Detection of human T lymphotrophic virus type I (HTLV-I) proviral DNA and analysis of T cell receptor V beta CDR3 sequences in spinal cord lesions of HTLV-I-associated myelopathy/tropical spastic paraparesis.

Identification of the localization of human T lymphotrophic virus type I (HTLV-I) proviral DNA in the central nervous system (CNS) is crucial to the understanding of the pathogenesis of HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) pathogenesis. We have developed a sensitive detection method, called two-step polymerase chain reaction (PCR) in situ hybridization, which enabled us to detect the HTLV-I proviral DNA in paraffin-embedded spinal cord tissue sections from HAM/TSP patients. HTLV-I proviral DNA was detected only in the nucleus of lymphocytes that had infiltrated into the spinal cord. However, no proviral DNA was amplified in any neuronal cells, including neurons and glial cells. This indicates that the demyelination of the spinal cord by HTLV-I as a result of viral infection of oligodendrocytes or neuronal cells is unlikely. The T cell receptor V beta gene sequence from lymphocytes in the spinal cord lesions taken from the same HAM/TSP autopsy cases revealed unique and restricted CDR3 motifs, CASSLXG(G) (one-letter amino acid. X is any amino acid), CASSPT(G), and CASSGRL which are similar to those described in T cells from brain lesions of multiple sclerosis (MS) and in a rat T cell clone derived from experimental allergic encephalomyelitis (EAE) lesions. The present results suggest that T cells containing restricted V beta CDR3 motifs, which are also found in MS and EAE, become activated upon HTLV-I infection and infiltrate into the spinal cord lesions of HAM/TSP patients.

Flexible and wavelength-independent optical transmission for laser pointing stabilization and assembly error compensation.

A flexible light guide was developed for an ITER (International Thermonuclear Experimental Reactor) poloidal polarimeter which is a passive laser beam alignment and stabilization system for free-space propagation of a wide range of wavelengths. The advantages of using a flexible light guide are (1) to compensate the relative movement between the floor of a building and an optical table, (2) to negate assembly error of the optical transmission line, (3) to minimize the time required for assembly of the line and laser position alignment in a radiological environment, and (4) to transmit a wide wavelength range from visible to far-infrared. The authors fabricated a flexible light guide with an inner diameter of 120 mm and with a motion range of 10 cm. Pointing stability of the laser beam passing through the flexible light guide was less than 70 µrad when the support moved ±50 mm. A flexible light guide error of 70 µrad leads to a beam position displacement of 0.98 mm at a beam position steering mirror ITER poloidal polarimeter that is located 14 m from the flexible light guide. The achieved error is stable enough to guide the laser beam to its target in ITER.

Note: Lossless laser beam combiner employing a high-speed rotating half-wave plate.

We have developed a laser beam combiner employing a high-speed rotating half-wave plate based on the specific requirements of the Thomson scattering measurement systems in the ITER. The polarization extinction ratio of the output beam may exceed 1000 and was maintained for more than 1 h via feedback control of the half-wave plate rotation speed. The pointing fluctuations introduced by rotating the half-wave plate were in the order of microradians. The high-speed rotating half-wave plate provides a lossless means of combining laser beams together with stable beam pointing.

Validations of calibration-free measurements of electron temperature using double-pass Thomson scattering diagnostics from theoretical and experimental aspects.

This paper evaluates the accuracy of electron temperature measurements and relative transmissivities of double-pass Thomson scattering diagnostics. The electron temperature (Te) is obtained from the ratio of signals from a double-pass scattering system, then relative transmissivities are calculated from the measured Te and intensity of the signals. How accurate the values are depends on the electron temperature (Te) and scattering angle (θ), and therefore the accuracy of the values was evaluated experimentally using the Large Helical Device (LHD) and the Tokyo spherical tokamak-2 (TST-2). Analyzing the data from the TST-2 indicates that a high Te and a large scattering angle (θ) yield accurate values. Indeed, the errors for scattering angle θ = 135° are approximately half of those for θ = 115°. The method of determining the Te in a wide Te range spanning over two orders of magnitude (0.01-1.5 keV) was validated using the experimental results of the LHD and TST-2. A simple method to provide relative transmissivities, which include inputs from collection optics, vacuum window, optical fibers, and polychromators, is also presented. The relative errors were less than approximately 10%. Numerical simulations also indicate that the Te measurements are valid under harsh radiation conditions. This method to obtain Te can be considered for the design of Thomson scattering systems where there is high-performance plasma that generates harsh radiation environments.

