[Organization and regulation of cells].
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Animals↗
Biomedical subjects
Y Hamaguchi
Publications and source records attributed to Y Hamaguchi.
[Current status and problems of home parenteral nutrition for terminal cancer patients from the viewpoint of indication].
Since June 1993, we have been performing home parenteral nutrition (HPN) for end-stage cancer patients. We studied how many patients could be treated with HPN, why they were not able to be treated with HPN in the terminal stage cancer patients, 158 cases, who admitted and died in our ward from June 1993 to April 1997. Eighty-six patients (54.4%) were considered to not be indicated for HPN, due to general weakness (28 cases), dyspnea (17 cases), need for medical care other than HPN (34 cases), and inability to understand this treatment (1 case). Fifty-six patients (35.4%) were considered to have been able to be treated with HPN and could have had returned home. Fifteen patients (26.8%) were given HPN. Four cases wanted to stay in the hospital after they knew the truth and the prognosis of their disease. Another four patients became weak while preparing for HPN and could not go home. Eighty percent of patients could not to be treated with HPN because of the factors of their family. Eight patients had no family to cared for them. Twelve patients were refused to the truth of their disease. The families of 13 cases refused to accept and care for them in their home. We may conclude that co-operation among patients, their family and the medical staff is the most important factor in providing HPN for end-stage cancer patients.
Adult↗
Calcium-dependent bidirectional power stroke of the dynein arms in sea urchin sperm axonemes.
Active sliding between doublet microtubules of sea urchin sperm axonemes that were demembranated with Triton X-100 in the presence or absence of calcium was induced with ATP and elastase at various concentrations of Ca2+ to examine the effects of Ca2+ on the direction of the power stroke of the dynein arms. Dark-field light microscopy of microtubule sliding revealed that the sliding from the axonemes demembranated with Triton and millimolar calcium and disintegrated with ATP and elastase showed various patterns of sliding disintegration, including loops of doublet microtubules formed near the head or the basal body. These loops were often thicker than the remaining axonemal bundle. In contrast, only thinner loops were found from the axonemes demembranated with Triton in the absence of calcium and disintegrated with ATP and elastase at high Ca2+ concentrations. Electron microscopic examination of the direction of microtubule sliding showed that the doublet microtubules in the axonemes demembranated in the presence of millimolar calcium moved toward the base of the axonemes by the dynein arms on the adjacent doublet microtubule as well as by their own dynein arms. Doublet microtubules in the axonemes demembranated in the absence of calcium moved toward the base of the axonemes only by their own dynein arms. Similar observations have been obtained from the axonemes from which the outer dynein arms were selectively extracted. From these observations, we can conclude that the dynein arms generate force in both directions and this feature of the dynein arms arises from at least the inner dynein arms.
Animals↗
[Polymyositis with rimmed vacuoles in a case of chronic thyroiditis].
We report a 37-year-old woman who developed chronic thyroiditis, and polymyositis with rimmed vacuoles (RVs). She developed paresthesia of glove and stocking type, muscle weakness of the lower extremities, and slowness in motion. On examination, her lower legs were atrophic and weak, more prominent on the right, while proximal muscles in the upper and lower extremities were slightly affected. Deep tendon reflexes of the upper and lower extremities were normal except for the absent Achilles tendon reflexes. Serum CK was high (1,809 IU/l), and laboratory data indicated chronic thyroiditis and hypothyroidism. Muscle CT showed severe fatty changes in the gastrocnemius and soleus muscles, more marked on the right, and slight atrophy of the thigh muscles. Electromyography and nerve conduction velocity study revealed peripheral neuropathy. Muscle biopsy revealed numerous myofibers with RVs, together with myofiber necrosis, phagocytosis and lymphocyte infiltration, consistent with myositis. Nerve biopsy was indicative of slight axonal degeneration. A diagnosis of polymyositis with RVs in combination with chronic thyroiditis was made. Prednisolone combined with levothyroxine markedly improved muscle weakness though atrophy and weakness of the lower legs were barely improved. Distal-dominant asymmetric affection of the leg muscles and many myofibers with RV on the biopsied muscle are atypical and rare findings of polymyositis. Associated chronic thyroiditis and neuropathy may be related to the formation of marked rimmed-vacuolar degeneration of myofibers in the present case.
Adult↗
Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-J kappa.
