Vascular endothelial dysfunction resulting from L-arginine deficiency in a patient with lysinuric protein intolerance.

Although L-arginine is the only substrate for nitric oxide (NO) production, no studies have yet been reported on the effect of an L-arginine deficiency on vascular function in humans. Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of dibasic amino acid transport caused by mutations in the SLC7A7 gene, resulting in an L-arginine deficiency. Vascular endothelial function was examined in an LPI patient who was shown to be a compound heterozygote for two mutations in the gene (5.3-kbp Alu-mediated deletion, IVS3+1G-->A). The lumen diameter of the brachial artery was measured in this patient and in healthy controls at rest, during reactive hyperemia (endothelium-dependent vasodilation [EDV]), and after sublingual nitroglycerin administration (endothelium-independent vasodilation [EIV]) using ultrasonography. Both EDV and NO(x) concentrations were markedly reduced in the patient compared with those for the controls. They became normal after an L-arginine infusion. EIV was not significantly different between the patient and controls. Positron emission tomography of the heart and a treadmill test revealed ischemic changes in the patient, which were improved by the L-arginine infusion. Thus, in the LPI patient, L-arginine deficiency caused vascular endothelial dysfunction via a decrease in NO production.

Characterization of a human monoclonal antibody with broad reactivity to malignant tumor cells.

Lymphocytes from mediastinal lymph nodes of 9 patients with primary lung cancer were fused with murine myeloma cells (P3U1). One of the clones (4G12) was stable for secretion (10 micrograms/ml) of human IgM lambda for 24 months. The antigen detected by 4G12 was sensitive to both trypsin and periodic acid-Schiff treatment. It immunoprecipitated a glycoprotein with an Mr of 65,000 upon analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced conditions. Immunohistochemical staining demonstrated that 4G12 possessed a high reactivity to squamous cell carcinomas of the lung (29 of 29) and also reacted with other lung carcinomas [adenocarcinomas (14 of 20) and large cell carcinomas (3 of 8)] and with some nonpulmonary malignant tumors (15 of 56). However, it did not react with small cell carcinomas of the lung. No benign tumors (0 of 26) so far tested have been positive. 4G12 did not react with most of the normal tissues; an exception was that it was weakly reactive on the glandular cells of the trachea and bronchi and on the proximal tubular cells of the kidneys. Thus 4G12 showed a broad reactivity to malignant tumors (68% of lung carcinomas, 27% of nonpulmonary carcinomas, and 0% of benign tumors). The reactivity of 4G12 on tissues from squamous cell carcinomas of the lung indicated that the expression of the antigenic determinant was much more in the well-differentiated grade than in the poorly differentiated grade. Thus the antigen detected by 4G12 appears to be related to tumor differentiation. Moreover, fluorescence-activated cell sorter analysis demonstrated that the expression of the antigen epitope depended on the cell cycle (G2-M). These data suggest that the 4G12 monoclonal antibody detects a new tumor-associated antigen that is recognized by the human immune system.

Investigation of the cytotoxic effect of flavopiridol in canine lymphoma cell lines.

The cyclin-dependent kinase (CDK) inhibitor, flavopiridol, was tested as a potential new cancer therapeutic agent to treat canine lymphoma by examining its effect on cell growth of canine lymphoma cell lines in vitro. Flavopiridol induced profound cell death in all eight lymphoma cell lines at 400 nM, and in all cases cell death was due to apoptosis. Apoptosis was inhibited by caspase inhibitor, despite the variable sensitivities between cell lines. Analysis of the mechanism of flavopiridol-induced apoptosis showed that Rb phosphorylation was inhibited, possibly due to CDK4 or CDK6 inhibition. There was also decreased expression of Rb protein and anti-apoptotic proteins, Mcl-1 and XIAP, possibly through transcriptional regulation by inhibition of CDK7 or CDK9 activation. Canine lymphoma cell line-xenotransplanted mice were then treated with flavopiridol and profound tumour shrinkage was observed. This study describes a new therapeutic approach using flavopiridol for canine lymphoma treatment.

The lca as an onco-fetal gene: its expression in human fetal liver.

