Uric acid, indoxyl sulfate, and methylguanidine activate bulbospinal neurons in the RVLM via their specific transporters and by producing oxidative stress.

Patients with chronic renal failure often have hypertension, but the cause of hypertension, other than an excess of body fluid, is not well known. We hypothesized that the bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are stimulated by uremic toxins in patients with chronic renal failure. To investigate whether RVLM neurons are sensitive to uremic toxins, such as uric acid, indoxyl sulfate, or methylguanidine, we examined changes in the membrane potentials (MPs) of bulbospinal RVLM neurons of Wister rats using the whole-cell patch-clamp technique during superfusion with these toxins. A brainstem-spinal cord preparation that preserved the sympathetic nervous system was used for the experiments. During uric acid, indoxyl sulfate, or methylguanidine superfusion, almost all the RVLM neurons were depolarized. To examine the transporters for these toxins on RVLM neurons, histological examinations were performed. The uric acid-, indoxyl sulfate-, and methylguanidine-depolarized RVLM neurons showed the presence of urate transporter 1 (URAT 1), organic anion transporter (OAT)1 or OAT3, and organic cation transporter (OCT)3, respectively. Furthermore, the toxin-induced activities of the RVLM neurons were suppressed by the addition of an anti-oxidation drug (VAS2870, an NAD(P)H oxidase inhibitor), and a histological examination revealed the presence of NAD(P)H oxidase (nox)2 and nox4 in these RVLM neurons. The present results show that uric acid, indoxyl sulfate, and methylguanidine directly stimulate bulbospinal RVLM neurons via specific transporters on these neurons and by producing oxidative stress. These uremic toxins may cause hypertension by activating RVLM neurons.

Synthesis and evaluation of a difluoromethylene analogue of sphingomyelin as an inhibitor of sphingomyelinase.

A sphingomyelin analogue 2, in which the long alkenyl chain and the phosphodiester moiety of sphingomyelin were replaced by a phenyl and an isosteric difluoromethylenephosphonic acid, was prepared to evaluate its inhibitory potency to sphingomyelinase. The analogue non-competitively inhibited the neutral sphingomyelinase in bovine brain microsomes with an IC50 of 400 microM. The compound had the ability to suppress tumor necrosis factor alpha-induced apoptosis of PC-12 neurons at a low concentration of 0.1 microM.

A novel subtype of the prostacyclin receptor expressed in the central nervous system.

By use of several prostacyclin analogs and an in vitro autoradiographic technique, we have found a novel subtype of the prostacyclin receptor, one having different binding properties compared with those of the known prostacyclin receptor in the rat brain. Isocarbacyclin, which is a potent agonist for the known prostacyclin receptor, had high affinity for the novel subtype (dissociation constant (Kd) of 7.8 nM). However, iloprost, which is usually used as a stable prostacyclin analog, showed low affinity binding (Kd = 159 nM) for the subtype. Other prostaglandins showed no or little affinity for the subtype. [3H]Isocarbacyclin binding was high in the thalamus, lateral septal nucleus, hippocampus, cerebral cortex, striatum, and dorsal cochlear nucleus. Although the nucleus of the solitary tract and the spinal trigeminal nucleus showed a high density of [3H]isocarbacyclin binding, [3H]iloprost also had high affinity in these regions, and the binding specificity was similar to that for the known prostacyclin receptor. Hemilesion studies of striatal neurons lesioned by kainate or of dopaminergic afferents lesioned by 6-hydroxydopamine revealed that the binding sites of the novel subtype exist on neuronal cells in the striatum, but not on the presynaptic terminal of afferents or on glial cells. Electrophysiological studies carried out in the CA1 region of the hippocampus revealed that prostacyclin analogs have a facilitatory effect on the excitatory transmission through the novel prostacyclin receptor. The widespread expression of the prostacyclin receptor in the central nervous system suggests that prostacyclin has important roles in neuronal activity.

In vitro positron emission tomography (PET): use of positron emission tracers in functional imaging in living brain slices.

Positron-emitting radionuclides have short half-lives and high radiation energies compared with radioisotopes generally used in biomedical research. We examined the possibility of applying positron emitter-labeled compounds to functional imaging in brain slices kept viable in an oxygenated buffer solution. Brain slices (300 microns thick) containing the striatum were incubated with positron emitter-labeled tracers for 30-45 min. The slices were then rinsed and placed on the bottom of a Plexiglas chamber filled with oxygenated Krebs-Ringer solution. The bottom of the chamber consisted of a thin polypropylene film to allow good penetration of beta+ particles from the brain slices. The chamber was placed on a storage phosphor screen, which has a higher sensitivity and a wider dynamic range than X-ray films. After an exposure period of 15-60 min, the screen was scanned by the analyzer and radioactivity images of brain slices were obtained within 20 min. We succeeded in obtaining quantitative images of (1) [18F]fluorodeoxyglucose uptake, (2) dopamine D2 receptor binding, (3) dopa-decarboxylase activity, and (4) release of [11C]dopamine preloaded as L-[11C]DOPA in the brain slice preparation. These results demonstrate that positron emitter-labeled tracers in combination with storage phosphor screens are useful for functional imaging of living brain slices as a novel neuroscience technique.