Expression and purification of human angiotensinogen in Chinese hamster ovary cells.

We have produced human angiotensinogen in Chinese hamster ovary (CHO) cells. The expression products were purified to homogeneity by a single column chromatography and its 17 amino-terminal sequences were identical to those of the native protein. We demonstrated the recombinant human angiotensinogen to be a substrate for human renin.

Direct measurement of endosomal pH in living cells of the rat yolk sac epithelium by laser confocal microscopy.

Endocytosis of dual-fluorescent dextran by epithelial cells was observed in the perfused rat yolk sac using a confocal laser scanning microscope. Endosomal pH was quantified from confocal images using a dual-fluorescence ratiometry technique. Changes in endosomal pH were followed over time up to 32 min with and without Brefeldin A treatment. After 4 min of endocytosis without Brefeldin A, endosomes had a pH of 6.1 +/- 0.3. After 16 min and 32 min, their pH levels varied widely from 4.0 to 6.6. When Brefeldin A (10 microM) was added to the perfusion medium, endosomal pH remained fairly stable in the range of 6.0 to 6.2 from 4 min to 32 min. The corresponding endosomal structures were examined by electron microscopy after endocytic labeling with horseradish peroxidase. After 4 min, small endocytic vesicles and large endosomal vacuoles with tubular extensions were labeled. After 16 min and 32 min, multivesicular bodies and dense lysosomal structures were progressively labeled, but their labeling was almost undetectable after Brefeldin A treatment. These results suggested that the pH within the sorting compartment of early endosomes is 6.1 +/- 0.3. This is the first quantitative measurement of pH within sorting endosomes in intact cells of the living yolk sac epithelium.

The regional distribution and cellular localization of mRNA encoding rat prostacyclin synthase.

The cloned cDNA for rat prostacyclin synthase was found to contain a 1503-bp open reading frame which encoded a 501-amino acid protein sharing 84.0% identity with the human enzyme. RNA blot analysis revealed that the rat prostacyclin synthase mRNA, as a single species of 2.1 kb, is expressed abundantly in the aorta and uterus. High levels of expression were also observed in the stomach, lung, heart, testis, liver, and skeletal muscle. Low but significant expression was also seen in the brain and kidney. Furthermore, the regional distribution and cellular localization of prostacyclin synthase mRNA were examined by in situ hybridization analysis of rat tissue sections. The definitive signals for the mRNA were localized in smooth muscle cells of the arteries, bronchi and uterus, and in the cells of the fibrous tunic surrounding the seminiferous tubules, which are characterized as smooth muscle cells. Besides smooth muscle cells, signals were also detected in the fibroblasts of the heart myocardium, lung parenchyma cells and kidney inner medulla tubules and interstitial cells.

Site-directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys-pocket, and Glu347 and Arg350 of the EXXR motif.

The possible active site Cys441 in the Cys-pocket and Glu347 and Arg350 of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441 Ala, Cys441 Ser, Cys441 His, Glu347 Ala and Arg350 Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF1alpha/min per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys441 in the Cys-pocket, and Glu347 and Arg350 of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

Expression of CD23 in the germinal center of thymus from myasthenia gravis patients.

