Biomedical subjects
Publications and source records attributed to M Kuroki.
A new series of 2-arylmethyl-1,4-benzoquinones (2) was synthesized for evaluation of their pharmacological activities. These compounds showed significant inhibition of platelet aggregation induced by arachidonic acid (AA) and some of them possessed a protective effect against endothelial cell injury caused by hydrogen peroxide.
Two types of chicken egg yolk antibody samples for oral passage trials in calves were prepared: (1) hydroxypropyl methylcellulose phthalate (HPMCP) antibody powder (HAP)--a powder produced by spray-drying a supernatant obtained after precipitation of lipids from egg yolk with HPMCP and (2) control antibody power (CAP)--a powder produced from an antibody solution with HPMCP. Antibody activity and pattern of distribution of both antibody preparations in the gastrointestinal tract of calves were compared by enzyme-linked immunosorbent assay. At 2 hr post administration, anti-K99 fimbrial antibodies from both the CAP and the HAP were detected in the abomasum of calves with titers of 1:128 and 1:256, respectively. However, at 4 hr, anti-K99 fimbrial titers of the CAP and the HAP were reduced to 1:2 and 1:64, respectively, due to digestion in the abomasum. These results indicated that the egg yolk antibody powder with HPMCP was more resistant against gastric juice in the stomach, thereby, ensuring a transfer of functional antibodies to the small intestine of calves after oral administration.
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We have established a new chemiluminescent enzyme immunoassay for carcinoembryonic antigen (CEA), designated ACCESS CEA, which is adapted to the fully automated ACCESS immunoassay analyzer. The assay is based on a one step sandwich-type method using two monoclonal antibodies, one of which is immobilized on micrometer-size paramagnetic particles and the other is conjugated to alkaline phosphatase. Ten microliters of calibrators or sera are incubated for 5 minutes at 37 degrees C with the particles and with the alkaline phosphatase conjugate. The particles are then magnetically separated and washed to remove unbound components. Time needed to obtain the first result is less than 15 minutes. The assay range was 0.04-1000 micrograms/l of CEA, and the possible high-dose hook effect was prevented at CEA concentrations up to 100000 micrograms/l in this working range. The coefficient of variation (CV) for intra-assay precision was 3.0 to 4.7%, and inter-assay CV was 3.4 to 5.6%. The sample carryover was less than 0.001%. The analytical recovery ranged from 98 to 104% and a dilution linearity was demonstrated. No interference was detected in any sample with levels up to 300 mg/l for bilirubin, 12000 mg/l for haemoglobin, 50000 mg/l for human serum albumin, 8500 mg/l for triacylglycerol, and 500000 IU/l for rheumatoid factor. The ACCESS CEA assay also showed very homogeneous reactivity with purified CEA preparations from different tumours and could discriminate CEA from four CEA-related normal antigens tested. Serum samples (n = 362) from patients with malignant or non-malignant disease, as well as from healthy individuals, were analyzed by the ACCESS CEA assay and by the established IMx CEA assay. The CEA values determined by the ACCESS CEA assay were in good agreement with those determined by the IMx CEA assay, and the ACCESS CEA assay significantly increased the sensitivity and specificity of tumour diagnosis as compared with the IMx CEA assay.
Thermosensitive liposomes are microscopic vesicles that can contain drugs and release them effectively in response to hyperthermia. To deliver an antitumor drug specifically to brain tumor, the authors used thermosensitive liposomes containing cis-diamminedichloroplatinum (CDDP) in conjunction with localized brain heating. The authors then investigated the antitumor effect on rat malignant glioma. Rous sarcoma virus-induced malignant glioma cells were transplanted into the brains of Fisher rats. Ten days after tumor inoculation, the rats were assigned to one of six treatment groups: control, free CDDP, hyperthermia, free CDDP + hyperthermia, liposomes containing CDDP (CDDP-liposome), and CDDP-liposome + hyperthermia. Liposomes containing CDDP or free CDDP were injected via the tail vein. Brain tumor heating was administered by means of a radiofrequency antenna designed at our institute. The rats treated with CDDP-liposome + hyperthermia had the longest survival time and the tumor CDDP level of this group was the highest when compared to the other groups. Histopathological examination showed that tumor cells were necrotized but surrounding normal brain tissue remained undamaged. On the basis of these findings we suggest that the combination of thermosensitive liposome and localized hyperthermia may better focus antitumor drugs to the tumor, providing a significantly greater antitumor effect.
Unilateral Moyamoya disease presents as unilateral stenosis or obstruction of the supraclinoid internal carotid artery, which causes cerebral hypoperfusion resulting in seizures or TIA-like attacks. In severe cases, surgical treatment is performed with superficial temporal artery-middle cerebral artery anastomosis. In mild cases, conservative management is the treatment of choice. Flunarizine is a calcium ion anti-blocking agent, whose primary effect is that the cerebral vessels have been used for the treatment of postcerebrovascular disorders. Recently, it has been suggested that flunarizine could be used to treat Moyamoya disease. This report documents the efficacy of flunarizine to improve regional cerebral perfusion in Moyamoya disease.