To map regions important for DNA binding of the mouse homologue of Suppressor of Hairless or RBP-J kappa protein, mutated mouse RBP-J kappa cDNAs were made by insertion of oligonucleotide linkers or base replacement. DNA binding assays using the mutated proteins expressed in COS cells showed that various mutations between 218 Arg and 227 Arg decreased the DNA binding activity drastically. The DNA binding activity was not affected by amino acid replacements within the integrase motif of the RBP-J kappa protein (230His-269His). Replacements between 291Arg and 323Tyr affected the DNA binding activity slightly but reproducibly. These results indicate that the region encompassing 218Arg-227Arg is critical for the DNA binding activity of RBP-J kappa. This region did not show any significant homology to motifs or domains of the previously described DNA binding proteins. Using a truncation mutant protein RBP-J kappa was shown to associate with DNA as a monomer.
Alternative Splicing↗
Recognition sequence of a highly conserved DNA binding protein RBP-J kappa.
DNA binding specificity of the RBP-J kappa protein was extensively examined. The mouse RBP-J kappa protein was originally isolated as a nuclear protein binding to the J kappa type V(D)J recombination signal sequence which consisted of the conserved heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences separated by a 23-base pair spacer. Electrophoretic mobility shift assay using DNA probes with mutations in various parts of the J kappa recombination signal sequence showed that the RBP-J kappa protein recognized the sequence outside the recombination signal in addition to the heptamer but did not recognize the nonamer sequence and the spacer length at all. Database search identified the best naturally occurring binding motif (CACTGTGGGAACGG) for the RBP-J kappa protein in the promoter region of the m8 gene in the Enhancer of split gene cluster of Drosophila. The binding assay with a series of m8 motif mutants indicated that the protein recognized mostly the GTGGGAA sequence and also interacted weakly with ACT and CG sequences flanking this hepta-nucleotide. Oligonucleotides binding to the RBP-J kappa protein were enriched from a pool of synthetic oligonucleotides containing 20-base random sequences by the repeated electrophoretic mobility shift assay. The enriched oligomer shared a common sequence of CGTGGGAA. All these data indicate that the RBP-J kappa protein recognizes a unique core sequence of CGTGGGAA and does not bind to the V(D)J recombination signal without the flanking sequence.
Animals↗
Different reactivity with monoclonal anti-tubulin antibodies between native and fixed mitotic microtubules in sea urchin eggs.
The effect on fixation on the reactivities of mitotic microtubules with monoclonal anti-tubulin antibodies was investigated by the indirect immunofluorescence procedure. All of the seven antibodies used intensely stained mitotic microtubules in sea urchin eggs lysed and fixed with methanol at -20 degrees C, whereas only two of them stained the stabilized microtubules in the lysed eggs before the fixation. The other five did not stain the mitotic microtubules even after microtubule components other than tubulin were removed by treating the lysed eggs with 0.4 M KCl solution containing taxol. These results exclude the possibility that the fixation affects proteins, which interact with microtubules including microtubule-associated proteins (MAPs) and interfere with the binding of monoclonal antibodies with tubulin, and strongly suggest that the fixation directly affects the three-dimensional conformation of tubulin. Furthermore, microinjection of these antibodies indicated the results as follows [combining the results reported previously; Oka et al., 1990: Cell Struct. Funct. 15: 373-378]: The antibodies which stained mitotic microtubules stabilized in the lysed eggs induced disassembly of native mitotic microtubules in the living eggs, but those which did not stain the stabilized microtubules did not disassemble the native microtubules. From these results, it is suggested that the monoclonal antibodies which stain microtubules in the eggs lysed but not fixed are useful for microinjection experiments.
Animals↗
Sperm-egg fusion in the sea urchin is blocked in Mg(2+)-free seawater.
Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al., Dev. Growth Differ. 22, 531-41, 1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2+ to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+ concentration of the egg was observed after insemination in the absence of Mg2+, although both could be observed after the addition of Mg2+. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2+ are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane.
Animals↗
Quantitation and immunocytological identification of interleukin-1 in nasal polyps from patients with chronic sinusitis.