The lca-transforming DNA was isolated from human hepatocellular carcinomas. This gene has no homology with known transforming DNA from human sources, and its role in neoplastic tissue formation has been left unanswered. In this communication, we report that RNAs prepared from human fetal livers hybridize to the lca DNA probe. The RNA is 1.8 kilobase in size and appears in the fetal liver only for a limited period during its development, viz. 19 weeks through 24 weeks of gestation. No other tissues carry detectable levels of the lca messenger RNA. Fetal hepatocytes at 5 weeks of gestation showed no transcripts of lca, but upon culturing for 2 more weeks in vitro, the cells became producers of the lca messenger RNA. These results suggest that the lca plays some role in the proliferative stage of the liver.

High-normal blood pressure is associated with new-onset electrocardiographic left ventricular hypertrophy.

Whether high-normal blood pressure (BP) is a predictor of new-onset electrocardiographic (ECG)-left ventricular hypertrophy (LVH) is not known. A total of 4112 subjects who underwent physical examinations were enrolled in this study. BP was measured on entry. Standard 12-lead ECG was recorded at initial evaluation and 3 years later. BP categories were defined on the basis of the 2013 European Society of Hypertension/European Society of Cardiology guidelines. Of the 4112 subjects, 133 developed ECG-LVH 3 years later. Crude cumulative prevalence rates of new-onset ECG-LVH were 2.0% for the optimal BP group, 3.2% for the normal BP group, 5.1% for the high-normal BP group and 5.0% for the hypertension group. After adjustment for age, gender, body mass index, smoking, low-density lipoprotein cholesterol, log-transformed high-density lipoprotein cholesterol, log-transformed triglycerides, estimated glomerular filtration rate, haemoglobin A1c, haemoglobin, uric acid and antihypertensive medication use, compared with the optimal BP group, the odds ratios of new-onset ECG-LVH for the normal BP, high-normal BP and hypertension groups were 1.52 (95% confidence interval (CI): 0.93-2.49, P=0.094), 2.38 (95% CI: 1.40-4.03, P=0.001) and 2.44 (95% CI: 1.43-4.18, P=0.001), respectively. Even high-normal BP was significantly associated with the presence of new-onset ECG-LVH.

Terrestrial gamma radiation dose rate in Ryukyu Islands, subtropical region of Japan.

In order to explain the distribution of natural radiation level in the Asia, in situ measurements of dose rate in air due to terrestrial gamma radiation have been conducted in a total of 21 islands that belong to Ryukyu Islands (Ryukyu Archipelago), subtropical rejoin of southwest Japan. Car-borne surveys have also been carried out in Okinawa-jima, the biggest island of the archipelago. Based on the results for these measurements, arithmetic mean, the maximum and the minimum of the dose rates at 1 m in height from the unpaved soil ground in the archipelago were estimated to be 47, 165 and 8 nGy h(-1), respectively. A comparative study of car-borne data obtained prior to and subsequent to the 2011 Fukushima nuclear accident, as for Okinawa-jima, indicated that the nuclear accident has no impact on the environmental radiation at the present time.

Regulation of insulin secretion by overexpression of Ca2+/calmodulin-dependent protein kinase II in insulinoma MIN6 cells.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in Ca2+-induced insulin secretion. We have previously reported that treatment of insulinoma MIN6 cells with secretagogues activated CaM kinase II and increased the phosphorylation of synapsin I, followed by insulin secretion. Here, we identified isoforms of CaM kinase II in MIN6 cells and rat islets. Immunoblot analysis suggested that the major isoforms of CaM kinase II were beta'e and delta2 at the protein level in MIN6 cells. Only the beta'e isoform was detected in rat islets by both RT-PCR and immunoblot analysis. We transiently overexpressed beta'e and delta2 isoforms in MIN6 cells and confirmed that treatment of cells with tolbutamide and glucose activated the isoforms. Immunoblot analysis with an antibody against synapsin I phosphorylated by CaM kinase II demonstrated that treatment with tolbutamide and glucose rapidly increased phosphorylation of synapsin I and that phosphorylation was potentiated by overexpression of the isoforms. The secretagogue-induced insulin secretion was potentiated by overexpression of the isoforms. Our results further support our conclusion that activation of CaM kinase II and the concomitant phosphorylation of synapsin I contribute to insulin secretion from pancreatic beta-cells.

