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For the functional analysis of mucin related glycan, we synthesized core 3 and 5 structures of mucin type O-glycan and investigated their binding interaction with lectins using sugar chip technology. The construction of Tn antigen moiety containing α-N-acetylgalactosamine residue was achieved by α-selective glycosylation of 2-azido-6-tert-butyldiphenylsilyl-3,4-di-O-chloroacetyl-2-deoxy-galctopyranosyl imidate and glucose moiety, which acts as a hydrophilic spacer when immobilized on a gold-coated sensor chip. Core 3 and core 5 structures were synthesized by the glycosylation of appropriate N-acetylglucosamine and N-galactosamine donors, respectively, and were converted into sugar-chain ligand conjugates according to the method reported. The interaction analysis of lectins with sugar chips coated with ligand conjugates was performed with a surface plasmon resonance (SPR) biosensor. The specific interaction was observed between the core 3 structure and Jacalin (JAC) and kinetic parameters were estimated as k a  = 1.5 × 10 4 , k d  = 5.8 × 10 -3 , and K D  = 3.8 × 10 -7 ., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Afatinib has been available in Japan for the treatment of epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) since May 2014. We conducted an observational study in patients treated with afatinib in Nagano prefecture, focusing on response and associated toxicities., Methods: We analyzed the clinical records of NSCLC patients treated with afatinib between May 2014 and February 2015., Results: The records of a total of 73 patients (27 men, 46 women) with a median age of 69 years (range: 42-85 years) were analyzed. Afatinib was administered to 11 patients as a first-line therapy, but it was predominantly administered as a fifth-line or beyond therapy (32 cases, 43.8%). The overall response rates for afatinib as a first-line therapy and beyond second-line therapy were 80% (95% confidence interval [CI]: 55.2-100.0%) and 27.1% (95% CI: 14.5-39.7%), respectively. The main toxicities grade >3 included diarrhea (8.2%), skin rash (6.8%), nausea (6.8%), and appetite loss (6.8%). A low body surface area (BSA) (<1.5m 2 ) was significantly associated with a higher frequency of diarrhea grade >2, compared with a higher BSA (≥ 1.5m 2 ). Forty-eight patients (63.0%) were treated without a dose reduction of afatinib., Conclusions: Although the survival benefit with afatinib remains unclear, our observational analysis demonstrated the feasibility of using afatinib for EGFR-mutated NSCLC in clinical practice. In particular, a relatively high level of drug delivery is possible. In addition, a lower BSA may be a predictor of diarrhea in patients treated with afatinib., (Copyright © 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Exclusion of the accessory renal arteries (ARAs) is required during endovascular aneurysm repair if they arise from the sealing zone or aneurysm sac. Here, we report a case of successful endovascular treatment for a common iliac artery aneurysm located close to the aortic bifurcation and associated with nephrotic syndrome in a 51-year-old man. The bilateral ARAs were successfully preserved using kissing stent grafts. During surgery, the proximal ends of endografts inserted from the bilateral femoral arteries were adjusted so that they met at the same level in the aorta, and simultaneous balloon dilatation was performed. This method can be a useful treatment option for common iliac aneurysms in cases with large ARAs., (Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the midterm outcomes of distal bypass surgery for very elderly patients, and to determine the ideal candidates for this procedure., Methods: Of 268 consecutive patients (328 limbs) with critical limb ischemia who were treated between 2006 and 2013, 106 (126 limbs) underwent distal bypass and were retrospectively reviewed. Nineteen patients (22 limbs) were aged ≥80 years (very elderly group) and 87 patients (104 limbs) were aged <80 years (control group)., Results: The baseline characteristics differed between the 2 groups in terms of regular hemodialysis rate (very elderly group, 4 [21%] vs control group, 60 [69%]; P = .0002) and the Charlson comorbidity index (very elderly group, 3.2 ± 1.7 vs control group, 5.0 ± 2.0; P = .0005). According to the Rutherford category of limb ischemia (4/5/6), the very elderly and control groups were classified as 5/17/0 and 11/87/6, respectively (P = .18). Before the surgery, 17 patients (77%) and 67 patients (64%) were ambulatory in the very elderly and control groups, respectively. At follow-up at 29 ± 22 months, the rates of primary (P = .33) and secondary patency (P = .14), limb salvage (P = .50), survival (P = .26), amputation-free survival (P = .42), major adverse limb event and also perioperative death (P = .11), and major adverse cardiovascular events (P = .36) did not significantly differ between the groups. In multivariate analysis, a history of coronary artery disease (hazard ratio [HR], 2.7; 95% confidence interval [CI], 1.3-5.9; P = .005), preoperative nonambulatory status (HR, 4.2; 95% CI, 2.1-8.1; P < .0001), and serum albumin levels <3 g/dL (HR, 2.7; 95% CI, 1.3-5.4; P = .01) were significantly related to poor amputation-free survival. Thirteen patients (59%) remained ambulatory at the latest follow-up. In 91 patients (110 limbs) with tissue loss, the Society for Vascular Surgery lower extremity threatened limb classification system: risk stratification based on Wound, Ischemia, and foot Infection classification stages 3 and 4 negatively affected complete wound healing, according to multivariate analysis (HR, 0.34; 95% CI, 0.20-0.61; P = .0005)., Conclusions: A very elderly age should not preclude a patient from undergoing distal bypass surgery. A history of coronary artery disease, a nonambulatory status, and hypoalbuminemia, along with the Wound, Ischemia, and foot Infection classification for patients with tissue loss, should be carefully considered to obtain the most benefit from distal bypass surgery., (Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Objectives: To verify the usefulness and limitation of intravascular ultrasound (IVUS) in endovascular aneurysm repair (EVAR)., Methods: A total of 112 consecutive patients, who underwent EVAR to treat abdominal aortic aneurysms, were examined retrospectively. Of these, 33 patients were assigned to the IVUS group because of renal failure, a suspected allergy to contrast agents or anatomical difficulties; the remaining 79 patients were assigned to the non-IVUS group., Results: Patients in the IVUS group required fewer intra-arterial contrast agents (IACAs) than those in the non-IVUS group (67±34ml vs. 123±50ml; p<0.01). Blood loss and operation time were comparable between the two groups. No patients died within 30 days of the operation. Three major renal complications occurred in the non-IVUS group. Renal deterioration evaluated by chronic kidney disease (CKD) stage was found to a greater extent in the non-IVUS group., Conclusions: IVUS is a powerful auxiliary method in EVAR for reducing the required volume of contrast agents. The combination of IVUS and IACA usage showed good overall performance; thus, we propose the routine use of IVUS in EVAR procedures., (Copyright © 2010 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.)

