Okayama University Medical School

We performed a cytogenetic study on 140 nonpolymalformed patients with mental retardation of clinically undefined origin, using a high resolution banding technique, to determine how much chromosome abnormalities contribute to the etiology of this condition. A total of 15 patients (10.7%) were found to have autosomal or sex chromosomal abnormalities. Autosomal abnormalities included partial monosomy (5 cases), reciprocal translocation (one case), 13/14 robertsonian translocation (3 cases), unbalanced translocation (one case), inverted duplication of 15q (one case) and mosaic trisomy 21 (one case). Sex chromosomal abnormalities comprised structural rearrangement of the short arm of the X chromosome (one case) and 47, XXY in a pure or mosaic form (two cases). It should be noted that four out of the 5 cases of partial monosomy had subtle interstitial deletions, which might have been unidentified by the conventional G-banding method alone. In one case of the robertsonian translocation 46,XY,t(13;14)/45,XY,t(13;14), a small deletion was thought to have occurred in the cells with a chromosome number of 45. Comparison of clinical features of the 15 chromosomally abnormal patients with those of patients with normal karyotypes did not show any clinical parameter indicative of chromosome imbalance. These results suggest that a subtle chromosomal deletion is specific to mental retardation associated with few malformations. We believe that diagnostic evaluation of mentally retarded patients, even if nonmalformed, should include chromosome analysis using a high resolution banding technique.

A case of ovarian leiomyoma is reported, together with histologic, immunohistologic and electron microscopic findings. A solid firm tumor, measuring 6.5 X 5 X 5 cm, was found in the right ovary of a 65-year-old woman. The tumor had an obvious whorled pattern on the cut-surface. Well-differentiated, long spindle-shaped neoplastic cells revealed positive immunoreactivity for anti-desmin. Ultrastructural observations included numerous microfilaments with dense patches in the cytoplasm, micropinocytotic vesicles beneath plasma membranes and continuous basal laminae around neoplastic cells. These findings were compatible with leiomyoma. The possible histogenesis of ovarian leiomyoma was discussed.

A simple cell-free system for studying a priming factor involved in the repair of bleomycin-damaged DNA was established. The template-primer used for the repair DNA synthesis was prepared by treating the closed circular, superhelical form of pUC19 plasmid DNA with 2.2 microM bleomycin and 20 microM ferrous ions. Single-strand breaks were introduced into pUC19 DNA by the bleomycin treatment, and the DNA was consequently converted largely into the open circular form. A system for repair of this bleomycin-damaged DNA was constructed with a priming factor, DNA polymerase (DNA polymerase beta or Klenow fragment of DNA polymerase I), ATP, T4 DNA ligase and four deoxynucleoside triphosphates. After incubation, the conformation of the DNA was analyzed by agarose gel electrophoresis and electron microscopy. The open circular DNA was largely converted to the closed circular DNA, indicating that the single-strand breaks of DNA were repaired. When the priming factor was omitted, DNA repair did not occur. The present system seemed to be applicable to the study of priming factors involved in the repair of DNA with single-strand breaks caused not only by bleomycin but also by ionizing radiation or active oxygen.

Murine adrenal tumor cells (Y-1 clone) were stimulated by adrenocorticotropic hormone (ACTH) and cyclic adenosine 3',5'-monophosphate (cyclic AMP) to produce steroid hormone (delta 4, 3-keto steroids). The steroids were secreted into the medium immediately after synthesis. The optimum concentrations of ACTH and cyclic AMP for stimulation of steroid production were 10(-2) U/ml and 1.0 mM, respectively. In serum-free medium, ACTH and cyclic AMP stimulated steroidogenesis in Y-1 cells, but the amount of steroid hormone in the culture medium was low. However, a high level of steroid production was maintained with medium containing 10 mg/ml bovine serum albumin (BSA). In culture medium containing a higher concentration of BSA, Y-1 cells did not become spherical as is usually the case when steroid production is stimulated by ACTH or cyclic AMP. The morphological changes did not always correlate with steroid secretion by Y-1 cells.