Alternative 5' splice site selection induced by heat shock.

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression.

Stump problems in traumatic amputation.

Stump problems in amputations resulting from employment related injuries were investigated in 397 cases in the Chugoku and Shikoku districts of Japan between 1987 and 1991. Ninety-seven patients (24%) had stump problems which interfered the prosthetic fitting. Stump problems of the upper extremity were seen in about 9% (17 amputees), two thirds of which were skin troubles. Stump problems of the lower extremity were seen in about 37% (80 amputees). Certain complaints were associated with specific methods of amputation; abnormal keratosis in Syme's amputation, equinus deformity in Chopart's amputation, reduced muscle power in above the knee (A/K) amputation and joint dysfunction in below the knee (B/K) amputation. Adequate prosthetic fitting was achieved by the modification of the socket and alignment in almost all amputees with stump problems. In only two cases, Chopart's amputation required subsequent Syme's amputation due to equinus deformity with abnormal keratosis. In almost every case, stump problems are avoidable by means of surgeons' deliberate evaluation of the affected limb and adequate choice of the amputation level.

Incidence and prognosis of dysvascular amputations in Okayama Prefecture (Japan).

This survey analysed the clinical characteristics of subjects who first underwent major amputation of lower limbs necessitated by dysvascular disease during the 5 year period from 1984 to 1988. All were residents of Okayama Prefecture, Japan, and have been issued with a Physically Disabled Person's Certificate. In total, 114 dysvascular amputees, representing 58.2% of all lower limb amputations performed in the resident population during the study period, were surveyed. The underlying diagnosis was arteriosclerotic obstruction in 64.9% of the subjects, diabetic gangrene in 22.8%, acute embolism in 7.0% and Buerger's disease in 5.3%. The yearly incidence of new dysvascular amputees per 100,000 people was estimated to be 1.2 among the general population and 5.7 among those aged over 65 years. At three years after primary amputation, the survival rate was 52.3% in arteriosclerotic obstruction, and 66.7% in diabetic gangrene. Secondary amputation was performed in 17.0% of the entire group. The concurrent incidence of hemiplegic stroke was 19.8%. Among 36 amputees due to arteriosclerotic obstruction, who survived 3 years postoperatively, 10 (27.8%) were fitted with prosthetic limbs.

Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

History of prostheses and orthoses in Japan.

Until the first contact with European civilization in 1543, prostheses and orthoses were not seen in Japanese medical history. Some physicians and surgeons who studied medicine in the Dutch language understood about prostheses and orthoses before the opening of the country in 1868. From 1868 to the end of World War II (1945), prostheses and orthoses were influenced by German orthopaedic surgery. From the latter half of the 1960s the research and development of these have been advanced, because of the establishment of a domestic rehabilitation system, international cultural exchange and economic development.

Molecular cloning of a mouse 47-kDa heat-shock protein (HSP47), a collagen-binding stress protein, and its expression during the differentiation of F9 teratocarcinoma cells.

A 47-kDa heat-shock protein (HSP47) is a major collagen-binding stress protein residing in the endoplasmic reticulum, and is assumed to be a molecular chaperone specific to collagen. Two-dimensional gel electrophoresis and immunoprecipitation studies showed that the expression of HSP47 was significantly induced during the differentiation of mouse teratocarcinoma F9 cells by treatment with retinoic acid alone or with retinoic acid and dibutyryladenosine 3',5'-phosphate. The induction of type-IV collagen was also observed during F9-cell differentiation. For further analysis, we cloned cDNA encoding mouse HSP47 from a cDNA library of BALB/c 3T3 cells and performed Northern-blot analysis. The cDNA contained a signal sequence at the N-terminus and an endoplasmic-reticulum-retention signal, RDEL, at the C-terminus. An homology search revealed that mouse HSP47, as well as chick HSP47, belonged to the serine protease inhibitor superfamily. While chick HSP47 mRNA was 4.5 kb with a long (2-kb) 3' untranslated region, mouse and human HSP47 mRNA were 2.5 kb, with a 0.8-kb 3' untranslated region. Northern-blot analysis revealed that the concurrent induction of HSP47 and type-IV collagen during F9-cell differentiation, and the transient induction of HSP47 after heat shock was regulated at the level of mRNA accumulation. These results suggested that HSP47 was closely related to collagens in terms of its expression as well as in its functional relevance.

HSP47: a tissue-specific, transformation-sensitive, collagen-binding heat shock protein of chicken embryo fibroblasts.