In order to investigate a pathogenic role of germinal centers which appear in the hyperplastic thymus of myasthenia gravis (MG) patients, we performed an immunohistochemical study using various monoclonal antibodies including CD23. In contrast with tonsilar germinal centers from non-MG individuals, CD23 was strongly and diffusely expressed in the whole area of germinal centers of MG thymi, including the outer zone. In addition, we measured the serum level of soluble CD23 (sCD23) in MG patients at various clinical stages. The high serum sCD23 levels, which were noted in the unthymectomized patients, fell to within normal range over 5 years after thymectomy, and the decline of serum sCD23 correlated well with clinical improvement. CD23 is thought to be responsible for preventing unselected germinal center B cells from entering apoptosis and, in turn, leads to the survival of auto-reactive B cell clones.

Immunocytochemical localization of 67 KD Ca2+ binding protein (p67) in ventricular, skeletal, and smooth muscle cells.

By using immunocytochemical techniques, we examined the localization of a 67 kDa Ca2+ binding protein (p67) and calpactin I heavy chain (p36) in ventricular myocytes, skeletal myocytes, and intestinal smooth muscle cells. Immunofluorescence microscopy revealed that the p67 was expressed in all these muscle cells, whereas anti-p36 antibody stained cells in connective tissues but failed to stain these muscle cells. Immunogold electron microscopy was carried out to examine the subcellular localization of the p67 in muscle cells. The results showed that the p67 was exclusively confined to the plasma membrane of muscle cells and the presumptive transverse tubules of the striated myocytes. Immunoblot analysis with anti-p67 antibody showed that the p67 was indeed a constitutive protein of the sarcolemma isolated from rat hearts. These results indicate that the p67 is a sarcolemma-associated Ca2+ binding protein expressed in both striated myocytes and intestinal smooth muscle cells.

Localization of gap junction proteins, connexins 32 and 26, in rat and guinea pig liver as revealed by quick-freeze, deep-etch immunoelectron microscopy.

By use of site-specific antibodies against synthetic oligopeptides, we examined the localizations of the gap junction proteins connexin 32 (Cx32) and connexin 26 (Cx26) in rat and guinea pig liver. Double-labeling immunofluorescence microscopy revealed that in guinea pig liver both proteins were spread throughout the liver lobules and seemed to localize together within the same gap junction plaque. In rat liver, co-localization of both Cx32 and Cx26 in the same plaques was also suggested in periportal zones. Quick-freeze, deep-etch immunoelectron microscopy showed that immunolabeling of isolated guinea pig liver gap junction plaques with either Cx32 or Cx26 antiserum yielded complete and dense antibody decoration of the cytoplasmic surface of the plaques. In isolated rat liver plaques, the cytoplasmic surfaces were densely decorated with Cx32 antiserum, whereas Cx26 labeling yielded diffuse decoration with variable intensity of the plaques. In both species we did not observe any focal or patchy clusters of the labeling in any plaques examined. Double-labeling immunoelectron microscopy confirmed that both Cx32 and Cx26 are co-localized in the same gap junction plaques. These results suggest that in hepatocytes expressing both Cx32 and Cx26, both types of gap junction proteins are not segregated but intermingle randomly within the same plaques.

Cytoplasmic surface ultrastructures of gap junctions in bovine lens fibers.

PURPOSE: To examine the cytoplasmic surface ultrastructures of lens fiber gap junctions, where the cytoplasmic domains of connexons were expected to be exposed. METHODS: Bovine lens fiber gap junctions, both in situ and in the form of isolated membranes, were examined with the deep etching replica methods. Isolated membranes were also examined with the same methods after the treatment with endoproteinase glu-C, which is known to cleave off the cytoplasmic domain of a putative lens fiber connexin MP70 to determine whether any structural changes should occur between proteolyzed and nonproteolyzed gap junctions. In addition, both proteolyzed and nonproteolyzed gap junctions were studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunolabeling with the monoclonal antibody that recognized cytoplasmic domain of MP70 to clarify whether MP70 lost its cytoplasmic domain by the treatment with endoproteinase glu-C. RESULTS: Gap junctions were shown to have particulate substructures on their cytoplasmic surfaces; the distributions of the particles were restricted within gap junctional plaques and the non-gap-junctional areas showed smooth cytoplasmic surfaces. Although the treatment with endoproteinase glu-C failed to remove the cytoplasmic particles of gap junctions in deep etching replica study, MP70 was shown to have lost its cytoplasmic domain in sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunolabeling studies. CONCLUSIONS: Each particle revealed on the cytoplasmic surfaces of lens fiber gap junctions corresponded to the cytoplasmic domain of a connexon. The particles were not removed by the treatment with endoproteinase glu-C, whereas MP70 was cleaved by the same treatment.