Objective: We evaluated the current clinical experience of temporary inferior vena cava (IVC) filter placement and its related complications., Methods: From January 2000 to December 2005, we enrolled 33 patients (8 men and 25 women) who underwent percutaneous insertion of a temporary IVC filter in the Department of Vascular Surgery of Tokyo University Hospital. Deep vein thrombosis (DVT) was proven in 78.8% of the patients. The indications for filter insertion were contraindication to anticoagulation therapy (9.1%), thrombolytic therapy (12.1%), perioperative prophylactic implantation (84.8%), pregnancy with DVT (3.0%), and prophylactic implantation in the absence of DVT (15.2%). A Neuhaus Protect was used in 13 patients, and an Antheor was used in 20 patients., Results: The mean +/- SD duration of filter placement was 10.6 +/- 7.0 days. There was no case of pulmonary embolism during filter protection and retraction. Filter thrombosis (capture of thrombus) was observed in four patients (12.1%), who then received additional thrombolytic therapy. Thrombi were dissolved by thrombolysis in three, one of whom had replacement with a permanent filter. The thrombus was not dissolved in one patient and was removed under venotomy at the insertion site. Major filter-related complications occurred in nine patients (27.3%), including filter dislocation in four patients (12.1%), catheter fracture in three (9.1%), and catheter-related infection in one (3.0%). In a patient with giant ovarian cancer, the IVC was nearly occluded with massive thrombus around the filter 2 days after operation, and the vena cava was then ligated under open laparotomy. No patients died during filter protection and retraction., Conclusions: Temporary IVC filters were effective for the prevention of fatal pulmonary embolism. However, our experience of a high incidence of complications related to temporary filters suggests that this device has limited indications and supports the need for innovative design of temporary filters.

Objective: Inflammatory responses are considered to represent a unique property after stent implantation, and we previously demonstrated that inflammatory signaling involving tumor necrosis factor receptor-associated factor 6 (TRAF6) contributes to neointimal formation in a balloon injury model of rabbit carotid artery. The purpose of this study was to examine the role of TRAF6 in in-stent lesion formation after stent implantation in the rabbit carotid artery., Methods: Rabbit carotid arteries were injured with a 2F Fogarty catheter, and 28 days later, the same arteries were implanted with a 3-mm-diameter Palmaz-Schatz stent. A dominant negative (DN) form of TRAF6 (pME-FLAG-T6deltaRZ5) was then transferred using a plasmid-based electroporation method. Its effects were evaluated compared with the findings in arteries treated with control plasmid (pME-FLAG)., Results: Immunostaining with anti-FLAG tag antibody showed that an expression plasmid vector containing the DN-TRAF6 sequence was successfully transferred to the arterial intima and media. Morphometric analyses revealed that the increase of intimal area in in-stent lesions was significantly inhibited by DN-TRAF6 14 days after stent implantation (DN-TRAF6 group, 3.01 +/- 0.25 x 10(5) microm2 vs control group, 4.25 +/- 0.23 x 10(5) microm2, P < .01), and the cell density was increased compared with that in the control group. In the DN-TRAF6 plasmid-treated vessels, cell replication was prevented in both the intima and media, and fewer leukocytes adhered to the luminal surface. Moreover, DN-TRAF6 suppressed macrophage infiltration, activation of proteases, and proteoglycan accumulation in the in-stent intima., Conclusions: These findings suggest that TRAF6 plays an important role in cell replication, inflammatory cell infiltration, protease activity, and extracellular matrix accumulation that contributes to in-stent lesion development.