Frequency of intron 14 splicing defect of cholesteryl ester transfer protein gene in the Japanese general population--relation between the mutation and hyperalphalipoproteinemia.

Cholesteryl ester transfer protein (CETP) deficiency, which has been found only in Japan, is characterized by marked hyperalphalipoproteinemia (HALP) and abnormalities of both low density and high density lipoproteins. We have reported that this deficiency is commonly associated with a G-->A mutation at the intron 14 splice donor site of the CETP gene (Yamashita et al., Biochem. Biophys. Res. Commun., 170 (1990) 1346-1351). In the current study, we determined the frequency of this mutation in Japanese subjects by using polymerase chain reaction. A single primer-template mismatch of one base pair from the CETP gene mutation permitted the introduction of a cleavage site for Nde I in mutant alleles but not in normal ones. Out of 171 patients with marked HALP whose serum HDL-cholesterol was more than 100 mg/dl, 6 (3.5%) subjects were homozygous and 48 (28.1%) were heterozygous for this mutation. Furthermore, in unrelated 512 healthy Japanese subjects, 5 (0.98%) were identified as heterozygotes. Relative allelic frequency of A at the intron 14 splice donor site was 0.0049 and the frequency of homozygous CETP deficiency was estimated to be approximately 1/42,000. These results demonstrate that this common mutation may be frequent in the Japanese population. Although HALP is very heterogenous, this mutation could be one of the major causes of marked HALP.

Localization of CD36 and scavenger receptor class A in human coronary arteries--a possible difference in the contribution of both receptors to plaque formation.

CD36 and scavenger receptor class A types I and II (SR-AI/II) are major receptors for oxidized low density lipoproteins (OxLDL) expressed on macrophages. To elucidate the role of these two macrophage scavenger receptors in the development of coronary atherosclerosis, we examined the localization of CD36 and SR-AI/II in human coronary atherosclerotic lesions. Serial cryostat sections of 49 coronary arteries obtained from 43 autopsied cases were examined immunohistochemically. Regarding the relationship between the severity of atherosclerosis and immunoreactivities to CD36, there was almost no immunoreactivity to CD36 in regions with diffuse intimal thickening, while the expression of CD36 was accelerated in parallel with the progression of atherosclerosis. In contrast, SR-AI/II was expressed persistently from regions with diffuse intimal thickening to atherosclerotic plaques. We also clarified the differential distribution of CD36 and SR-AI/II in atheromatous plaques. Close to the luminal surface of the intima, macrophages were relatively small in size, contained lesser lipids, and expressed SR-AI/II more abundantly than CD36. In contrast, macrophages in the core region were larger in size, contained more lipids, were strongly positive for CD36 and showed a weaker immunoreactivity to SR-AI/II than those in the luminal surface of the intima. In conclusion, the expression of CD36 and SR-AI/II on macrophages may be regulated differently in the process of coronary atherogenesis.

Increased serum remnant lipoproteins in patients with apolipoprotein E7 (apo E Suita).

Apolipoprotein (apo) E7 was originally identified by Yamamura et al. in subjects with atherosclerotic cardiovascular diseases (J. Clin. Invest. 1984;74:1229). However, the lipoprotein abnormalities associated with apo E7 phenotype have not been elucidated. In the current study, to clarify the physiological roles of apo E7, lipoprotein abnormalities were studied in 12 apo E7 heterozygotes. A total of seven subjects were hyperlipidemic and five subjects were normolipidemic. The apo E phenotype was apo E7/3 in 11 subjects and apo E7/4 in one subject. Polymerase chain reaction revealed that all of the subjects with apo E7 phenotype had the same mutation as that of apo E(Suita) as reported previously (J. Biochem. 1989;105:249). All the hyperlipidemic subjects were over 40 years of age and two of them also had and severe coronary heart disease. Ultracentrifugal analysis revealed that the cholesterol level both in very low density lipoprotein and in intermediate density lipoprotein (IDL) was substantially higher in hyperlipidemic apo E7 heterozygotes, compared with control subjects and that the IDL cholesterol was also increased even in normolipidemic apo E7 heterozygotes. Polyacrylamide gel electrophoresis of lipoproteins showed a midband, which implies the increase of remnant lipoproteins, in 11 subjects out of 12, irrespective of the presence or absence of hyperlipoproteinemia. In two cases, a broad beta pattern was observed similar to that seen in type III hyperlipoproteinemia. Dietary therapy was dramatically effective for the treatment of hyperlipidemia in patients with apo E7. These findings confirm that apo E is crucial for remnant lipoprotein metabolism and that apo E7 is related to the increase in serum remnant lipoproteins, which leads to hyperlipoproteinemia in association with obesity, aging and impaired glucose metabolism.

