Biomedical subjects
Publications and source records attributed to T Hatae.
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.
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.
Angiotensinogen, the precursor of a vasoactive octapeptide angiotensin II, is the only known natural substrate of renin, and its reaction exhibits strict species specificity and is the rate-limiting step in the renin-angiotensin system that controls the blood pressure. We measured blood pressure and heart rate of the transgenic mice with overproduced human angiotensinogen, and showed no significant difference in these parameters between transgenic and nontransgenic mice. We also provided evidence that mouse renin could not cleave human angiotensinogen, indicating a lack of angiotensin production from the human substrate. These results suggested that the blood pressure of transgenic mice is normally maintained, probably due to the inability of mouse renin to release angiotensin from the transgene products.
To examine whether expression of human renin in the transgenic mouse kidney is regulated in a cell-specific manner, we have characterized monoclonal antibodies against human renin and determined the renin-production site by immunohistochemistry. By using a monoclonal antibody specific for human renin, A6-11-6, we demonstrated that human renin in the transgenic mouse kidney is localized to the juxtaglomerular cells of afferent arterioles.
A 30-year-old man showed exercise-induced myalgia, calf muscle hypertrophy and high serum creatine kinase level, without muscular weakness. The symptoms began in childhood and did not progress. Electromyographic findings were consistent with myopathy, and the muscle histology showed nonspecific myopathic changes without evidence of storage of glycogen or lipid. Immunohistochemical staining with antibodies raised against three different dystrophin peptides revealed proper subcellular localization of dystrophin at the sarcolemma of all myofibers, but the intensity of the stain was decreased. Western blot analysis using the same antibodies revealed normal dystrophin in size, but showed the reduced amount of the protein. The DNA obtained from the patient's peripheral leukocyte showed a deletion of the dystrophin gene including exon 45 by using PCR technique. Though the detailed size and portion of the deleted gene are not ascertained, the deletion in this case may not severely affect the function of dystrophin, unlike cases of Duchenne or Becker muscular dystrophy. Dystrophin analysis is useful for the etiologic diagnosis in cases of myalgia and high CKemia.
The organization of the endosomal compartment in the rat kidney proximal tubule cell was studied by computer-aided three-dimensional reconstruction based on tracing of serial thin sections. Besides the well-documented endocytic elements such as small vesicles, tubules and large vacuoles, the endosomal compartment contained a prominent structural complex consisting of a central vacuolar element (with diameters up to 2000 nm) and associated systems of radiating tubular profiles. The large endosomes examined had from two to twenty-four tubules as straight or bending extensions directed randomly from the central vacuole. The tubules were 70-90 nm in diameter and varied with the length up to 1500 nm. Quantitative evaluation from examples of the large endosomes with more than ten tubular extensions, showed that 25-40% of the membrane resided in the tubular portion in contrast to 5-10% of the volume. These results indicate the high efficiency of the role of the endosomal tubules as a mediator of the membrane recycling, because the tubules simultaneously allow retention of most of the content in the central vacuole during the recycling process.
This paper reports the nature of abnormally expressed Forssman (F) antigen in the lymph node cells of MRL/MpJ-lpr/lpr, autoimmune mice, and also reports its autoantibody in sera. By acetylation study of the F antigen with [14C]acetic anhydride, we concluded that the F antigen was not a glycolipid but a glycoprotein. Several bands of F-active glycoproteins were identified on a nitrocellulose sheet after purification by an anti-F antibody affinity column. Hemolysis of SRBC by some sera from MRL/MpJ/lpr/lpr was inhibited by purified F glycoprotein and also by F glycolipid. The antibody in the serum, however, seemed to be more specific for F glycoproteins than F glycolipid, but the opposite was the case for rabbit anti-F glycolipid antibody. No significant difference of the SRBC hemolysis levels was observed between the sera from MRL/MpJ-lpr/lpr and its congenic MRL/MpJ-+/+ mice.