Effect of ionic sites of surfactants on leukocyte metabolism.

The effect of ionic and nonionic surfactants on the metabolism of leukocytes was studied. Among anionic surfactants, sodium laurylsulfate, sodium laurate, and sodium lauroyllactate stimulated oxygen uptake and glucose oxidation through the hexose monophosphate pathway. The magnitude of the effect of anionic surfactants seemed to be related to the electrostatic strength of the ionic site of each compound. Cationic, amphoteric, and nonionic surfactants were inhibitory to leukocyte metabolism.

Metabolic pattern of polymorphonuclear leucocytes induced by trypsin-digested microsomes.

The addition of trypsin [EC 3.4.21.4]-digested liver microsimes induced cyanideinsensitive respiration in guinea pig polymorphonuclear leucocytes with concomitant acceleration of the hexose monophosphate oxidative pathway. The respiration was insensitive to inhibitors of mitochondrial respiration but sensitive to glycolytic inhibitors. These metabolic alterations are similar to those associated with phagocytosis, though the digested mocrosomes were apparently not taken up by the cells and prpbably trigger the netabolic changes by interaction with the cellular membrane. Intact microsomes or microsomes treated with chymotrypsin [EC 3.4.21.1], bacterial proteinase, ribonuclease [EC 3.1.4.22], or neuraminidase [EC 3.2.1.18] could not induce such respiration.

Microheterogeneity and oligosaccharide chains on the beta chains of HLA-DR, human major histocompatibility complex class II antigen, analyzed by the lectin-nitrocellulose sheet method.

The beta chain of human histocompatibility complex class II antigen, HLA-DR, showed 4 to 5 microheterogeneous spots on a gel obtained by two-dimensional polyacrylamide gel electrophoresis. The types of oligosaccharide chains on the beta chains were analyzed by the lectin-nitrocellulose sheet method for each microheterogeneous spot with 3 cell lines of two haplotypes (HLA-DR 4,4, and 3,3). Two kinds of oligosaccharide chains were observed and were essentially the same in the microheterogeneous spots from all three cell lines. One, the oligosaccharide chain on the most basic spot (beta 1), was stained with peroxidase-coupled concanavalin A (Con A-P.O.) but not with peroxidase-coupled wheat germ agglutinin and was sensitive to endo-beta-N-acetylglucosaminidase H (endo H), indicating that it was a high-mannose type. The oligosaccharide chains on other spots that were not stained with Con A-P.O. but were stained with peroxidase-coupled Ricinus communis agglutinin were resistant to endo H. beta 2 and beta 3 were stained with E-PHA. Thus, they probably had bisected biantennary and others probably had multiantennary complex-type oligosaccharides. Sialidase experiments showed that the charge heterogeneity was due to post-translational sialylation of the oligosaccharide chains. In pulse-chase experiments, the most basic spot of beta chain (beta 1) was labeled first, beta 2 and beta 3 were labeled next, and beta 4 was labeled last. These labeling characters accorded well with the results on the oligosaccharide types mentioned above.

Rapid freezing replica studies of membrane specializations in the proximal tubules of mouse kidney.

The membrane specializations of the fresh unfixed kidney cortex of adult and neonatal ICR mice were examined by using rapid freezing replica methods. In proximal tubular cells, numerous apical intracellular tubules exhibited helical patterns on the E face with a pitch of about 12 nm. This regular pattern was often continuous with similar striped indentations on the edge of the vacuoles connecting with the tubules. On the luminal surface (ES) of these vacuoles, membrane surface particles were arranged regularly in striped patterns with a center-to-center spacing of about 12 nm. We could not identify differentiations on the PF or PS of the same membrane systems. Another membrane specialization was a plaque or patch of clear pits in tilted lattice alignments on the P face of the large vacuoles with a center-to-center spacing of about 20 nm. This type of specialization was often observed in the neonatal mice proximal tubular cells. These membrane specializations may indicate the active membrane functions in the proximal tubules and suggest the functional continuity and structural relationship of these apical endocytic membrane systems.