Immunoreactive interleukin-1 (IL-1) alpha and beta levels in nasal polyp (NP) extracts were measured from 22 adult patients with chronic sinusitis (CS) in order to determine their significance in the pathogenesis of NP. The NP sample was separated into mononuclear and polymorphonuclear fractions. In the mononuclear fraction, the mean value of IL-1 beta was 21.5 pg/ml/g, which was significantly higher than that of IL-1 alpha (8.4 pg/ml/g, p < 0.01). In younger adults, the IL-1 alpha and IL-1 beta levels were 21.2 and 59.4 pg/ml/g, which were significantly higher than those in older CS patients (p < 0.01). There was a significant reverse correlation between patient's age and IL-1 beta levels in the mononuclear fraction (r = -0.509, p < 0.01). Immunoreactive IL-1, mainly IL-1 beta, was identified in the cytoplasmic area of monocytes. A certain amount of immunoreactive IL-1 is produced in mononuclear leukocytes, particularly activated monocytes, and IL-1 beta production is greater than IL-1 alpha. In younger adult CS patients, NPs contain larger amounts of IL-1 beta in monocytes, compared to those of older patients.
Adolescent↗
Human Jk recombination signal binding protein gene (IGKJRB): comparison with its mouse homologue.
The mouse Igkjrb protein specifically binds to the immunoglobulin Jk recombination signal sequence. The IGKJRB gene is highly conserved among many species such as human, Xenopus, and Drosophila. Using cDNA fragments of the mouse Igkjrb gene, we isolated its human counterpart, IGKJRB. The human genome contains one functional IGKJRB gene and two types of processed pseudogenes. In situ chromosome hybridization analysis demonstrated that the functional gene is localized at chromosome 3q25, and the pseudogenes (IGKJRBP1 and IGKJRBP2, respectively) are located at chromosomes 9p13 and 9q13. The functional gene is composed of 13 exons spanning at least 67 kb. Three types of cDNA with different 5' sequences were isolated by rapid amplification of cDNA ends, suggesting, the presence of three proteins. The aPCR-1 protein, which possessed the exon 1 sequence, was the counterpart of the mouse RBP-2 type protein. The aPCR-2 and 3 proteins may be specific to human cells because the mouse counterparts were not detected. The amino acid sequences of the human and mouse IGKJRB genes were 98% homologous in exons 2-11, whereas the homology of the human and mouse exon 1 sequences was 75%.
Amino Acid Sequence↗
Quantification of interleukin-1 in nasal polyps from patients with chronic sinusitis.
We have measured interleukin-1 alpha (IL-1 alpha) and beta levels in nasal polyps (NP) from patients with chronic sinusitis (CS) in order to determine their significance in the pathogenesis of NP. NP in ten cases (five male and five female; age range, 17-63 years) were removed to separate mononuclear (M) and polymorphonuclear (PMN) fractions through Ficoll-Paque separation media. ELISA measurements of the M fraction showed that the mean value of IL-1 beta was 17.8 pg/ml per gram, which was significantly higher than that of IL-1 alpha (7.04 pg/ml per gram, P < 0.01, Wilcoxon test). In the PMN fraction, the mean value of IL-1 alpha was 8.79 pg/ml per gram and that of IL-beta was 7.85 pg/ml per gram, which was not significantly different. Mononuclear leukocytes, particularly activated monocytes, seem to be a major source of IL-1 in the NP taken from patients with CS.
Adolescent↗
Calcium ion regulation of chirality of beating flagellum of reactivated sea urchin spermatozoa.
Near an interface, sea urchin spermatozoa swim almost in circles. The direction is usually clockwise at the lower surface of a coverslip and counterclockwise at the upper surface of a glass slide, when viewed from above. Examination of demembranated spermatozoa has shown that Ca2+ regulates the direction of the circular motion of spermatozoa reactivated with adenosine triphosphate (ATP). This finding suggests that Ca2+ changes the chirality of the three-dimensional bending waves of sperm flagella.
Animals↗
A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs.
Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins. C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the cleavage furrows. C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22,000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human platelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively involved in the organisation and maintenance of the contractile ring.
ADP Ribose Transferases↗
[A case of oral tuberculosis suspected malignancy].
Oral tuberculosis with pulmonary tuberculosis is very rare in Japan. A 45-year old man admitted to our hospital because of spontaneous teeth extraction and pain in oral cavity for the past 3 months. The painful granulation in palate and fistel of 7th tooth root defect in right upper gum were observed. The diagnosis of oral tuberculosis was made by the histological examination of biopsy material and positive smear test for M. tuberculosis in surface of granulation. Chest X-ray showed multicavitary lesions in bilateral upper lobs and spread shadows in bilateral lower lung fields. He was treated with chemotherapy (INH, RFP, SM and EB) and with tube feeding. Five month's chemotherapy was needed to achieve cured granulation and negative smear test for M. tuberculosis in sputum. He was discharged 10 months after admission.