It has been assumed that the in vivo reduction of 3-mercaptopyruvate, an intermediate of cysteine metabolism, to 3-mercaptolactate is catalyzed by lactate dehydrogenase (EC 1.1.1.27) though no definitive evidence has been presented. In order to examine this assumption, reduction of 3-mercaptopyruvate and its inhibition were studied using rat liver homogenate, lactate dehydrogenase purified from rat liver and anti-lactate dehydrogenase antiserum. Reduction of 3-mercaptopyruvate was actively catalyzed by rat liver homogenate and by the purified lactate dehydrogenase. This reducing activity was completely inhibited by anti-lactate dehydrogenase antiserum. These results indicate that the reduction of 3-mercaptopyruvate to 3-mercaptolactate in rat liver is catalyzed by lactate dehydrogenase.

To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He) cells was established and characterized. SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution. The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin. Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates. The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III. The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl. Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta) and DNA polymerase beta were involved in the repair process. The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s).

The major gag protein (p34) of squirrel monkey retrovirus-H was purified in one chromatographic step by anion-exchange high performance liquid chromatography. The virus in a crude fraction was disrupted with Brij 35 in the presence of three kinds of protease inhibitors. The soluble virus lysate was injected into a Polyanion SI column, and p34 was eluted with a linear salt gradient. The recovery of the protein was about 60%. The purified p34 was nearly homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining.

To clarify the initiation, development and recovery processes of disseminated intravascular coagulation (DIC), rat glomerular capillaries and fibrin thrombi were examined under transmission and scanning electron microscopes. DIC was induced in rats by a single intraperitoneal injection of endotoxin (Et., 7.5 mg/kg lipopolysaccharide:B, E. coli 026:B6). At 2 h after Et. injection, the endothelial surface of the glomerular capillary became irregular with projections like a sea anemone. At 4 h after Et. injection, agglomerated fibrin thrombi composed of fibrin fiber bundles with fine cross-striated fibriform structures were observed in the capillary lumen. The fibrin thrombi gradually changed into fine reticular systems suggesting a degradation process by 6 h after Et. injection, and formed a coarse granular agglomerate by 8 h after Et. injection. These fibrin thrombi disappeared within 12 h of Et. injection, but the endothelial surface remained edematous. At 24 h after Et. injection, the microstructure of the glomerular capillaries returned normal. Based on these observations, we concluded that DIC was primarily initiated by injury to the capillary endothelium, and that changes on the endothelial surface contributed to the development of DIC.

Three types of traditional Chinese herb medicine were used to treat 98 patients with advanced esophageal squamous cell carcinoma prior to surgical treatment. Forty-two patients with the same diagnosis were treated with these herbs plus cyclophosphamide (endoxan). One hundred similar patients received surgical treatment without herbs or endoxan treatment as controls. Histologic examinations of surgical specimens were made on all of these patients. Stromal lymphoid-cell infiltration and cancer tissue degeneration were more prominent in Menispernum dehuricum DC- or Chelidonium majus L-treated patients, and were less clear in patients treated with herbs plus endoxan and the controls. The antitumor action of herbs is thought to be brought about by the activation of an immunological rejection mechanism. Herbs plus endoxan may result in the masking of the immunological response of hosts without obviously damaging cancer tissues.

Sprague-Dawley rats, 6 with aminonucleoside nephrosis and 6 controls, were intravenously injected with human liver ferritin isolated from post mortem liver, and their 24-h urine samples were examined for human ferritin by immunoradiometric assay. In rats with aminonucleoside nephrosis, the amount of excreted ferritin in urine was forty times greater than in control rats. Much more monomeric ferritin was excreted than that of polymeric ferritin. We are the first to have utilized human liver ferritin as a tracer to measure a minor amount of ferritin by a commercially available kit. Our present study seems to indicate a critical role for glomerular basement membrane as a size barrier.