Carcinoembryonic antigen (CEA) is recognized as being important not only as a tumour marker, but also as a cell adhesion molecule in the metastasis of malignant tumour cells, and in the development of inflammatory disorders in some organs. However, the role played by this antigen in inflammatory dermatoses has not been studied previously. To investigate the expression of CEA in inflamed skin, we performed immunohistochemical studies to detect the antigen in both frozen and formalin-fixed, paraffin-embedded biopsy specimens of inflammatory dermatoses, using five different monoclonal antibodies and one polyclonal antibody against human CEA or CEA-related molecules. CEA and CEA-related antigens were expressed by epidermal keratinocytes and by some infiltrating inflammatory cells in inflamed skin. The expression was observed primarily in the upper part of the malpighian layers of the epidermis. On the basis of the distribution of this expression, we suggest an additional role for CEA-related molecules, differing from their roles as tumour markers and adhesion molecules, namely that these molecules may be associated with the state of differentiation of epidermal keratinocytes. Unexpected expression of CEA-related antigens was seen on normal follicular keratinocytes. Thus, it is conceivable that CEA-related antigen may also play a role in the biology of hair follicles.
To determine the association of a hypoxia-induced, vascular endothelial cell selective growth factor: vascular endothelial growth factor (VEGF) on proliferative diabetic retinopathy (PDR), undiluted vitreous fluids were collected from 10 eyes with PDR at the time of vitrectomy. Vitreous fluids from 10 eyes without PDR were used as control. All samples were electrophoresed with a 14% sodium dodesyl sulfatepolyacrylamide gel, and electrically transferred to a Durapore membrane. Immunoblot detection of VEGF was done by standard western blotting using rabbit polyclonal anti-human VEGF antibody. Positive immunoreaction bands were observed on lanes of 7 samples from PDR, and they were comigrated with recombinant human VEGF. No reactions were observed on lanes of all samples without PDR. The presence of VEGF within the vitreous fluids of eyes with PDR was determined, and suggesting the association of VEGF with PDR.
The Klippel-Feil syndrome is one of the spinal malformations characterized by fusion of the cervical vertebrae. It is well known that the malformation can cause some neurologic disorders. However, an aneurysm in the vertebral artery associated with the Klippel-Feil syndrome is extremely rare, with only one case having been reported in the literature. We present a 39-year-old female with a sudden onset of disturbed consciousness. Lateral cervical x-ray films showed the Klippel-Feil syndrome and hypermobility between C1 and C2. MRI showed multiple infarctions in the posterior circulation, including bilateral thalami. Right vertebral angiogram identified the extracranial vertebral artery aneurysm as the source of the emboli. The patient gradually recovered with conservative therapy. The aneurysm was thought to be produced by chronic arterial trauma secondary to excessive movement between C1 and C2. We conclude that an extracranial vertebral artery aneurysm is a serious complication in a patient with the Klippel-Feil syndrome.
The N-linked sugar chain structures of the carcinoembryonic antigen (CEA) produced by liver metastases of colon cancers and the normal counterpart of CEA purified from human adult feces (NFA-2) were previously determined comparatively (K. Fukushima, T. Ohkura, M. Kanai, M. Kuroki, Y. Matsuoka, A. Kobata, and K. Yamashita. Glycobiology, 5:105-115, 1995). Seventy-five % of NFA-2 contained complex type sugar chains with Gal beta 1-->3GlcNAc residues, in contrast to the sugar chains of CEA, in which over 90% of the oligosaccharides contained Gal beta 1-->4GlcNAc residues, and Sia alpha 2-->6Gal beta 1-->4GlcNAc residues were detected in 18 to 65% of the oligosaccharides. The expression of Sia alpha 2-->6Gal beta 1-->4GlcNAc residues on CEA molecules in sera and tissues was investigated using Trichosanthes japonica agglutinin I (TJA-I), which interacts with Sia alpha 2-->6Gal beta 1-->4 GlcNAc residues. Ten purified CEA samples bound to a TJA-I column while seven NFA-2 samples passed through the column. Various concentrations of serum CEA samples from patients with metastatic colon cancers exclusively bound to the TJA-I column, reflecting that CEA molecules exfoliated into the blood circulation comprise sugar chains with Sia alpha 2-->6Gal beta 1-->4GlcNAc residues. In histochemical studies involving biotinylated TJA-I, normal mucosa (n = 20) and benign adenomas (n = 20) were not stained, and 83% of well and moderately differentiated colon adenocarcinomas (n = 53) reacted with TJA-I, although poorly differentiated ones (n = 9) and mucinous specimens (n = 10) were negative. Because over 90% of colon adenocarcinomas can be differentiated, TJA-I staining might be applicable to the early diagnosis of colon cancers.