Activation of monocytes in vivo causes intracellular accumulation of lipoprotein-derived lipids and marked hypocholesterolemia--a possible pathogenesis of necrobiotic xanthogranuloma.

Necrobiotic xanthogranuloma (NXG) is a rare histiocytic disease with generalized xanthomatosis. However, most cases with NXG are normolipidemic or hypolipidemic. The mechanism for the formation of xanthoma in NXG has not yet been clarified. We observed a case of NXG with severe hypocholesterolemia (total cholesterol: 1.69 mmol/l) and analyzed the function of monocytes in this case. Histological examinations by light microscopy revealed a large amount of lipid deposition in the patient's freshly isolated monocytes. The patient's monocytes showed a 3-fold increase in cholesteryl ester content and a 3-fold enhancement of acetyl low density lipoprotein (LDL) uptake compared with the control monocytes. However, no significant difference was noted in the expression of CD36 protein and the mRNA levels of scavenger receptor-class A (SR-A) between the monocytes of the patient and the control. The phagocytotic ability of the patient's monocytes was enhanced 1.5-fold compared with that of the control monocytes. These findings suggest that the activated monocytes may have degraded the modified LDL via a pathway other than CD36 or SR-A, and accumulated a great amount of lipids in vivo. In conclusion, the present study has demonstrated a possible pathogenesis of NXG that the activation of monocytes in vivo may contribute to the intracellular accumulation of lipoprotein-derived lipids leading to non-inherited xanthomatosis and the marked hypocholesterolemia.

Prognostic significance of intraperitoneal free cancer cells in gastric cancer patients.

We performed intraoperative peritoneal cytology in 171 gastric cancer patients undergoing curative surgery. Intraperitoneal free cancer cells were demonstrated in almost all patients in whom the area of serosal cancer invasion exceeded 15-20 cm2. In patients with both serosal cancer invasion and free cancer cells the 5-year survival rat was 13% as compared with 85% for patients who had neither, and 40% for patients who had serosal invasion but no free peritoneal cancer cells. Peritoneal metastasis was the most frequently observed recurrence pattern. Therefore, in gastric cancer patients with marked serosal invasion, intraoperative IP administration of cytocidal anticancer drugs should be considered.

Large and cholesteryl ester-rich high-density lipoproteins in cholesteryl ester transfer protein (CETP) deficiency can not protect macrophages from cholesterol accumulation induced by acetylated low-density lipoproteins.

High-density lipoprotein (HDL) has been speculated to have an anti-atherogenic function. Many in vitro studies have demonstrated that HDL has the ability to remove cholesteryl ester (CE) from lipid-laden macrophages. However, the effect of alteration in chemical composition and particle diameter on the in vivo function of HDL is unknown. In the study described here, we have isolated the HDL from patients homozygous for cholesteryl ester transfer protein (CETP) deficiency and examined its function in vitro, in order to clarify the anti-atherogenic property of HDL in CETP-deficient subjects. Apolipoprotein (apo) E-free HDL2 from the patients, separated by heparin-Sepharose column chromatography, was rich in CE, poor in triglycerides (TG), and enlarged in size on 4-30% nondenaturing polyacrylamide gradient gel electrophoresis. In contrast, HDL3 from the patients was normal in size and in its chemical composition. First, we examined the effect of HDL on CE accumulation in macrophages. After mouse peritoneal macrophages had been incubated with both acetylated low-density lipoproteins (Ac-LDL) and HDL, cellular CE content was determined by an enzymatic, fluorometric method. Ac-LDL alone induced a 9-fold accumulation of CE. The addition of apo E-free HDL2 and HDL3 from controls and patients' HDL3 prevented CE accumulation in macrophages, while patients' HDL2 had no preventive effect. We next investigated the in vitro ability of HDL to remove cellular CE from lipid-laden macrophages after incubation with Ac-LDL. After loading of macrophages with cholesterol by Ac-LDL, HDL was added to the culture medium and the cellular CE content was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Polydisperse low-density lipoproteins in hyperalphalipoproteinemic chronic alcohol drinkers in association with marked reduction of cholesteryl ester transfer protein activity.