Suppression of the cellular immune system appears to be a prerequisite for the manifestation of adult T-cell leukemia (ATL). In other words, ATL will develop when impairment of the immune system is caused by the infection of human T-lymphotropic virus type I (HTLV-I). This defect of immune surveillance against virus-infected cells may be a result of the impairment of the function of cytotoxic T-cells (CTLs) specific for the HTLV-I-infected cells. The manifestation of ATL could be predicted by examining the function of CTLs in HTLV-I carriers. A new strategy of prevention and therapy for ATL would include an attempt to restore and fortify the CTL function of the host.

We investigated the effects of fractionated sera obtained from cancer patients by double filtration plasmapheresis (DFPP) plus antitumor agents on murine pulmonary metastasis. Fractions of the sera, in combination with natural human tumor necrosis factors (nTNF) and cyclophosphamide (Cy), were systemically administered to Lewis lung carcinoma-bearing mice. When the second filtrate (a plasma fraction containing substances composed of smaller molecular weight compounds) combined with low-dose nTNF (1,000 U/kg) and Cy (250 micrograms/kg) was administered to the mice, the degree of metastasis was significantly suppressed compared with the control group (p less than 0.01). In contrast, the discarded fluid (a plasma fraction containing larger molecular weight compounds) combined with the same doses of nTNF and Cy caused little inhibition of metastasis. Also, the discarded fluid significantly suppressed natural killer activity compared with normal sera (p less than 0.01). The results suggested that DFPP combined with nTNF and Cy is an efficient procedure to remove immunosuppressive factors from the sera of cancer-bearing hosts, to enhance the host antitumor immunity, and to suppress tumor proliferation.

In vivo inactivation of cystathionine gamma-lyase by D,L-propargylglycine, a suicide inhibitor, was found to be less profound in rat kidney than in the liver. We investigated the cause of this difference using rat tissues. We fractionated kidney extract to characterize the substance which protected enzyme, and found that cysteine exhibits protecting action. Addition of 0.3 mM L-cysteine to the incubation mixture containing dialyzed kidney supernatant and 0.5 mM D,L-propargylglycine resulted in the protection of cystathionine gamma-lyase from the inactivation by the inhibitor. The content of cysteine in the kidney was six-fold higher than that in the liver. Thus, we have concluded that one of the reasons why the in vivo inactivation of cystathionine gamma-lyase in rat kidney was less than that in the liver is the presence of a higher concentration of cysteine in the kidney. S-Carboxymethylcysteine, a cysteine derivative, exhibited a similar, but weaker, protective effect.

In vivo inactivation of cystathionine gamma-lyase by D,L-propargylglycine, a suicide inhibitor, was found to be less profound in rat kidney than in the liver. We investigated the cause of this difference using rat tissues. We fractionated kidney extract to characterize the substance which protected enzyme, and found that cysteine exhibits protecting action. Addition of 0.3 mM L-cysteine to the incubation mixture containing dialyzed kidney supernatant and 0.5 mM D,L-propargylglycine resulted in the protection of cystathionine gamma-lyase from the inactivation by the inhibitor. The content of cysteine in the kidney was six-fold higher than that in the liver. Thus, we have concluded that one of the reasons why the in vivo inactivation of cystathionine gamma-lyase in rat kidney was less than that in the liver is the presence of a higher concentration of cysteine in the kidney. S-Carboxymethylcysteine, a cysteine derivative, exhibited a similar, but weaker, protective effect.

The effect of various factors and substrates on the growth of a human hepatoblastoma cell line, HuH-6, which was inoculated at low density in a serum-free medium was examined. Several supplements were required to enhance cell growth of HuH-6. These included cholera toxin (CT), glucagon (Glu) and selenium (Se). Type IV collagen (C-IV) provided the most conductive environment tested for cell growth. These results suggest that CT, Glu, Se, and C-IV are important stimulators for the continuous growth of HuH-6 in a serum-free medium at low density.