Objective: Surgical treatment of arterial lesions associated with Behçet disease (BD) is often complicated by graft occlusion and recurrence of aneurysms. The purpose of this study was to clarify the long-term outcome of surgical intervention for arterial involvement in BD., Methods: Ten patients with BD (9 men, 1 woman) who underwent surgical treatment for arterial aneurysms between 1980 and 2004 were included in the study. The age of patients at the first operation ranged from 36 to 69 years (mean, 50.4 +/- 9.0 years). The mean period between the onset of BD and that of arterial manifestations was 8.0 +/- 5.0 years. We retrospectively reviewed their postoperative courses, including survival, graft occlusion, formation of anastomotic false aneurysms, and the development of aneurysms at different sites. The Kaplan-Meier method was used to calculate the chronologic incidence of complications after surgery., Results: The mean follow-up period was 133 +/- 92 months, ranging from 5 to 285 months. One patient died of rupture of a dissecting aortic aneurysm after undergoing several surgical interventions for multiple aneurysms. There were five graft occlusions among 21 grafts. The cumulative primary graft patency rate in the infrainguinal region was 83.9% at 3 years. Five anastomotic false aneurysms formed among 49 anastomoses between grafts and host arteries. The overall cumulative incidence of formation of anastomotic pseudoaneurysm was 12.9% at 5 and 10 years. All of them formed within 18 months after surgery. Development of new aneurysms in different arteries was observed in two patients., Conclusions: Early occurrence of anastomotic false aneurysm is characteristic of BD. Further investigation is necessary to establish effective postoperative treatment.

Interleukin-4 (IL-4), an anti-inflammatory cytokine, has been shown to inhibit osteoclast differentiation. Therefore, this cytokine is considered to be a promising therapeutic applicant for bone-resorbing diseases such as rheumatoid arthritis (RA). Recently NFATc1, a transcription factor, has been shown to play critical roles in osteoclastogenesis. The aim of this study was to clarify the role of IL-4 on the intracellular signaling of NFATc1. A RAW264.7 monocyte/macrophage cell line and murine bone marrow precursors were differentiated into osteoclasts in the presence of receptor activator of nuclear factor kappaB ligand (RANKL) and/or macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining and a pit assay using dentine were used for the identification of activated osteoclasts. The protein expression of IL-4 receptor, NFATc1, and c-Fos was determined by Western blot analysis. In addition, the gene expression of NFATc1 and c-Fos was determined by reverse transcription and polymerase chain reaction. The IL-4 receptor was constitutively expressed in RAW264.7 cells. RANKL induced osteoclast generation, as determined by TRAP staining and pit assay. IL-4 inhibited RANKL-induced osteoclastogenesis at low concentrations of 10ng/ml and more. Interestingly, IL-4 potently inhibited RANKL-induced expression of NFATc1 at mRNA level. Furthermore, IL-4 inhibited c-Fos expression, which is shown to be responsible for NFATc1 expression, in time- and dose-dependent manners. In addition, IL-4 inhibited the RANKL-induced expression of NFATc1 and c-Fos in murine bone marrow cells. Thus, we suggest that IL-4 may downregulate osteoclastogenesis in part through inhibition of the expression of transcription factors, NFATc1 and c-Fos. These findings provide new insight into development of new medication for osteoporosis and RA.

We previously reported that cadmium (Cd) induced prostaglandin E2 (PGE2) biosynthesis through the activation of cytosolic phospholipase A2 (cPLA2) and induction of cyclooxygenase 2 (COX-2) in primary mouse osteoblastic cells. In the present study, we further investigated the mechanism of PGE2 production by Cd focusing on the main mitogen-activated protein kinase (MAPK) subfamilies that mediate prostaglandin synthesis, extracellular signal-regulated kinase (ERK1/2 MAPK), c-jun-amino-terminal kinase (JNK MAPK) and p38 MAPK, and protein kinase C (PKC) which is activated by Cd in several kinds of cells. Cd at 2 microM and above stimulated PGE2 production in osteoblastic cells and its production was inhibited by the kinase-specific inhibitors PD98059, SB203580, curcumin, and calphostin C. Calphostin C also inhibited the production of PGE2 by phorbol 12-myristate 13-acetate (PMA), which is a potent activator of PKC. PD98059 inhibited PGE2 production stimulated by PMA as well as Cd, indicating that activation of PKC by ERK1/2 MAPK was necessary for Cd-stimulated PGE2 production. Moreover, Cd stimulated the phosphorylation of these three MAPKs, and inhibition of the phosphorylation of ERK1/2 MAPK by calphostin C was also observed. On the other hand, Cd was found to phosphorylate cPLA2 and the phosphorylation was inhibited by PD98059, indicating that cPLA2 was activated by Cd through ERK1/2 MAPK and released arachidonic acid (AA), a substrate of COX-2, from membranous phospholipids. From these results, it was suggested that activation of each of the ERK1/2, p38, and JNK MAPK cascades in addition to that of PKC and cPLA2 played an important role in the Cd-stimulated biosynthesis of PGE2 in mouse osteoblastic cells.