Diagnosis, Differential↗
Genomic organization of mouse J kappa recombination signal binding protein (RBP-J kappa) gene.
We have isolated a cDNA clone (RBP-2) for the protein (RBP-J kappa) which binds to immunoglobulin recombination signals with 23-base pair spacers (Matsunami, N., Hamaguchi, Y., Yamamoto, Y., Kuze, K., Kangawa, K., Matsuo, H., Kawaichi, M., and Honjo, T. (1989) Nature 342, 934-937). During further screening of a cDNA library from the same mouse pre-B cell line (38B9), we have isolated a second cDNA clone (RBP-2N) which differs from RBP-2 in its 5' sequence. RNase protection assays indicated that the RBP-2N type mRNA was produced in 10-20 times the quantity as RBP-2 mRNA. To elucidate the relationship between these two mRNAs, we analyzed the genomic organization of the RBP-J kappa gene. Southern hybridization of mouse genomic DNA detected at least 7 EcoRI fragments hybridizing to an RBP-2 cDNA probe, suggesting a complex structure for the RBP-J kappa gene. Cloning of each EcoRI fragment revealed one functional RBP-J kappa gene and three related genes. The functional gene was composed of 11 exons and spanned at least 50 kilobase pairs. The sequence of exon 1 and its 5'-flanking region contained a GC-rich promoter-like region but no apparent TATA box. The initiation site of transcription was heterogeneous, and the two types of mRNA are produced from the same exon by transcription initiation at different sites and by different usage of splice signals. Two of the three related genes were processed pseudogenes with scattered stop codons. The other was also a processed gene with a sequence exactly the same as that of RBP-2, except that this gene lacked the sequence corresponding to the first exon of the functional gene.
Animals↗
Mitotic apparatus formation and cleavage induction by micromanipulation of the nucleus and centrosome: the centrosome forms a spindle together with only the chromosomes at a short distance.
We micromanipulated the nucleus and centrosomes in the zygote of the starfish, Asterina pectinifera, in order to investigate their roles in mitotic apparatus formation and cleavage induction. The zygote cleaved without spindle formation when its nucleus was removed. When one or two centrosomes were transplanted, they formed asters in the recipient cell, which cleaved into three or four blastomeres so that each blastomere might contain one centrosome or aster. When one centrosome was removed, a half-spindle formed in the manipulated cell, which did not cleave until the other centrosome was duplicated. When both centrosomes were removed, no microtubular structures such as the spindle and the aster appeared in the manipulated cell, which failed to cleave. These results indicate that two centrosomes or more in the cell induce cleavage with or without the nucleus and that one centrosome or less does not induce cleavage. It is also concluded that the centrosome(s) together with the nucleus forms a half-spindle or bipolar spindle. However, from the experiments of nucleus transplantation and displacement, spindle formation is found to depend on the distance between chromosomes and centrosomes. The half-spindle formed when the distance from the centrosome to the chromosomes was shorter than 22 microns; on the other hand, when the distance was longer than 22 microns, the nucleus remained apart from the aster, which means that the functional range of the astral microtubule's ability to engage chromosomes was 22 microns from the centrosome.
Animals↗
Biochemical and immunological characterization of the DNA binding protein (RBP-J kappa) to mouse J kappa recombination signal sequence.
We have investigated whether J kappa recombination signal sequence (RS) binding protein (RBP-J kappa) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA. Murine RBP-J kappa protein purified by J kappa-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity. To obtain a large amount of purified RBP-J kappa protein, recombinant RBP-J kappa was synthesized in Escherichia coli as a fusion protein and also in silkworm cells. Although recombinant RBP-J kappa produced in silkworm cells could bind J kappa-RS, it failed to show either ligase or DNA bending activity. Since the DNA affinity-purified RBP-J kappa has the ligase activity, the RBP-J kappa protein may form a complex with a ligase in vivo. We have raised monoclonal antibodies against the RBP-J kappa fusion protein which was synthesized in E. coli and unable to bind J kappa-RS. Using the anti-RBP-J kappa monoclonal antibody we have shown that the RBP-J kappa protein is expressed ubiquitously in mammalian tissues. The ubiquitous expression of the RBP-J kappa protein is consistent with the hypothesis that the RBP-J kappa protein may have dual function [Furukawa et al. (1991) J. Biol. Chem. 266, 23334-23340].
Animals↗