In the jejunal absorptive cells of suckling rats, a distinctive membranous body was found in their lateral cytoplasm and the Golgi region. This organelle was ovoid in contour (1 to 2 microns in diameter) and consisted of highly convoluted cisternae of a rough endoplasmic reticulum (RER). The individual cisternae were round (approximately 55 nm in diameter) with variable electron-dense content and separated by uniformly narrow channels (approximately 25 nm in width) which were continuous to the cytoplasmic matrix. In a proper section, the cisternae arranged in an approximately hexagonal pattern, and one of the cisternae was seen occupying the center of the hexagon. The membranous body was in continuity at its margin with the cisternae of a smooth endoplasmic reticulum (SER), and with the dilated cisternae which often contain chylomicron lipid droplets. It is possible that this structure serves as a kind of "reservoir" of the SER membranes for the lipid synthesis of the jejunal absorptive cells.
Computer-aided three-dimensional models of the helical structure within an apical tubule (AT) of several absorbing epithelia (kidney proximal tubule, visceral yolk sac, and ductuli efferentes) were constructed using ray-tracing graphics software to further understand the highly ordered structural configuration. Our previous electron microscopic studies using thin-section technique have first revealed the helical structure within the AT fixed in situ with a mixture of formaldehyde, glutaraldehyde, and osmium tetroxide. In the present study, we construct a computer-aided three-dimensional model of the helical structure in the AT to explain quantitative data obtained by the electron microscopy. The model could well explain several aspects of electron microscopical images and enables us to understand more clearly the three-dimensional configuration of the unique structure associated with the AT.
Transgenic mice carrying human renin gene were produced by microinjection of 15 kilobases (kb) DNA molecules with up to 3 kb of 5'-flanking sequence and 1.2 kb of 3'-flanking sequence. The transgenes have been shown to be stably transmitted to progeny. It was revealed by RNase protection assay that the human renin gene in a transgenic mouse is expressed preferentially in the kidney. The human renin RNA was also detected at a small level in a variety of tissues such as brain, heart, lung, pancreas, spleen, stomach, testis, and thymus. The direct radioimmunoassay using a monoclonal antibody specific for the active site of human renin demonstrated the synthesis of human active renin in the transgenic mouse kidney. These results suggest that the human renin gene in the transgenic mouse is regulated in a tissue-specific manner.
The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a 'bisecting' acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain.
1. The microheterogeneous alpha molecules of class II antigen, DR molecules obtained from human B cell line and I-A molecules from mouse B cell hybridoma cell line, were separated by 2-D PAGE, transferred onto NC sheets and N-linked oligosaccharide types were analyzed by staining with P.O./lectins. 2. This is the first report to show directly the type of oligosaccharide chain corresponding to each spot separated by 2-D PAGE. The glycosylation patterns of class II alpha chains in human and mouse were compared.
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.
The ileal absorptive cells of suckling rats exhibit high levels of endocytic activity being engaged in nonselective uptake of macromolecules from the intestinal lumen. The apical cytoplasm usually contains an extensive network of small, membrane-limited tubules (apical tubules: AT), in addition to newly formed endocytic vesicles and large endocytic vacuoles. To determine whether the AT are directly involved in the endocytic process by carrying the tracer into the cell, we have analysed movements of the apical cell membrane of the ileal absorptive cells by using a membrane-bound tracer (horseradish peroxidase-labelled concanavalin-A: Con-A HRP). The ileal absorptive cells were exposed in vitro to Con-A HRP for 10 min at 4 degrees C, incubated for different times in Con-A free medium at 37 degrees C, and prepared for electron microscopy. After 1 min incubation at 37 degrees C, invaginations of the apical cell membrane, including coated pits, and endocytic vesicles were labelled with HRP-reaction product, whereas the AT and large endocytic vacuoles were negative. After 2.5 min, almost all the large endocytic vacuoles were labelled with reaction product, which was seen in their vacuolar lumen and along the luminal surface of their limiting membrane. A few AT with reaction product were seen in the apical cytoplasm; they were in frequent connection with the reaction-positive large endocytic vacuoles. With increasing incubation time, the number of the labelled AT increased. Thus, after 15 min at 37 degrees C, the apical cytoplasm was fully occupied by the reaction-positive AT. The ends of these AT were often continuous with small spherical coated vesicles. No reaction product was detected in the Golgi complex at any time after incubation. These observations indicate that the AT located in the apical cytoplasm probably originate by budding off from the large endocytic vacuoles, rather than being involved in the process of endocytosis.