Antidiuretic action of tachykinin NK-3 receptor in the rat paraventricular nucleus.

Studies were performed on the central antidiuretic actions via the tachykinin NK-3 receptor in the rat hypothalamic paraventricular nucleus (PVN). Microinjections of the selective tachykinin NK-3 receptor agonist senktide (2-200 pmol) into the PVN resulted in prolonged inhibition of urine output in water-loaded rats, its effect being dose-dependent. The antidiuretic action of senktide was blocked by pretreatment with the vasopressin V2 receptor antagonist OPC-31260 (1 mg/kg, i.v.), but not by microinjection of the angiotensin II AT-1 receptor antagonist losartan (1 nmol) into the PVN. NK-3 receptor mRNA was strongly detected in the magnocellular part of the PVN and the supraoptic nucleus (SON) of the hypothalamus as detected by in situ hybridization histochemistry. Moreover, [3H]senktide binding sites were also detected in the PVN and the SON by receptor autoradiography. These findings suggest that NK-3 receptors in the PVN may be involved in water regulation by stimulation of vasopressin secretion from the posterior pituitary gland, and that vasopressin caused water reabsorbtion via the kidney V2 receptor.

The effect of rolipram on the production of cytokines in HTLV-I infected cell lines and peripheral blood mononuclear cells of patients with HTLV-I-associated myelopathy (HAM).

Previous studies have reported that the levels of pro-inflammatory cytokines, such as TNF-alpha and IFN-gamma, are elevated in the serum as well as in the cerebrospinal fluid of HAM/TSP patients. To evaluate the effect of the phosphodiesterase type IV inhibitor, rolipram on cytokine production, peripheral blood mononuclear cells (PBMCs) of HAM/TSP patients or HTLV-I infected T-cell lines (HUT102, MT2) were cultured in the presence of different doses of rolipram. The amount of cytokines in the supernatants of the cultured cells was determined by ELISA for TNF-alpha, IFN-gamma and TGF-beta. Rolipram inhibited TNF-alpha production by HUT102 and PBMCs from all the HAM/TSP patients in a dose-dependent manner. The suppression of IFN-gamma varied and was weaker in some HAM/TSP patients compared to that of TNF-alpha. The concentration of TGF-beta in the culture supernatants was not influenced by rolipram. The levels of TNF-alpha mRNA determined by competitive PCR were not changed in the cultured cells in the presence of rolipram, suggesting that rolipram inhibits TNF-alpha production at the post-transcriptional level. These findings suggest the possible benefit of rolipram as a therapeutic agent for HAM/TSP patients.

Collagen types in human posterior capsule opacification.

The fibrous type of human posterior capsule opacification was examined by electron microscopy and immunoelectron microscopy to determine which types of collagen were present. The opacification consisted of lens epithelial cells and a large amount of extracellular matrix. The extracellular matrix comprised collagen fibrils and basal lamina-like material. Immunoelectron microscopy revealed that collagen types I, III, and IV were present. Types I and III were localized to the collagen fibrils. Type IV was present in the basal lamina of the lens epithelial cells and in the basal lamina-like material of the extracellular matrix.

Radioimmunoassay of a cancer-related glycoprotein. Circulating levels.

Circulating levels of (a) tumor-related glycoprotein(s) were determined by radioimmunoassay for a variety of patients and controls, and correlated with sialic acid concentration. Levels were highest in patients with metastatic disease and progressively declined to those with localized disease receiving therapy. Values for normal, adjuvant, and cured patients were significantly lower. Sialic acid concentrations correlated best for the metastatic group but not for the normals.