To evaluate the availability of the serum neurotrophins for the diagnosis of the patients with neurodevelopmental disorder, we measured the serum concentration of brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) in the patients diagnosed with autism (n=18) and mental retardation (n=20), or healthy controls (n=16), using enzyme-linked immunosorbent assay. There tended to be a higher concentration of serum BDNF found in the autistic group ( P <0.05 by analysis of variance (ANOVA)) and the mental retardation group ( P <0.001 by ANOVA) compared to the control group. Serum NT-4 concentration tended to be increased in the mental retardation group (P <0.05 by ANOVA). We conclude that measuring the serum concentration of two neurotrophins, BDNF and NT-4, might be helpful to diagnose or classify disorders such as autism or mental retardation.

To examine acute cardiovascular and autonomic responses to microgravity (microG), arterial pressure (AP), aortic flow velocity (AFV), central venous pressure (CVP), and renal nerve activity (RNA) were measured in anesthetized rats during 4.5 s of microG produced by free drop. A smooth and immediate reduction in gravity occurred during free drop, microG being achieved 100 ms after the start of the drop. Acute microG elicited an immediate and striking, but transient, decrease in RNA with no significant change in AP and AFV, but a significant decrease in CVP. The decrease in RNA lasted 2 s, then RNA recovered to the control level despite the G value remaining at < 0.001 for 4.5 s. The RNA decrease was attenuated or completely abolished by sinoaortic denervation, vagotomy, or sinoaortic denervation plus vagotomy. These results suggest that acute microG conditions stimulate sinoaortic and cardiopulmonary mechanoreceptors and suppress RNA.

Seventeen DL-amino acids labeled with a fluorescent chiral labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(-)-DBD-PyNCS), were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). The reagent reacted with amino functional group in dl-amino acids under basic medium. The thiocarbamoyl derivatives were converted to thiohydantoin via thiazolinone in trifluoroacetic acid (TFA) solution. The epimerization ratios during the reaction of the cyclization were less than 37% in all dl-amino acids tested. The resulting thiohydantoin derivatives of individual dl-amino acids were completely separated with isocratic elutions using acidic mobile phase involving 0.1% TFA. The separations of the thiohydantoins yielded from acidic, basic, neutral, hydroxyl, and aromatic amino acids were good enough for the identification of dl-amino acid. The method using the reagent was adopted to identification of dl-amino acid sequences in eight peptides. The separation and identification of the thiohydantoin derivatives liberated from the peptides labeled were performed by the isocratic elutions. The applicability of the proposed procedure to sequential analysis of peptide was demonstrated with [D-Ala(2)]-leucine enkephalin, [D-Ala(2)]-deltorphin II, d-Phe-Met-Arg-Phe-amide, and Phe-D-Met-Arg-Phe-amide. D-Ala, D-Phe, and D-Met in the peptides were positively identified with the proposed procedures. [L-Ala(2)]-leucine enkephalin, beta-lipotropin, Asp-Ser-Asp-Pro-Arg, and Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-amide were also analyzed as the references without D-amino acid., (Copyright 1999 Academic Press.)

D-Amino acids in food and biological samples labeled with R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles (DBD-PyNCS) were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). DL-Amino acids were efficiently labeled at 55 degrees C for 20 min in basic medium. The resulting thiocarbamoyl-amino acids were resolved by an isocratic elution using water:30% methanol in acetonitrile (72:28) containing 0.1% trifluoracetic acid as mobile phase for hydrophilic amino acids and gradient elutions using sodium acetate buffer (pH 5. 2)/acetonitrile as gradient solvent mixture for hydrophobic amino acids, respectively. The detection limits (S/N = 3) of DL-amino acids tested were in the range of 0.16-0.75 pmol. The proposed method was applied to determine the D-amino acid(s) in milk, cream, fermented dairy products (yogurt and yakult), tomato products (juice, puree, and catchup), fermented beverages (beer and red wine), and human urine. The existence of D-amino acid(s) was demonstrated in all the samples tested. Furthermore, the identification of the D-amino acid(s) was performed using both isomers of DBD-PyNCS and by on-line HPLC-electrospray ionization-MS., (Copyright 1999 Academic Press.)

Total resolution of 17 DL-amino acids after derivatization with a fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfony l)-2,1,3- benzoxadiazole [R(-)-DBD-PyNCS], was studied by reversed-phase liquid chromatography. The reaction of the reagent with amino acids proceeds effectively at 55 degrees C for 20 min in the presence of 1% TEA to produce the corresponding fluorescent diastereomers (excitation at 460 nm, emission at 550 nm). Each pair of the resulting derivatives was efficiently separated with water-acetonitrile containing 1% acetic acid as the mobile phase. Peak resolution was in the range of 0.92 (DL-Arg)-9.8 (DL-Cys). Although mutual separation of some DL-amino acids was possible using the elution solvent, simultaneous resolution of 17 DL-amino acids was difficult with a single chromatographic run, even if some gradient elutions were adopted. Therefore, both gradient and isocratic elution systems were used for total resolution of the DL-amino acids. Thus, 17 DL-amino acids were well resolved by a gradient and an isocratic elution systems. The proposed derivatization and elution methods were applied to the determination of DL-amino acids in yogurt. The results showed that some of the L-amino acids, i.e., Glu, Asp, Ser, Gly, Ala, Thr, Pro, Lys, Phe and Met, were found in the methanol extracts of yogurt. On the other hand, the D-amino acids that were identified in the extracts were D-Glu, D-Asp and D-Ala, and the mean % to each L-amino acid were 11.9% (D-Glu), 27.6% (D-Asp) and 56.7% (D-Ala), respectively.