The cells of the kidney proximal segment of the migrating arctic lamprey, Lampetra japonica, contain particles of the same size, electron-density and intracellular location as particles identified by others as very low-density lipoproteins (VLDL) in the liver and intestine of teleost fishes and lampreys. These particles are synthesized within the cisternae of the smooth endoplasmic reticulum and elements of the Golgi complex. They are transferred to the lateral intercellular space and lamina propria by way of the Golgi vesicles and an intracellular channel system. Some particles are discharged into the lumina of the sinusoidal capillaries of the lamina propria. Although the physiological role of lipoprotein secretion in the renal proximal segment cells is unknown, the present observations provide morphological evidence that the kidney of the arctic lampreys synthesizes lipoproteins and releases them into the circulation at the time when they are undertaking their anadromous migration.
Using horseradish peroxidase (HRP) as a tracer, we have investigated if the so-called apical tubules (AT) in the kidney proximal tubule cells are directly involved in the endocytic process by carrying the tracer into the cells, or if they are derived from the intracellular membrane compartments. Rat kidney was fixed by vascular perfusion at different time intervals after intravenous injection of HRP and prepared for electron microscopy. An analysis revealed that 0.5 min after injection, invaginations of the plasma membrane and small apical endocytic vesicles, including coated vesicles, were labelled with reaction product, whereas almost all large apical endocytic vacuoles and the AT were negative. The endocytic vacuoles and about 18% of the AT were labelled 1 min after injection. The reaction product in the large endocytic vacuoles was usually seen along the luminal surface of the vacuoles. The AT with reaction product appeared as a branched network, and were frequently connected with the labelled endocytic vacuoles. Three min after injection, reaction product was detected in about 38% of the AT, and thereafter, the percentage increased to about 74% after 7 min. No reaction product was detected in the Golgi complex at any time after HRP-injection. These findings indicate that the AT are probably formed by budding off from the large endocytic vacuoles, rather than being directly involved in the endocytic process.
Unique and highly ordered structures were discovered in the so-called apical tubules of several absorbing epithelia (kidney proximal tubule, visceral yolk sac and ductuli efferentes) fixed in situ with a mixture of formaldehyde, glutaraldehyde and osmium tetroxide. The apical tubules were especially numerous in the apical cytoplasm, in addition to the invaginations of the apical plasma membrane, newly formed endocytic vesicles and large endocytic vacuoles. They showed a cylindrical structure (approximately 80 nm in diameter) limited by a smooth membrane. Helically wound parallel rows of particles (approximately 11 nm in diameter) were found in the apical tubules in close proximity to their limiting membrane. The structure of the helix was determined by following the rows through serial sections and semithin sections, and was found to be a left-handed quadruple helix. These particles surround an electron-lucent cylinder (approximately 35 nm in diameter), containing at its center a single row of particles (approximately 9 nm in diameter). The apical tubules with the luminal specializations were not seen in continuity with the apical plasma membrane, but were frequently connected with the large endocytic vacuoles, which were present in the deeper levels of the apical cytoplasm. From these observations, it is suggested that the apical tubules are not derivatives of the apical plasma membrane; rather, they represent an intracellular compartment, which is morphologically related to the large endocytic vacuoles.