Long-term heavy alcohol intake is well known to increase serum high-density lipoprotein (HDL) cholesterol concentrations. Epidemiologic studies have shown that the protective effect of alcohol intake against coronary heart disease (CHD) is observed in moderate alcohol drinkers, but not in heavy ones. To clarify whether heavy alcohol intake may cause abnormalities in lipoprotein metabolism, we analyzed the plasma lipoproteins in eight male chronic heavy alcohol drinkers with marked hyperalphalipoproteinemia. Although their serum HDL cholesterol levels were remarkably high, ranging from 2.67 to 3.58 mmol/L, three patients had CHD and corneal arcus was present in seven patients. Cholesteryl ester transfer protein (CETP) activity was reduced in all subjects (7.3% +/- 4.2%/10 microL/18 h in alcohol drinkers v 20.5% +/- 2.4%/10 microL/18 h in control; mean +/- SD, P < .001). The CETP mass levels were also markedly reduced in these subjects. The analysis of low-density lipoprotein (LDL) on nondenaturing polyacrylamide gradient gel electrophoresis revealed that four subjects with severely low CETP activity (< 25% of control) had polydisperse LDLs, similar to those observed in genetic CETP deficiency. The other four subjects with approximately half the normal CETP activity had homogeneous but smaller-sized LDLs, as compared with control subjects. Particle size of HDL was larger than that of normal control HDL in all subjects. After cessation of alcohol intake, plasma HDL cholesterol levels were decreased and LDLs became more homogeneous and normal in size, in parallel with elevation of CETP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmid transformation in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer.

A transformation system with plasmids was developed for Bacillus subtilis NB22, an antibiotic iturin producing strain. Treatment of B. subtilis NB22 with 4 M KCl was effective for the induction of competence, followed by uptake of plasmid DNA in the presence of polyethylene glycol. The efficiency of transformation of this bacterium with pC194 and pUB110 was 4.1 X 10(3) and 1.5 X 10(3) transformants per micrograms DNA, respectively and the transformation frequency was 3.3 X 10(-3) and 7.2 X 10(-4), transformants per viable cell, respectively. This method was much faster and three orders of magnitude more efficient in transformation efficiency than protoplast transformation methods.

Low-density lipoproteins in hyperalphalipoproteinemic heavy alcohol drinkers have reduced affinity for the low-density lipoprotein receptor.

Heavy alcohol intake causes a marked inhibition of cholesteryl ester transfer protein (CETP) activity resulting in cholesterol ester enrichment of HDL. In this study we have characterized LDL of 35 chronic heavy alcohol drinkers with hyperalphalipoproteinemia to clarify the effect of alcohol on the metabolism of LDL. Serum concentrations of LDL-cholesterol and apolipoprotein B were normal, while the chemical composition of LDL was characterized by depletion of cholesteryl ester and enrichment of triglyceride. The LDL particles of the drinkers were significantly smaller in size than those of controls and had reduced affinity for LDL receptors of normal human fibroblasts. After cessation of alcohol, these abnormal characteristics returned toward normal along with elevation of CETP activity. These results suggest that heavy alcohol intake alters the compositions and particle size of LDL, consequently reducing their affinity for LDL receptors. This may be attributed, at least in part, to the reduction of CETP activity.

Adhesion formation can be reduced by the suppression of transforming growth factor-beta1 activity.