To clarify whether hypercalcemia after injection of Pb to rats is due to biological bone resorption or physicochemical mineral dissolution, the effect of lead (Pb) on release of previously incorporated 45Ca in organ culture was investigated. Pb at 50 microM and above stimulated the release of 45Ca and hydroxyproline (Hyp). Pb did not stimulate 45Ca release from the bones inactivated by freezing and thawing. Eel calcitonin (ECT), bafilomycin A1 and scopadulcic acid B (SDB) inhibited Pb-stimulated 45Ca release. These results indicate that Pb-induced 45Ca release is due to osteoclastic bone resorption. Pb-stimulated bone resorption was inhibited by indomethacin and flurbiprofen. Pb stimulated the release of prostaglandin E2 (PGE2) from the bones into the media. There was significantly high correlation between 45Ca and PGE2 release. Pb-induced bone resorption was inferred to be mediated by PGE2. From these results, it was suggested that hypercalcemia after Pb injection might be caused by biological bone resorption.

HeLa S-3 cells were treated with 195mPt-radiolabeled cis-diammine(1, 1-cyclobutane-dicarboxylato)platinum(II) (carboplatin) under various conditions, and the relationship between lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with carboplatin at 37 degrees C for 1, 2 and 3 h were 553.4, 194.3 and 68.7 microM, respectively. By using identically treated cells, the numbers of Pt-atoms combined with DNA, RNA and protein molecules were determined after fractionation of the cells. In this way the D0 values (D0, dose that would give an average of one lethal event per member of the population), expressed as the drug concentration, were substituted for the number of Pt atoms combined with each fraction. The target volumes, the efficiency of Pt atom to kill cells expressed as the reciprocals of the D0 values, were then calculated with each fraction. The results suggested that DNA was the primary target for cell killing by carboplatin. The target volumes for DNA were 0.891 x 10(4), 2.01 x 10(4) and 3.96 x 10(4) nucleotides for 1, 2 and 3 h treated cells, respectively. The cell killing effects of carboplatin were lower than those of cis-diamminedichloroplatinum(II) (CDDP) by factors of 6.0, 2.8 and 2.6 for 1, 2 and 3 h treatments at 37 degrees C, respectively, in terms of the target volume, while those in terms of the mean lethal dose (D0) were 59.5, 29.0 and 21.5, respectively.

We previously reported that the organotin triphenyltin chloride (TPTC), which has been widely used as an anti-fouling coating for fishing nets and ship bottoms, potentiated clastogen-induced chromosome aberrations during the G2 phase of the cell cycle. In this communication, CHO cells treated with mitomycin C (MMC) were post-treated with TPTC in the presence and absence of other agents--cytosine-1-beta-D-arabinofuranoside (araC), hydroxyurea, or caffeine--having a similar effect during the G2 phase of the cell cycle. The potentiating effect of araC was completely inhibited in the presence of TPTC at the concentration at which TPTC showed its potentiating effect, suggesting that potentiating effects of TPTC and araC are antagonistic. On the other hand, combined treatment with TPTC and caffeine or hydroxyurea showed a potentiating effect almost equal to the sum of the potentiating effects of each given separately.

Mercury compounds are among the most serious environmental pollutants. In this communication, the potentiating effects of organic and inorganic mercuries on clastogen-induced chromosome aberrations were studied in Chinese hamster CHO K1 cells. Post-treatment with monoalkylated mercuries--methyl mercuric chloride (MeHgCl) and ethyl mercuric chloride (EtHgCl)--increased the number of breakage- and exchange-type aberrations induced by 4-nitroquinoline 1-oxide (4NQO) and methyl methanesulfonate. With the DNA crosslinking agents mitomycin C (MMC) and cisplatin, MeHgCl enhanced both types of aberrations while EtHgCl enhanced breakage-type aberrations only. Since these monoalkylated mercuries did not show clastogenic effects by themselves under the present experimental conditions, the increases in chromosome aberrations were not additive. Dialkylated mercuries (dimethyl mercury and diethyl mercury) and inorganic mercuries (HgCl and HgCl2) did not show any potentiating effects. When MMC- or 4NQO-treated cells were post-treated with MeHgCl during the G1 phase, both breakage- and exchange-type aberrations were enhanced. Treatment with EtHgCl during the G1 phase also enhanced both types of aberrations induced by 4NQO. With MMC, however, G1 treatment with EtHgCl did not show any potentiating effect. MeHgCl and EtHgCl treatments during the G2 phase enhanced breakage-type aberrations only. Based on these results, the following possible mechanisms for potentiation of clastogenicity by monoalkylated mercuries were suggested; (1) they interfere with repair of base lesions induced by 4NQO and MMS during the pre-replicational stage, thereby increasing unrepaired DNA lesions which convert into DNA double-strand breaks in S phase, (2) MeHgCl (but not EtHgCl) also inhibits repair of crosslinking lesions during the pre-replicational stage, and (3) their G2 effects enhance breakage-type aberrations only.