We report the isolation and characterization of a cDNA clone encoding HSP47, a transformation-sensitive heat shock protein that binds to collagen. A cDNA library was prepared from total RNA isolated from heat-shocked chicken embryo fibroblasts and screened by using oligonucleotide mixtures prepared on the basis of the N-terminal amino acid sequence of biochemically purified HSP47. The cDNA insert contained 3,278 bp, which encoded a 15-amino-acid signal peptide and a mature protein coding region consisting of 390 amino acid residues; it also included part of the 5' noncoding region and a long 3' noncoding region. The deduced amino acid sequence revealed an RDEL sequence at the C terminus, which is a variant of the KDEL retention signal for retention of proteins in the endoplasmic reticulum. Northern (RNA) blot analyses and nuclear run-on assays established that the induction of HSP47 by heat shock and its suppression after transformation of chicken embryo fibroblasts by Rous sarcoma virus are regulated at the transcriptional level. A homology search revealed that this protein belongs to the serpin family, the superfamily of plasma serine protease inhibitors. Although structurally homologous to the serpins, HSP47 lacks the active site thought to be essential for the inhibition of proteases and does not appear to bind to intracellular proteases. HSP47 is the first heat shock protein found to be a member of the serpin superfamily. Conversely, it is the first serpin family member that is not secreted from cells, which could be explained by acquisition of the RDEL retention signal during evolution.

Amplification of both c-myc and c-raf-1 oncogenes in a human osteosarcoma.

Fourteen human bone and soft part tumor tissues were screened by Southern blot hybridization using five oncogene probes (c-myc, c-K-ras, c-fos, c-raf-1, and N-myc). Amplification of c-myc was found in two osteosarcomas and one malignant fibrous histiocytoma. One of these osteosarcomas had amplified c-raf-1 gene. Rearrangement of the amplified gene was not observed. This is the first report of c-raf-1 amplification in human cancer tissues.

Follow-up study of cartilaginous bone tumors.

A series of clinical and pathological studies were performed on 74 cartilaginous bone tumors including osteochondromas, multiple cartilaginous exostoses, chondromas, chondromatoses, benign chondroblastomas and chondrosarcomas. Resection was adequate for the osteochondromas, and no recurrence was observed. Out of 14 multiple cartilaginous exostoses, three, all in flat bones showed malignant change. The predominant sites of chondroma were the finger and toe bones, and curettage and bone graft was adequate treatment. Neither recurrence nor malignant change was observed. Two cases of chondromatosis, one of Ollier's disease and one of Maffucci's syndrome, were included in our series. Leg length discrepancy and pathologic fracture were common problems in chondromatosis. Moreover, malignant change was suspected in a hemangioma of the Maffucci's syndrome patient. Benign chondroblastoma was treated by curettage and bone graft, with no recurrence. In our series, 4 primary and 3 secondary chondrosarcomas were observed. Metastasis was seen in only one case. Because of the discrepancy between the biological behavior and histological findings of cartilaginous bone tumors, the malignancy of tumors should be evaluated by clinical signs and symptoms as well as by histological findings.

Follow-up study of giant cell tumor of bone.

Forty-two patients with giant-cell tumor of bone were treated by various surgical procedures. Local recurrence and pulmonary metastasis were surveyed with respect to the operation performed. A high incidence of local recurrence was observed in the group who underwent curettage for the bone lesion. Four cases had pulmonary metastasis and three died of tumor. Adequate surgical treatment of giant-cell tumors of bone needs at least en bloc resection of the bone lesion with reconstruction. Although the microscopic findings in our malignant cases differed somewhat from benign ones, it was very difficult to predict later malignant change from the histological features of the initial stage.

Transfection with the proviral DNA of a type D retrovirus (Mason-Pfizer monkey virus).

Chromosomal DNA was isolated from CMMT cells which were producing Mason-Pfizer monkey virus (M-PMV), a type D simian retrovirus. Human embryonic cells were transfected with the DNA. About 2 weeks after transfection, 3 of 7 culture dishes began to produce M-PMV which could be detected by virus-mediated cell fusion in RSb human cells dually transformed by Rous sarcoma virus and simian virus 40, reverse transcriptase assay, and electron microscopy. Mock-transfected cultures did not produce virus.

Malignant fibrous histiocytoma with skeletal involvement.

Malignant fibrous histiocytoma of soft part is rather common but malignant fibrous histiocytoma of the bone is rarely encountered clinically. Authors present five cases of malignant fibrous histiocytoma with skeletal involvement and discuss their clinical course, x-ray findings and histological features. This tumor has marked tendency for local recurrence and metastasis. Other bone tumors such as giant cell tumor, aneurysmal bone cyst, non ossifying fibroma, osteosarcoma, fibrosarcoma of bone and metastatic cancer can be excluded by several characteristic findings observed in x-rays as well as histopathological features. All information on the patient should be carefully analysed, because it is difficult to decide whether bone involvement is primary or secondary. Four out of five cases definitely originated within the bone.