Surgery or trauma often results in restrictive adhesions around joints or tendons that cause severe functional impairment. The formation of adhesion is essentially a fibrogenetic process; therefore, peptide growth factors, such as transforming growth factor-beta, are assumed to play central roles in its development. The purpose of this study was to test the hypothesis that suppression of transforming growth factor-beta1 activity reduces adhesion formation. Sixty rabbits were prepared and randomly divided into six groups of 10. Intraarticular adhesions were created in the right knee joints by cortical bone shaving and subsequent cast immobilization for 4 weeks. In animals in three of the six groups, transforming growth factor-beta1 activity was suppressed by continuous administration of the neutralizing antibody in three graded doses; animals in the other three groups were used as controls. Four weeks after the surgery, the casts were removed and the adhesions were assessed macroscopically, histologically, biomechanically, and biochemically. Gross observation showed that the neutralizing antibody had suppressed adhesion formation in a dose-dependent manner. This is consistent with biomechanical measurement results demonstrating that the antibody reduced the flexion contractures. Histologically, the adhesion in our model was fibrous tissue and the adhesions in the animals in the antibody groups were thin and loose in comparison with the controls. Biochemical analyses further supported these results, demonstrating that administration of the antibody reduced collagen content in the adhesions with a predominance of type-I collagen. Thus, this study showed that suppression of the actions of transforming growth factor-beta1 reduced adhesion formation. Considering the various possible measures to control the activity of the growth factor, suppression of transforming growth factor-beta may be a novel, potent approach to preventing adhesions.

Macrosphelide, a novel inhibitor of cell-cell adhesion molecule. II. Physiochemical properties and structural elucidation.

New anticell-adherence compounds, macrosphelides A and B, were isolated from the fermentation broth of Microsphaeropis sp. FO-5050, and their structures were elucidated by spectroscopic methods and chemical transformations. Macrosphelides A (M.W. 342, C16H22O8) and B (M.W. 340, C16H20O8) with three esters in their molecules were classified as 16-membered macrocyclic compounds. Macrosphelide B was found to be a corresponding oxidative product of macrosphelide A at the C-14 position.

Macrosphelide, a novel inhibitor of cell-cell adhesion molecule. I. Taxonomy, fermentation, isolation and biological activities.

Potent anti-adherent activity was detected in fermentation extracts of microbial strain FO-5050. Active compounds designated macrosphelide A and B were isolated and the structure was determined to be 16-membered macrolide antibiotics possessing three ester bonds in the ring structure. Macrosphelide A dose-dependently inhibited the adhesion of HL-60 cells to LPS-activated HUVEC monolayer (IC50, 3.5 microM); macrophelide B also inhibited HL-60 adhesion but to a lesser extent (IL50, 36 microM). Macrosphelide A did not show any antimicrobial and cytocidal activities at the concentration of 1000 micrograms/ml and 100 micrograms/ml, respectively.

A modified system of stress radiography for patellofemoral instability.

Axial radiographs were obtained under valgus and external rotation stress at 45 degrees of knee flexion with and without contraction of the quadriceps muscle in order to assess the dynamics of patellar subluxation or dislocation. The radiography was performed on 82 knees in 61 patients with patellofemoral instability, and on 44 normal knees. The lateral patellofemoral angle and the congruence angle were measured and compared with the conventional Merchant views. Both parameters showed greater differences between symptomatic and normal knees on the stress radiographs obtained without quadriceps contraction. There was a major difference in the lateral patellofemoral angles between the groups, which clearly distinguished symptomatic knees from normal controls. Congruence angles on stress radiography had a significant correlation with the functional scores obtained after a period of conservative treatment and a positive correlation with the frequency of patellar subluxation. When the quadriceps contracted, two patterns of patellar shift were observed. While the patella reduced into the trochlear groove in all normal knees and about 70% of the symptomatic knees, contraction of the quadriceps caused further subluxation of the patella in the remaining symptomatic knees. All the knee joints which showed this displacement failed to respond to conservative treatment and eventually required surgical treatment. Thus, this technique of stress radiography is a simple, cost-effective and useful method of evaluating patellar instability and predicting the prognosis.