Immunohistochemical studies of the serotonergic supraependymal plexus in the mammalian ventricular system, with special reference to the characteristic reticular ramification

Distributional and morphological features, especially characteristics of the ramification of serotonin-containing supraependymal fibers (SEF), were studied in the ventricular systems of mammals (mouse, rat, guinea pig, rabbit, cat, dog, monkey) by means of a modified peroxidase antiperoxidase technique, using antiserotonin antiserum prepared in our laboratory. SEF were present in all ventricular systems, except on the third ventricle floor and in the choroid plexus. The density of SEF was higher in the smaller species. In the rat, light- and scanning electron microscopical SEF were almost completely abolished 1 week after intraventricular administration of 5,6-dihydroxytryptamine. Ramification of SEF was complicated; the SEF formed a true network with frequent anastomosing. In the ventricular system of rats rendered hydrocephalic by kaolin administration, the mode of axonal branching in the supraependymal plexus could best be analyzed by the scanning electron microscope because the meshes of the plexus were spread out.     

Transient glucose intolerance during attacks of thyrotoxic periodic paralysis

In a 19-year-old Japanese male (case 1) with thyrotoxic periodic paralysis (TPP), an increase of plasma glucose concentration together with abnormally high levels of serum immunoreactive insulin (IRI) was observed preceding a spontaneous attack of paralysis. Therefore, the plasma glucose, glucagon, epinephrine, norepinephrine, serum IRI, growth hormone and cortisol levels, and the erythrocyte insulin receptors were measured in case 1 and a 40-year-old Japanese male (case 2) with TPP during attacks of paralysis induced by prolonged glucose loading. In case 1, the serum IRI concentration was elevated to the extraordinarily high level of 655.0 microU/ml at the beginning of paralysis, and at that time, the plasma glucose concentration was 147 mg/dl. However, when paralysis was not induced by a similar glucose loading during methimazole treatment, the serum IRI and plasma glucose levels at the corresponding time after glucose loading were 20.9 microU/ml and 87 mg/dl, respectively. Furthermore, the affinity of the erythrocyte insulin receptors was decreased during the attack. In case 2, plasma glucose and serum IRI concentrations were increased in accordance with the initiation of paralysis although the blood levels of hormones counteracting insulin were not significantly changed. These findings suggest that there is something interacting with the normal action of the insulin in the early phase of paralysis.     

Structural characterization of novel complex oligosaccharides accumulated in the caprine beta-mannosidosis kidney. Occurrence of tetra- and pentasaccharides containing a beta-linked mannose residue at the nonreducing terminus

Four oligosaccharide fractions were isolated and purified from the kidney of goats affected with beta-mannosidosis by repeating Bio-Gel P-2 column chromatography. The structural characterization of the purified oligosaccharide fractions (oligosaccharides A, B, C1,2, and D) included sugar composition analysis by gas chromatography, sugar sequence analysis by mass spectrometry of their permethylated alditols, and by methylation analysis as well as anomeric configuration studies by exoglycosidase digestions. Oligosaccharides A and B were the major oligosaccharides accumulating in the kidney and were elucidated as Man beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4GlcNAc, respectively (Matsuura, F., Laine, R. A., and Jones, M. Z. (1981) Arch. Biochem. Biophys. 211, 485-493). Oligosaccharide C1,2 was a mixture of two tetrasaccharides and oligosaccharide D was a pentasaccharide. The proposed structures are: oligosaccharide C1, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc; oligosaccharide C2, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc; oligosaccharide D, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc beta 1-4GlcNAc. Tetrasaccharide C1 and pentasaccharide D are heretofore undiscovered oligosaccharides. There is no precedent for these structures in glycoproteins or other glycoconjugates. One possibility which accounts for the presence of oligosaccharide C1 and D is that a bisecting N-acetylglucosamine (the beta-N-acetylglucosamine residue linked at the C-4 position of the beta-mannosyl residue of the trimannosyl core of the asparagine-linked sugar chains) is linked by a beta-mannosyl residue. Moreover, the detection of oligosaccharides containing two N-acetylglucosamine residues at the reducing terminus, together with those containing a single N-acetylglucosamine residue, is further corroboration of species-specific differences in glycoprotein catabolic pathways (Hancock, L. W., and Dawson, G. (1984) Fed. Proc. 43, 1552) or in glycoprotein structures.     