The co-clastogenic effect of cadmium ion (Cd2+) was studied in Chinese hamster CHO K1 cells and excision repair-deficient human XP20SSV cells. Cd2+ at < or = 28.0 microM did not show any clastogenic effects under the experimental conditions used. Cd2+ post-treatment at < or = 3.50 microM, however, increased the number of both breakage- and exchange-type chromatid aberrations induced by mitomycin C (MMC) and 4-nitroquinoline 1-oxide (4NQO) in CHO K1 cells. Enhancement of chromosome aberrations induced by MMC was observed when CHO K1 cells were treated with Cd2+ during the G1 phase. Cd2+ was also co-clastogenic with MMC in XP20SSV cells. Its co-clastogenic effect, however, was not observed in 4NQO-treated XP20SSV cells. These results suggest that Cd2+ inhibits DNA pre-replicational repair, perhaps DNA excision repair, thereby causing co-clastogenic effects.

HeLa S-3 cells were treated with 195mPt-radiolabeled trans-diaminedichloroplatinum(II) (TDDP) under various conditions, and the relationship between lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with TDDP at 37 degrees C for 1, 2 and 3 h were 163.7, 65.8 and 24.9 microM, respectively. By using identically treated cells, the number of Pt atoms combined with DNA, RNA and protein molecules was determined after the cells were fractionated using the method of Schneider. In this way, the D0 values given as the drug concentration were substituted for the number of Pt atoms combined with each fraction, then the target volumes, expressed as the reciprocals of the D0 values, were calculated for each fraction. The results suggested that DNA and high molecular weight RNAs (except t-RNA), under some limited condition, could be the target molecules for cell killing by TDDP. The target volumes for DNA were 1.31 x 10(3), 3.01 x 10(3) and 6.26 x 10(3) nucleotides for 1, 2 and 3 h treated cells, respectively. Cell killing effects of TDDP were lower than CDDP by a factor of 39.5, 19.0 and 16.5 for 1, 2 and 3 h treatments at 37 degrees C, respectively, when viewed from the stand point of the target volume, while those from the mean lethal dose (D0) were 17.6, 9.8 and 6.7, respectively.

Effects of cadmium on bone resorption were investigated using neonatal mouse parietal bone culture system. Cadmium at 0.5 microM and above stimulated hydroxyproline release as well as 45Ca release. As cadmium-stimulated bone resorption was inhibited by calcitonin, bone resorption induced by cadmium is osteoclast-mediated bone resorption. CI-1, collagenase inhibitor, depressed cadmium-stimulated bone resorption in a dose-dependent manner. Osteoblasts are also involved in cadmium-induced bone resorption. Indomethacin-inhibited cadmium-stimulated bone resorption and cadmium-treated bones released prostaglandin E2 to a greater extent than untreated bones. Cadmium-stimulated bone resorption was shown to be dependent on the production of prostaglandin E2. 3-Isobutyl-1-methylxanthine potentiated cadmium-stimulated bone resorption and verapamil depressed it. It is possible that an increase in levels of cAMP and calcium ion in bone cells is involved in cadmium-induced bone resorption. From these results, cadmium was found to stimulate osteoclast-mediated bone resorption which is dependent on prostaglandin E2. Second messengers in cadmium-induced bone resorption may be cAMP and calcium ion.

To clarify the effects of dexamethasone (Dex) on metallothionein (MT) synthesis and calcification in osteoblastic cells, a clonal osteogenic cell line (MC3T3-E1) was used in the present study. Under culture conditions designed not to cause calcification, MT synthesis of cells at 3 days after inoculation increased with increasing concentration of Dex (2.5-50 nM) for a 24-h culture period. Cells at 6 or 9 days after inoculation also synthesized MT by a 24-h exposure to Dex. These show that undifferentiated osteoblasts have the ability to synthesize MT by Dex. Under culture conditions designed to cause calcification, cells at 6 days after inoculation were cultured with 50 nM Dex in the presence of 7 mM beta-glycerophosphate (beta-GP) for 7 days. Ca content of Dex-treated cells was about 7.5 times as high as that of untreated cells. Dex-treated cells showed a high activity of alkaline phosphatase (ALP). The Zn content of the MT fraction in Dex-treated cells was about 8 times as high as that of untreated cells. These results show that Dex has the ability to induce MT synthesis in osteoblastic cells and to cause a high calcification which is due at least partly to an enhanced activity of ALP.

To investigate the interaction between cadmium and copper in ossification, femurs from 9-day-old chick embryos were cultured for 6 days in the presence of 2 microM cadmium and/or 1 microM copper. It was found that cadmium + copper treatment caused interactively severe damage to osteogenic mesenchymal cells in the periosteum and a severe degenerative change in osteoblasts around the trabecula, resulting in severe impairment of ossification in the diaphysis. Cadmium content was increased by copper; however, copper content was unaffected by cadmium in the diaphysis. It was therefore considered that the copper-induced increase in cadmium content was a primary factor in the interactive toxic effect of the cadmium + copper treatment in ossification.

The effect of aluminum (Al) on bone metabolism was assessed in organ cultures of embryonic chick bone. Al of 10(-4)M and above caused an inhibitory effect on mineralization without inhibiting matrix formation and a stimulative effect on demineralization without stimulating matrix degradation. Therefore, Al was shown to influence the mineral metabolism of bone.