Effects of capsaicin on the circular muscle motility of the isolated guinea-pig ileum were investigated. Capsaicin produced a contraction followed by a relaxation of the circular muscle. Both responses were easily desensitized. As the late relaxation response was not sufficiently intense to be analyzed, the inhibitory effect of capsaicin on substance P-induced contractions was explored. Capsaicin abolished the substance P-induced contractions. This inhibitory effect was not affected by tetrodotoxin, and the effect was desensitized. Therefore, all effects of capsaicin on circular muscle motility seem to be due to the release of sensory neuropeptides, similarly to those elicited in the longitudinal muscle.

Changes in the hemodynamics of six patients having received Fontan-like operations were closely observed during the first 48 h after the operation. Catheterization studies and simultaneous angiocardiography were also performed before and after the operation. Hemodynamic derangement was particularly severe during the first 24 h postoperatively as indicated by a low cardiac output of less than 2.01/min/m2, which persisted in spite of very high central venous pressure. Furthermore, the central venous pressure needed to re-establish the circulation soon after the Fontan procedure significantly correlated with the angiocardiographically assessed preoperative size of distal pulmonary arteries. Accordingly, the preoperative evaluation of the distal pulmonary arterial size is very important, that provides a good guide-line for the degree of circulatory volume expansion necessary to elevate the central venous pressure and to sustain the circulation in the early postoperative period.

The effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the organization of cytoskeleton and growth of normal and established chick embryo cells (CEC) were studied. The cytoskeleton of normal CEC formed stress fibers, while that of the CEC lines established in our laboratory formed no stress fibers. TPA treatment of normal CEC resulted in disorganization of the stress fibers into amorphous structure, while that of the established CEC lines induced no reorganization of the cytoskeleton. TPA had no promotional effect in vitro or in vivo on tumor growth in normal or the established CEC.

Non-radioactive hybridization probes were prepared using the M13 phage vector and the universal sequencing primer. The probe sequence to be used was first cloned into the M13 vector, and the minus strand of the template DNA was then synthesized with the Klenow fragment of E. coli DNA polymerase I in the presence of the biotinylated nucleotide, biotin-11-dUTP, as a label. Resultant DNA was heavily biotinylated, and made up of the entire minus strand of the template DNA. The long tag sequence derived from the M13 vector may increase the sensitivity of the detection. The biotinylated hybrids were visualized with the streptavidin-alkaline phosphatase conjugate and chromogenic substrates. As shown by Southern hybridization, the probe prepared in this way could be used to detect less than 1 pg of target sequence and a single copy gene sequence in human genomic DNA within several hours of signal development.

Dynamic ergometer exercise in a supine position was applied to 64 patients more than 1 year after valvular heart surgery, and the left ventricular reserve was evaluated echocardiographically. The left ventricular reserve declined in the mitral stenosis-mitral valve replacement group, while it was better maintained in the mitral stenosis-mitral commissurotomy, aortic regurgitation and aortic stenosis groups. The patients were divided into 3 groups depending on whether the percentage increase during exercise of stroke index, an index of left ventricular pump function, increased, unchanged, or decreased. The percentage increase of mean velocity of circumferential fibre shortening (y) and that of left ventricular end-diastolic diameter (x) during exercise were plotted for each group. The increased group was isolated from the unchanged group by the line of y = -5.02x + 30.1; the unchanged group was isolated from the decreased group by that of y = -5.68x-10.0, and the increased and unchanged groups were clearly isolated from the decreased group by that of y = -6.86x-4.76. We conclude that dynamic ergometer exercise echocardiography is useful for evaluating the left ventricular reserve of postoperative patients with valvular heart disease. It was also thought that the subclinical state of cardiac failure can be effectively detected by the present method.</P>