Fetus-in-fetu: report of a case

A 5 month-old female was brought to our clinic because of diarrhea and abdominal distension. A plain radiograph demonstrated a mass with a vertebral column in the right upper quadrant of the abdomen. At operation a mass was found to be retroperitoneal, well encapsulated, and connected to the abdominal aorta of the host by two small vessels; no other connections and adhesions were seen between the mass and the host. The ovaries, uterus, and other pelvic and abdominal viscera of the host were normal. The mass was diagnosed as a fetus-in-fetu. The fetus-in-fetu, encapsulated with an amniotic capsule, was covered with skin and had a top with long hair, two protuberances, an amniotic hernial sac, upper limbs with syndactylic fingers, a gluteal region, and lower limbs with polysyndactylic toes. A brain mass and a spinal cord were identified in the cranial cavity and the vertebral canal. Several spinal ganglia and a nerve plexus were found. A noselike structure, upper lip, maxillalike bone with teeth, tonguelike structure, intestines, ribs, bones of the extremities, and skeletal muscles were also identified. A cloacalike cyst was observed to have an opening in the external female genitalia. Microscopically, a small number of motor neurons were seen in the brain mass and the anterior horn of the spinal cord. In the spinal ganglia, ganglion cells were differentiated. The submucosal and myenteric plexuses were seen in the intestinal wall. Well-differentiated muscle fibers were often accompanied with myelinated nerve fibers. Hematopoiesis was observed in the cranial bone marrow. The presence of the sex chromatin was confirmed in the nuclei of motor neurons and polymorphonuclear leukocytes. Thus, the present fetus-in-fetu, which was connected to the abdominal aorta of the host by two vessels, was a monozygotic twin which developed within its own amniotic cavity.     

Electron microscopical findings with special reference to cancer in rats caused by inhalation of nickel oxide

A special exposure system was used for the inhalation of nickel oxide (NiO) aerosol by Wistar male rats. The median aerodynamic diameter and the geometric standard deviation were 1.2 μm and 2.2, respectively. A histopathological study of the rats was performed immediately, and at intervals of 12 and 20 mo after a 1-mo expsoure to NiO. Electron microscopy showed that localization of NiO particles was restricted to the lungs and that each particle had been engulfed by the alveolar macrophages. Type II pneumocytes and nonciliated bronchiolar epithelial cells (Clara cells), as well as numerous tubular myelin (surfactant) in the alveoli were prominent. In rats dissected after 12 mo, clusters of NiO particles were still present within the terminal bronchioli, alveolar walls, and lysosomes of the alveolar macrophages. Pools of tubular myelin were observed in the peribron-chial lymphatics. The Clara cells, which project into the lumen of bronchioli, showed active secretion and were filled with smooth en-doplasmic reticulum (SER) in the apical cytoplasm. In the experimental group sacrificed after 20 mo, one rat had papillary adenocarcinoma and two rats showed adenomatosis in the peripheral portion of the lung, but none in the upper respiratory tract.     

A novel cell-surface antigen expressed on most leukocytes and a minor cortisone-resistant population of thymocytes in rats: characterization by monoclonal antibodies

A cell-surface antigen on rat lympho-hemopoietic cells was determined by using a monoclonal antibody, R2-1B3 (1B3). The 1B3 antibody, when tested for its reactivity with different hemopoietic cells by cytofluorography with a FACS analyzer, labeled more than 80% of lymph node, spleen, and bone marrow cells and 10-20% of thymus cells. Cytofluorographic analysis performed on purified rat T cells, B cells, macrophages, and granulocytes demonstrated that the antigen defined by 1B3 was readily detectable on all of these cell types, with the greatest expression on B cells. A minor population of thymocytes that were labeled by 1B3 appeared to be cortisone-resistant and were located mainly in the thymic medulla. These 1B3 positive thymic cells seemed to be functionally more mature than 1B3-negative thymus cells as suggested by the fact that the cytotoxic treatment of thymus cells with 1B3 antibody and complement (C) resulted in significant reduction of their responsiveness to phytomitogens and lymphokines derived from concanavalin A (con A) activated rat spleen cell cultures. Immunochemical data showed that 1B3 antibody recognized the broad ill-defined band with a molecular weight of 32K to 47K daltons as estimated by SDS-polyacrylamide gel electrophoresis. These data indicate that the 1B3 defined antigen is distinct from other, previously reported, antigens on rat lymphoid cells including leukocyte-common (L-C) and MRC OX-22 antigens, and that this 1B3 antibody is a useful reagent for analyzing the intrathymic differentiation of T cells in rats.     

Macrophage Activation by Tetrahymena pyriformis. I. Active Subcellular Fractions of Tetrahymena

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H₂O₂) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H₂O₂ release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Metabolism of apolipoprotein E-containing human plasma lipoproteins by rat and human cells in culture.

Cultured preadipocytes from rat epididymal fat pads were able to bind, internalize, and degrade human plasma very-low-density lipoproteins (VLDL) more efficiently than low-density lipoproteins (LDL). VLDL, but not LDL, activated acyl-CoA: cholesterol acyltransferase (ACAT) and increased cholesterol accumulation in these cells. However, trypsin-treated VLDL (T-VLDL) lost the capacity to bind, activate ACAT, and increase cholesterol accumulation. After the treatment of VLDL with trypsin, SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that apolipoprotein E (apo E) was completely degraded, whereas apolipoprotein CII (apo C-II) was preserved. ApoE complexed with dimyristoyl phosphatidylcholine (DMPC) was able to complete with VLDL for binding to the cells. Although T-VLDL did not bind to the preadipocytes, these cells accumulate triacylglycerols from T-VLDL, presumably after lipolysis, as efficiently as from native VLDL. Rat smooth muscle cells and skin fibroblasts also bind and metabolize human VLDL better than LDL. However, human skin fibroblasts and omental preadipocytes metabolized LDL better than VLDL. These studies indicate that rat tissues can recognize and metabolize apoE-containing human plasma VLDL although they cannot recognize human LDL.