Okayama University Medical School

A new gas chromatographic method for the determination of sulfate was developed. In this method, sulfate was quantitatively converted to a volatile derivative, dimethyl sulfate, by a two-step procedure. First, sulfate was converted to silver sulfate by reaction with silver oxide, and then to dimethyl sulfate by reaction with methyl iodide. The derivative was analyzed by gas chromatography. Methyl methanesulfonate was used as an internal standard. The method was applied to the determination of total urinary sulfate. Phosphate and chloride ions, which interfered with the present method, were eliminated with the use of basic magnesium carbonate and an excess of silver oxide, respectively. Recovery was over 96% when 5 to 40 mumol/ml of sulfate was added to human urine samples.

Serial left ventricular (LV) echocardiographic studies were performed in 21 patients before and after aortic valve replacement for chronic aortic regurgitation. The effect of valve replacement on LV dimensions, cross-sectional area of the LV muscle and LV function was determined from the echocardiographic data. The relation between degeneration of the myocardium and surgical outcome was also investigated. The average LV end-diastolic dimension decreased from 66.0 +/- 8.3 mm to 46.3 +/- 5.7 mm twelve months postoperatively. The average LV end-systolic dimension also fell from 43.4 +/- 8.1 mm to 31.1 +/- 5.0 mm. The muscle cross-sectional area decreased from 33.1 +/- 5.1 cm2 to 24.5 +/- 4.0 cm2, indicating a decrease in LV mass. The indices of contractility (fractional shortening, ejection fraction and mean velocity of circumferential fibre shortening) had a tendency to decrease one month after surgery, but they subsequently increased to the normal level 12 months after surgery. Nineteen out of 21 patients showed a favorable outcome as to the functional status. The remaining two patients had a large LV dimension and subnormal contractility, and they failed to show a significant reduction in the follow-up period. The muscle score in the two patients was greater than 8 points, which indicated irreversible impairment of the myocardium. Patients with persistent postoperative LV enlargement have a poor prognosis and should be identified so that aggressive medical treatment can be instituted.

We studied the effects of insulin and glucagon on energy and carbohydrate metabolism of rat hepatocytes in primary culture. The aim of this study is to elucidate the mechanism of the synergistic action of insulin and glucagon and to evaluate the combined effects of these hormones on liver injury. Insulin increased the level of adenosine triphosphate in hepatocytes in the presence of glucagon. Insulin increased the activities of glucokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40) type L and glucose 6-phosphate dehydrogenase (EC 1.1.1.49). Glucagon had no antagonistic effect on these increases. Glucagon increased the activity of glucose 6-phosphate (EC 3.1.3.9) (G6Pase) in the presence or absence of insulin, while insulin had no effects on the levels of G6Pase and fructose 1,6-bisphosphatase (EC 3.1.3.11) in the presence or absence of glucagon. Metabolite analysis of cultured hepatocytes indicated that insulin and glucagon have antagonistic effects on the glycolytic activity of hepatocytes. These combined effects of insulin and glucagon may partially explain the preventive effects of these hormones on liver injury.

The effect of alpha-tocopherol acetate (VE) on the toxicity and tissue distribution of adriamycin (ADM) in mice was studied. After the administration of ADM in 2 doses of 15 mg/kg, mice pretreated with olive oil survived 7.1 +/- 1.0 days, while mice pretreated with VE in ten doses of 500 mg/kg/day (subcutaneously) survived 5.5 +/- 1.7 days (p less than 0.01). The total concentration of ADM and its major metabolite, aglycone I in the liver (1, 3, 5 h), kidneys (1, 3 h), and heart (3 h), as determined by high performance liquid chromatography was significantly higher in the VE-pretreated group (four doses of 500 mg/kg/day) than in the olive oil-pretreated group. The aglycone levels of the VE-pretreated group were significantly higher than those of the olive oil-pretreated group in the liver, kidney and heart, but there was no significant difference between the groups in the levels of the unmetabolized form. Considering these results, the administration of VE concomitant with anti-tumor drugs, including ADM, requires great caution.

The effects of prolonged weak anodal direct current (DC) on the electrocorticogram (ECoG) were investigated in awake rabbits. When the current (20-40 microA) was applied to the motor region of the cerebral cortex, seizure activity in the ECoG appeared from the frontal cortex. Repeated application of the DC decreased the threshold current for producing the seizure activity. Diazepam significantly elevated the threshold of the seizure activity. In contrast to the marked changes in the ECoG, no behavioral changes were observed during or after the application of weak anodal DC. The changes in the ECoG are discussed in relation to the intensity and duration of the DC.