Serum lipid and apolipoprotein concentrations were measured in 37 male survivors of cerebral infarction (CI) and in 30 healthy controls. Both groups had similar total cholesterol levels, but the HDL-cholesterol level was significantly lower and the serum triglyceride level was significantly higher in the CI patients than in the controls. The ApoB level was significantly higher in the CI patients but there was no significant difference between the 2 groups in the levels of the other apolipoproteins (ApoA-I, A-II, C-II, C-III, and E). The HDL-cholesterol/ApoA-I ratio was significantly lower in the CI patients. Both the VLDL-triglyceride and VLDL-cholesterol levels were higher in the CI patients but the VLDL-cholesterol especially its cholesterol ester level was conspicuously high. A population of VLDL particles that bound to heparin on heparin-Sepharose columns was increased in the CI patients. We suggest that cholesterol ester is excessively transferred from HDL to VLDL during the disturbed catabolism of VLDL in CI patients.

To clarify the effects of cadmium (Cd) on bone formation, a clonal osteogenetic cell, MC3T3-E1, was used in the present study. After 24 h of culture, Cd at 1 ppm and above decreased DNA synthesis and alkaline phosphatase activity, but Cd at 1.5 ppm caused no significant decrease in collagen content. The cells treated with Cd (0.03-1.0 ppm) for 24 h showed the dose-dependent effects on metallothionein-like protein synthesis. The marked increase of Cd content unbound to metallothionein (MT)-like protein with cadmium at 1 ppm may be responsible for the toxic effects of cadmium. After 10 days of culture, the accumulation of 45Ca to the cell layer decreased with increasing level of cadmium at 0.03 and 0.1 ppm. The cadmium-treated cell layer showed a weaker reaction to histochemical staining for mineral compared with control culture. This result suggests that Cd inhibits an initial process of calcification.

Attempts were made to prepare antisera monospecific for Lp(a) lipoprotein and to investigate the distribution of subjects according to plasma levels of Lp(a) in Japanese controls and patients with coronary heart disease or cerebral infarction. Positive plasma reactions to the double diffusion test for Lp(a) (Ouchterlony) were observed in 32.3% of the healthy Japanese subjects, which is similar to results previously reported in western countries. The plasma threshold level of 17 mg/dl was considered an appropriate point for dividing subjects into positive and negative groups depending on reactions to the double diffusion test. When subjects were divided into two groups at 17 mg/dl, a significant association was found between a high plasma level of Lp(a) and either coronary heart disease or cerebral infarction in the distribution of the cortical artery. These results suggest that Lp(a) may play an important part as a risk factor for coronary heart disease and cerebral infarction.

The effect of cadmium (Cd) on the solubility of bone collagen was examined in cultured tibiae from 9-day chick embryos. The solubility of collagen was not significantly increased by 8.9 microM Cd, but showed a significant increase at 13.4 microM Cd. As the [3H]hydroxyproline (Hyp) formation was inhibited by Cd at 8.9 and 13.4 microM [3H]Hyp formation and collagen solubility were investigated in the presence of 200 microM Fe2+, a metal which is known to prevent the inhibitory effects of Cd on prolyl hydroxylase in vitro. Fe affected neither a decrease in [3H]Hyp formation nor an increase in collagen solubility caused by Cd. This suggests that a Cd-induced increase in collagen solubility may be due to the decrease of lysyl oxidase activity, not to the formation of underhydroxylated collagen. Zn at 48 and 134 microM depressed the Cd-induced increase in collagen solubility, but caused no increase in collagen solubility. Our present and previous results suggest that Zn can protect the disturbance of both the collagen synthesis and the collagen cross linking caused by Cd.

2,4-Dinitrotoluene (2,4-DNT) and its metabolites (2A4NT, 4A2NT, 2,4-DNB, 2A4NB, 4A2NB, 2,4-DAT, 2N4AAT, 2A4AAT, 2A4AABA and 2,4-DNBA), and 2,4-dinitrobenzaldehyde (2,4-DNA1), putative metabolite of 2,4-DNT, were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100. 2,4-DAT, 2N4AAT, 2A4AAT, 2A4AABA and 2,4-DNBA were inactive for strains TA98 and TA100. 2,4-DNT itself was only a weak mutagen. Two aminonitrotoluenes (2A4NT and 4A2NT), two aminonitrobenzyl alcohols (2A4NB and 4A2NB) and 2,4-dinitrobenzyl alcohol (2,4-DNB) were increasingly mutagenic in that order, in both strains, however, they were only weak or weak mutagens at mM concentrations. In contrast with these compounds, 2,4-DNA1 was mutagenic even at microM concentrations in both strains. These results suggest that a high mutagenicity of 2,4-DNA1 may be correlated to the carcinogenicity of 2,4-DNT.

