate a 36-kDa protein, the findings suggest that the 39-kDa HLA might be the product of alternatively spliced mRNA with deletion of the exon coding for transmembrane domain. By using polymerase chain reaction and DNA sequencing, the presence of alternatively spliced mRNA with deletion of the transmembrane domain exon was identified in mononuclear leukocytes of peripheral blood. This alternatively spliced HLA mRNA was not detectable in mononuclear leukocytes of an individual who had no 39-kDa plasma HLA. This finding indicates that the alternatively spliced mRNA in mononuclear leukocytes is responsible for the synthesis of a secretable class I HLA.

Inhibition of an early event in the cell division cycle of Escherichia coli by FL1060, an amidinopenicillanic acid.

Analysis of exponential and synchronous cultures of Escherichia coli B/r after the addition of FL1060 indicates a block point for division by this agent some 15 to 20 min before the end of the preceding cell division cycle, a time corresponding to the beginning of the C period of the cell division cycle. Morphological examination of FL1060-treated synchronous cultures of E. coli /r was consistent with inhibition by FL1060 of a very early event in the cell division cycle. This event appears to be essential for normal cell surface elongation in a rod configuration. Temporary treatment of synchronous cultures of E. coli B/r with FL1060 resulted in division delay, the extent of which was a function of the duration of exposure to FL1060. However, even after relatively long times of FL1060 treatment the delayed divisions were still synchronous. Although FL1060 had no direct effect on deoxyribonucleic acid (DNA) synthesis, the synchronous delayed division occuring after temporary treatment with FL1060 were accompanied by a delay in the attainment of resistance of cell division to inhibitors of DNA, ribonucleic acid, and protein synthesis. These results suggest aht an FL1060-sensitive event initiates at the beginning of the C period of the cell division cycle of E. coli and is responsible for normal cell elongation. This cell elongation pathway procedes independently of DNA synthesis, but there is an interaction between this pathway and termination of a round of DNA replication in which a normal rod configuration is necessary to allow a signal for cell division to be generated upon completion of DNA replication.

Fibroepithelial ureteral polyps.

Two cases of fibroepithelial polyps of the ureter are reported. In one case the polyp was protruding through the meatus of the urethra whereas in the other case no clinical manifestations were observed. Both benign tumors were treated by local surgery. Etiology, clinical features, diagnosis and therapy are discussed.

Nucleoid condensation and cell division in Escherichia coli MX74T2 ts52 after inhibition of protein synthesis.

The reorganization of the bacterial nucleoid of an Escherichia coli mutant, MX74T2 ts52, was studied by electron microscopy after protein synthesis inhibition by using whole mounts of cell ghosts, ultrathin-sectioning, and freeze-etching. The bacterial nucleoid showed two morphological changes after chloramphenicol addition: deoxyribonucleic acid (DNA) localization and DNA condensation. DNA localization was observed 10 min after chloramphenicol addition; the DNA appeared as a compact, solid mass. DNA condensation was observed at 25 min; the nucleoid appeared as a cytoplasm-filled sphere, often opened at one end. Ribosomes were observed in the center. Giant nucleoids present in some mutant filaments showed fused, spherical nucleoids arranged linearly, suggesting that the tertiary structure of the nucleoid reflects the number of replicated genomes. Inhibitors which directly or indirectly blocked protein synthesis and caused DNA condensation were chloramphenicol, puromycin, amino acid starvation, rifampicin, or carbonyl cyanide m-chlorophenyl hydrazone. All inhibitors that caused cell division in the mutant also caused condensation, although some inhibitors caused condensation without cell division. Nucleoid condensation appears to be related to chromosome structure rather than to DNA segregation upon cell division.

Percutaneous chemolitholysis of cystine stones. Possibilities for ambulatory procedure.

We report on a patient with symptomatic cystine calculi in a solitary kidney in whom percutaneous pelviocaliceal irrigation with tromethamine-E was performed as an ambulatory procedure for a significant period of time. Our experience demonstrates that in selected cases this form of treatment is well-tolerated.

Quantification of the passive mechanical properties of the resting platelet.

Sudden coronary artery occlusion is one of the leading causes of death. Several in vitro models have been used to study the relationship between hemodynamic forces and platelet function. However, very few in vivo studies exist that fully explore this relationship due to the lack of rheologic data for the platelet. For this purpose, micropipette aspiration techniques were used in the present study to determine the mechanical properties of platelets. The data were analyzed by two mathematical models: (1) an erythrocyte-type membrane model which yielded a platelet shear modulus of 0.03+/-0.01 dyn cm[-1] (mean+/-SD) and a viscous modulus of 0.12+/-0.04 dyn s cm[-1]. (2) An endothelial-type cell model which approximated the platelet Young's modulus to be 1.7+/-0.6 x 10(3) dyn cm(-2) with a viscous modulus of 1.0+/-0.5 x 10(4) dyn s cm(-2). The endothelial-type cell model more accurately describes the mechanics occurring at the micropipette tip and permits more appropriate assumptions to be made in quantifying the rheologic properties of a platelet. Results from this study can be integrated into numerical models of blood flow in stenosed coronary arteries to elucidate the impact of local hemodynamics on platelets and thrombus formation in coronary artery disease.

A case of pancreatic oncocytic tumor.

An uncommon case of potentially malignant oncocytoma arising in the pancreatic tail of 54-year-old woman is reported. The tumor was encapsulated and measured 11 x 7 x 6 cm. The tumor cells were uniform in appearance, plump and polyhedral, with distinct finely granular eosinophilic cytoplasm, and were arranged in solid acinar groups. Electron microscopy revealed that the tumor cells contained numerous mitochondria in the cytoplasm. In the present case, the tumor cells showed perineural and small venous invasion in the pancreas and potentially malignant characteristics. However, neither recurrence nor metastasis has been detected 3 years after resection. These findings indicate that the present tumor had apparent low-grade malignancy.

Estimating the long-term effects of storage at -70 degrees C on cholesterol, triglyceride, and HDL-cholesterol measurements in stored sera.

We estimated the effects of long-term storage at -70 degrees C on serum total cholesterol, HDL-cholesterol, and triglycerides in specimens that had been stored for up to 7 years. These estimates were made using measurements in serial specimens collected from the placebo control group of the Air Force/Texas Coronary Atherosclerosis Prevention Study over a period of approximately 5 years. We compared the group means for pairs of serial specimens taken at 6- and 12-month intervals, assuming that (a) a negligible placebo effect occurred between the serial specimen pairs; (b) in the absence of storage effects, the variation in the group means would reflect only normal biological variation and would not materially affect the group means for the serial specimens; (c) any systematic changes in these group means would reflect storage-related changes; and (d) storage-related changes are cumulative, i.e., the overall changes for a given storage period are the sum of the changes during previous storage periods. We observed average decreases of 2.0% per year for total cholesterol over 7 years and 2.8% per year in triglycerides for the first 5 years. HDL-cholesterol decreased by 1.3% per year, but this change was not statistically significant. This approach may be useful for estimating storage-related changes for studies in specimens stored for a period of years and for which stability data may not be available.