We studied the correlation between the cell surface markers and prognosis of non-Hodgkin's lymphoma (NHL) patients treated in the Shikoku Cancer Center Hospital from 1980 to 1986. Thirty-one cases were selected on the basis of having a lymphnode as a primary lesion, having been immunophenotyped before chemotherapy, being in the intermediate histologic grade and being in stage II, III or IV. Thirteen cases of the T-cell type (T-lymphomas) and 18 cases of the B-cell type (B-lymphoma) were identified. The complete remission rate was 54% among T-lymphoma patients and 78% among B-lymphoma patients. The median length of survival was 12+ months in T-lymphoma and 26+ months in B-lymphoma. The survival rate of T-lymphoma patients was significantly lower than that of B-lymphoma patients. The importance of making surface marker studies was reappraised in our study.

The cartilage-synovium junction of knees afflicted with rheumatoid arthritis was observed light microscopically using formalin-fixed, decalcified and immunohistochemically stained tissues. Decalcification had little or no influence on immunoreactivity for lysozyme and S-100 protein. All the specimens had pannus formation, which was classified into four types: A) cellular pannus with homogeneous cell pattern, B) cellular pannus of inflammatory cells, C) fibrous pannus with many fibrous bundles, D) fibrous pannus including round cells with scattered fibrous bundles. Type A pannus may be responsible for extensive cartilage degradation, and may occur at the first stage of pannus formation. Type B pannus may occur afterwards, and may be followed by type C pannus at a later stage. Type D pannus was found in two out of 19 specimens. Round cells in type D were positive for S-100 protein and lysozyme, and were probably chondrocytes. The findings indicated that chondrocytes were responsible for cartilage degradation and pannus formation.

The rearrangement of breakpoint cluster region (ber) was examined in leukemic cells obtained from 3 patients initially diagnosed as having Ph+ acute leukemia, 2 with acute lymphocytic leukemia (ALL) and one with acute mixed leukemia. DNA was digested with Bgl II and BamH I. The ber rearrangement was present in the case of acute mixed leukemia (Case 1), but was absent in the 2 cases of ALL (Cases 2 and 3). These results suggest that Case 1 represented a type of blast crisis of chronic myelocytic leukemia which was unusual in the sense of the occurrence of a myeloid-lymphoid conversion and lack of an apparent chronic phase. Cases 2 and 3 appeared to be de novo Ph+ ALL.

In order to explore the pathogenic mechanism of proteinuria in glomerulonephritis, ultrastructural changes of the glomerular basement membrane were investigated in rats with chronic serum sickness induced by repeated intravenous injections of bovine serum albumin (experimental rats). Rats injected with saline served as controls. The animals were sacrificed and examined 13 weeks after treatment, when the mean urinary protein of experimental animals reached 206 mg/24h/100g body weight. Enhanced transcapillary passage of anionic ferritin was observed in experimental rats. Purified glomerular basement membranes of control and experimental rats were examined by electron microscopy after negative staining. The glomerular basement membrane of experimental rats had enlarged pores. The results suggest that an increase in the radius of glomerular pores may be responsible for proteinuria in glomerulonephritis.

Interleukin-2 (IL2) is the obligatory signal for both T cell mitogenesis and in vitro generation of alloreactive cytotoxic T lymphocytes (CTL). An investigation was made to determine whether an antibody directed against IL2 would suppress the rejection reaction of rat cardiac allografts. Rabbit anti-interleukin 2 (anti-IL2) antiserum was obtained by immunizing at 2 week intervals over a period of 8 weeks with 10(6) U of recombinant human IL2 along with complete Freund's adjuvant. The bioassay for inhibition of IL2 activity by anti-IL2 antiserum was carried out in conjunction with the IL2-dependent cytotoxic T cell (CTLL cell) assay. Cardiac allografts of F344 rats were heterotopically transplanted into ACI rats. Seven daily doses of 1 ml of anti-IL2 antiserum were administered intravenously following transplantation. IL2-driven [3H]thymidine incorporation in CTLL cells was significantly inhibited by rabbit anti-IL2 antiserum. Graft survival in the anti-IL2 serum-treated group was significantly prolonged in a dose-dependent fashion compared to control groups. In conclusion, these results indicate that rabbit anti-IL2 antiserum may prove to be of significant value as an immunosuppressive agent in clinical organ transplantation.