The effect of cadmium (Cd) on bone collagen synthesis was assessed in organ cultures of embryonic femur by measuring the incorporation of [3H]-proline(Pro) into collagenous-digestible protein (CDP) using purified bacterial collagenase. Cd produced a decrease when [3H]Pro ws incorporated in CDP. There was little alteration in the hydroxylation of [3H]Pro to [3H]hydroxyproline(Hyp) in CDP of Cd-treated bone. An accumulation of underhydroxylated collagen and a decrese in the activity of prolyl hydroxylase (EC 1.14.11.2) in Cd-treated bone was not observed. These reslts indicated that the inhibitory effect of Cd on collagen synthesis was largely due to inhibition of collagenous peptide synthesis without inhibition of its hydroxylation.

To clarify a possible mechanism of zinc (Zn)-induced tolerance to cadmium (Cd) toxicity on bone formation, femurs from 9-day-old chick embryos were cultured for 6 days by the roller-tube method in the presence of Cd (2, 4 or 9 microM) and/or Zn (60 microM). Zn prevented a decrease in bone growth caused by Cd at 4 and 9 microM. An increase in calcium (Ca) content of diaphysis was inhibited by Zn in both the presence and absence of Cd. Histologically, Zn protected a Cd-induced degenerative change of mesenchymal cells in the periosteum and that of osteoblasts around the trabecula at each Cd level. At 60 microM Zn, Cd accumulated less in the bone at 2 microM but more at 9 microM. From these results, it was concluded that Zn prevented Cd-induced toxicity in the process of ossification except calcification in a culture system by two different mechanisms, i.e. a decreasing Cd accumulation at a low level of Cd and probably an induction of metallothionein (MT)-like protein at a high level of Cd.

Subcutaneous (s.c.) and intraperitoneal (i.p.) injection and oral administration of calcium (Ca) into rats increased Ca content of liver compared with control rats. When liver cytosol was filtered through a Sephadex G-75 column, zinc (Zn) content of metallothionein (MT)-like protein fraction was several times higher in Ca treatment than in control. Zn and copper (Cu) content of high-molecular (HM) and moderate-molecular (MM) fractions and Cu content of MT fraction was unaffected by Ca treatment. Zn in MT fraction showed two peaks in the direct-connection method of high-performance liquid chromatography to atomic absorption spectrophotometer (AAS). The retention time of these two peaks agreed with those of Zn or cadmium (Cd) in MT fraction of liver cytosol obtained from Zn- or Cd-administered rat. These results show that MT-like protein containing Zn is induced by Ca. In conditions showing a slight increase in liver Ca and a significant decrease in serum Ca by synthetic [Asu1,7] eel calcitonin (CT) injection, gel filtration of liver cytosol obtained from CT-treated rats showed a higher content of Zn and a higher radioactivity of [3H]cystine than that from control injection. This suggests that CT causes an increase in liver Ca and results in induction of MT-like protein containing Zn by Ca.

The mutagenic activities of 2,6-dinitrotoluene (2,6-DNT) and its 6 metabolites, and their 8 related compounds were examined using Salmonella typhimurium strains TA98 and TA100 in the absence or presence of S9 mix. 2,6-DNT itself showed no mutagenicity toward either strain, but 2,6-dinitrobenzaldehyde (2,6-DNBAl), one of the metabolites of 2,6-DNT, showed the highest mutagenic activity in strain TA100. 2,6-DNBAl was a direct-acting mutagen, not requiring metabolic activation. The other compounds containing nitro groups showed weak or no mutagenic activity. This result suggests that the direct-acting mutagenicity of 2,6-DNBAl is mainly due to the aldehyde group of the 2,6-DNBAl molecule.

We examined the effects of several forms of vitamin D added to renal brush-border membrane suspensions on phosphate and glucose transport and on membrane fluidity. The 1,25-D stimulated and the other vitamin D decreased phosphate uptake. In contrast, glucose uptake was not affected by the treatment of vitamin D. The 1,25-D resulted in a significant shift of the lower transition temperature in Arrhenius plots for phosphate, but not for glucose uptakes, from 15 degrees C to 11.5 degrees C. These data indicate that the 1,25-D may alter membrane fluidity, limited to the phosphate transporter, thus affecting the phosphate uptake.

Femurs from 9-day-old chick embryo were cultivated for 6 days by the roller-tube method in the presence of Cd, Cu or Zn. Cd (5.0 microM and above) and Cu (2.5 microM and above) caused a decrease in collagen content of both diaphysis and epiphysis, mainly due to inhibition of collagen synthesis. In addition, Cd and Cu each showed a tendency to inhibit an increase in Ca content of diaphysis, where intraperiosteal ossification could be observed. Alkaline phosphatase (ALP) activity was decreased by Cd (5.0 microM and above) or Cu (10 microM and above) in diaphysis. On the other hand, Zn at 50 microM and above inhibited an increase in Ca content of the diaphysis with a remarkable elevation of ALP activity in the medium. At this time, Zn did not decrease the collagen content of the diaphysis so strongly. Histological observations revealed that Cd and Cu each decreased both calcified and uncalcified osteoid tissue at 2.5 microM, while Zn at 100 microM decreased calcified tissue but increased uncalcified osteoid tissue. As Zn accumulated particularly in diaphysis and deposited at the edge of calcified tissue, it was suggested that Zn inhibited calcification physicochemically. It was concluded that Cd or Cu would induce bone damage represented by osteoporosis, whereas Zn would induce osteomalacia.