The development of useful therapy for intraabdominal carcinomatosis originating from gastrointestinal cancer is an important theme in cancer therapy. We developed recently an experimental model of intraabdominal carcinomatosis in nude mice by intraperitoneal transplantation of human colon cancer cells (RPMI 4788). Using this model, we investigated the antitumor effects of recombinant human interferon (rIFN)-beta and rIFN-gamma administered singly or in combination. Treatment was initiated 2 days after CD-1 nude mice were inoculated intraperitoneally with 5 X 10(6) RPMI 4788 cells. Intraperitoneal administration for 10 consecutive days of either rIFN-beta (2.5 X 10(5) IU/mouse/day) or rIFN-gamma (2.5 X 10(5) JRU/mouse/day) resulted in a significant prolongation of survival compared with the saline control group [survival in the control: 41.8 +/- 5.6 days (mean +/- SD)]. Combined administration of rIFN-beta and rIFN-gamma for 10 days yielded a marked synergistic effect on the prolongation of survival (114.0 +/- 8.2 days). However, combined administration of rIFN-beta and rIFN-gamma in a single dose equal to the total dose given fractionally over 10 days did not yield a synergistic effect. These results suggest that daily administration of rIFN-beta and rIFN-gamma combined may provide a highly potent antitumor effect against human peritoneal carcinomatosis.

In vitro release of atrial natriuretic peptide (ANP) from atria was examined by ANP radioimmunoassay. Isolated right rat atria were incubated in Krebs-Ringer bicarbonate buffer, and test substances were added to the incubation medium. The fluid was assayed for rat ANP by a radioimmunoassay method recently developed in our laboratory. We produced an antiserum to human ANP(99-216) (alpha-hANP(1-28)) which showed a good cross-reactivity of 63% with rat ANP(99-126) (alpha-rANP(1-28)) and was useful for measuring rat ANP concentrations of the medium. Application of the medium to a reverse phase high performance liquid chromatography (HPLC) system resulted in a single peak of immunoreactive rat ANP corresponding to a small molecular weight synthetic rat ANP of 28 amino acid residues. Catecholamines (epinephrine, norepinephrine and isoproterenol) reduced the basal secretion of ANP, whereas acetylcholine stimulated the release of ANP. Forskolin and dibutyryl cyclic AMP did not affect the release of ANP. These results suggest the possibility that the regulation of ANP release may be partially associated with adrenergic and cholinergic mechanisms.

The surface ultrastructure of larval Anisakis type I, Anisakis type II, Raphidascaris, Contracaecum type A, Thynnascaris type A and Thynnascaris type B was examined by scanning electron microscopy. These species were identified clearly by the presence of a boring tooth, a mucron, and other morphological features. The means of the distances between transverse striations (DBTS) of larval Anisakis type I (5.45 +/- 0.125 micron), larval Raphidascaris (2.92 +/- 0.051 micron), and larval Contracaecum type A (1.68 +/- 0.056 micron) are significantly different (p less than 0.05). There was a correlation between the diameter of worm trunk (DOWT) and DBTS among these three larval types. In most cases a larva could be identified from the mean value of DBTS and DOWT even if obtained as a fragment from a patient.

The plantar metatarsal arteries of some mammals were studied. In the dog, raccoon dog and cat, the second proximal perforating branch was fully developed and produced the plantar metatarsal arteries. These plantar metatarsal arteries ran on the plantar surfaces of the interosseous muscles along the metatarsal bones or intermetatarsal spaces, and gave rise to the digital arteries of the second to fifth toes. In the rabbit, a branch of the medial plantar artery ran transversely on the plantar surfaces of the metatarsal bones at a level distal to the bases of these bones, and produced the plantar metatarsal arteries. These plantar metatarsal arteries ran deep in the interosseous muscles along the metatarsal bones or intermetatarsal spaces, and joined with the digital arteries which were derived from the medial plantar artery. The plantar metatarsal arteries could be classified into four kinds of arteries (sM, sI, dM and dI) in relation to the interosseous muscles and metatarsal bones. This classification largely coincides with that of the human hand and foot (Murakami, 1969, 1971), the monkey hand and foot (Nakai et al., 1987; Hinenoya et al., 1987), and the forepaws of some animals, including the dog and cat (Murakami et al., 1987).

The effect of the heart rate and myocardial contractile force on the extravascular resistance to blood flow of the left anterior descending coronary artery (LAD) was evaluated in 15 mongrel dogs anesthetized with sodium pentobarbital. The LAD was maximally dilated by intracoronary infusion of adenosine, which precluded the influence of vasomotor tone. Increases in the heart rate and myocardial contractile force decreased coronary blood flow in the absence of a change in coronary perfusion pressure. The changes in mean coronary resistance showed a significant linear relationship to changes in developed tension. The changes in coronary resistance caused by varying the heart rate and contractile force were so small that a normal coronary vascular tree could easily compensate for the increase in resistance. However, it is supposed that with critical stenosis of the vascular tree even a small increase in resistance might cause deleterious effects on coronary blood flow.

A case of malignant lymphoma associated with complete heart block in a 30-year-old woman is reported. The patient progressively deteriorated despite temporary pacing and died 24 days after being admitted. Microscopic examination of the heart revealed marked infiltration by lymphoma cells in the atrioventricular node and the bundle of His. A diffuse lymphoma (large cell type, B cell) was diagnosed. This case is considered to be rare, since complete heart block was the first and only manifestation of the malignant lymphoma.

Eradication of immunologically-syngeneic tumors was achieved by adoptive chemotherapy using effector cells induced by Corynebacterium parvum-Pyridine Extract Residue (CP-PER). A mixture of 2 X 10(6) Meth A cells and 0.1 mg CP-PER was subcutaneously inoculated into the back of donor BALB/c mice, with the result that their spleen cells showed an antitumor effect 10 to 13 days after the inoculation. These cells were used as immune cells. Recipient mice were inoculated with 1 X 10(6) Meth A cells, and 2 days later were administered cyclophosphamide. On the following day, 1 X 10(8) immune cells were adoptively transferred into the recipient mice. As a result, the tumor began to regress 7 to 12 days after the adoptive transfer. An immuno-histochemical study of the donors' spleens and the recipients' regressing tumors revealed that the ratio of L3T4+ T cells to Lyt-2+ T cells in the donors' spleens was increased and that the infiltrating cells in the recipients' tumors were mainly composed of L3T4+ T cells. This confirmed that the transfer of L3T4+ T cells led to the infiltration of L3T4+ T cells into the recipients' tumors, causing their eradication.

The interosseous and lumbrical muscles in twenty-five hands of Japanese adult cadavers were dissected. The palmar and dorsal interosseous muscles continued, with few exceptions, into the wing tendons. The dorsal interosseous muscles gave off tendons which pierced the transverse laminae or passed deep to the transverse laminae, and attached to the bases of the proximal phalanges. The palmar interosseous muscles seldom had such attachments. The palmar and dorsal interosseous muscles sometimes gave off additional tendons which passed superficial to the transverse laminae and attached to the bases of the proximal phalanges. These latter attachments were typical in the contrahentes muscles. Thus, the present findings suggest that the human dorsal interosseous muscles are composite muscles derived from the dorsal abductor, flexor brevis and contrahens muscles, and that the human palmar interosseous muscles are composite muscles derived from the flexor brevis and contrahens muscles. The lumbrical muscles rarely gave off accessory slips with atavistic attachments to the proximal phalanges.

Isolated rat glomerular basement membrane was treated with elastase and observed by transmission electron microscopy. The treatment with elastase revealed the fundamental structure of the glomerular basement membrane quite clearly, and enabled the observation of a sieve structure within the glomerular basement membrane. This sieve structure may play a major role in the filtration of blood as well as in the production of urine. Treatment with antibody showed that the sieve was mainly constituted of type IV collagen.

A mixture of glycol methacrylate (GMA) and Quetol 523 was examined as an embedding medium for atrial tissue to be selectively stained for specific atrial granules. Semi-thin sections of rat atrial tissue embedded in this resin were stained with lead hematoxylin and observed under a light microscope. Atrial granules were found to be specifically stained blue black with lead hematoxylin. The same semithin sections stained with OsO4 vapor were examined electron microscopically and the atrial granules could be distinguised clearly from other cytoplasmic components. The GMA-Quetol 523 mixture is a useful embedding medium for studying the distribution of specific atrial granules by light and electron microscopy.