To reduce heterophilic antibody interference in a two-site immunoassay for carcinoembryonic antigen (CEA), we utilized a human/mouse chimeric antibody to CEA as the tracer. One mouse monoclonal antibody (MAb), F82-61, which reacts with an epitope present on the domain N of CEA, was immobilized on 96-well polystyrene microtiter plates. A human/mouse chimeric antibody (Ch F11-39), which recognizes an epitope present on the domain B3 of CEA, was biotinylated for the tracer (Ch F11-39 system). Another MAb F11-39, the parental MAb of Ch F11-39, was also biotinylated and used as the control tracer (F11-39 system). For a fair comparison, the same 503 serum samples from healthy individuals were simultaneously assayed in the present study. When a tentative common reference limit of 5 ng/ml was used, the false positive rate with the Ch F11-39 system was only 2.8% (14/503) and that with the F11-39 system was 29.0% (146/503). Adding normal mouse serum (NMS; 1%) or a mixture of purified mouse IgG subclasses (heterophilic blocking reagent (HBR, 15 micrograms/test)) to the F11-39 system reduced the false positive rate from 29.0% to 6.2% (31/503) or 4.8% (24/503), respectively, suggesting that heterophilic antibodies reactive with mouse IgG gave rise to the high positive rate in normal populations with the F11-39 system. On the other hand, the false positive rate with the Ch F11-39 system was only slightly reduced from 2.8% to 2.6% (13/503) or to 2.0% (10/503) by adding NMS or HBR to the Ch F11-39 system. The false positive rates with two commercially available assay systems, CEA Roche EIA.DM or Abbott IMx CEA, were 5.4% (27/503) and 5.8% (29/503), respectively, which both corresponded roughly to that with the F11-39 system including NMS or HBR. These results indicate that the application of human/mouse chimeric antibodies in two-site immunoassays is more effective for reducing interference from heterophilic antibodies than the adding of NMS or purified mouse IgG in the assay using conventional MAbs.
The epitopes of 42 well-characterized monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) from 10 different research groups were mapped in terms of domain structure (domains N, A1-B1, A2-B2, and A3-B3) of the CEA molecule on the basis of the reactivities with recombinant CEA proteins expressed in Chinese hamster ovary cells. Thirty-six of the 42 MAbs tested have previously been classified into 5 essentially nonoverlapping epitope groups (GOLD 1-5) by cross-competition assays among MAbs for CEA binding (Hammarström S, et al.: Cancer Res. 1989; 49:4852-4858). The epitopes recognized by GOLD 2 MAbs were all present on domain A2-B2, those for GOLD 5 MAbs were all on domain N, and those for GOLD 4 were mapped around domains A1-B1 and A2-B2. On the other hand, the epitopes for GOLD 1 MAbs were distributed into domains N, A2-B2, and A3-B3, and those for GOLD 3 MAbs were separated into domains N and A3-B3. Although the exact reasons for the dispersed patterns of GOLD 1 and 3 MAbs on the domain structure of the CEA molecule are unclear at present, several factors, such as a spatial relation or a close proximity of epitopes, conformation dependency, and repetitivity of epitopes, may be considered as possible explanations. The epitope mapping reported here helps form the basis for understanding the relation between the chemical structure and antigenic activities of the CEA molecule and may be useful to study the functions of the CEA molecule, especially those of the respective domains.
Normal faecal antigen-2 (NFA-2) and non-specific cross-reacting antigen-2 (NCA-2), cross-reacting with anticarcinoembryonic antigen (CEA) antibodies, were found in normal human faeces and meconium, respectively. Because NFA-2, NCA-2 and CEA are considered as the same gene products, NFA-2 and NCA-2 should be normal counterparts of CEA produced by colon epithelial cells of normal adults and fetuses, respectively. Comparison of sugar chain structures of these three antigens is indispensable in order to unravel the structural alteration induced by malignant transformation and development of colon epithelial cells. The sugar chain structures of CEA (Yamashita, K. et al., Cancer Res., 47, 3451-3459, 1987) and NCA-2 (Yamashita, K. et al., J. Biol. Chem., 264, 17873-17881, 1989) were previously reported. In this paper, the structures of the oligosaccharides released from four NFA-2 samples by hydrazinolysis were studied by means of lectin-affinity column chromatography, endo- and exo-glycosidase digestion, methylation analysis, hydrazinolysis-nitrous acid deamination and electrospray ionization mass spectrometry. NFA-2 contains 24-27 mol of N-linked sugar chains/molecule, which is similar to NCA-2 (27 mol) and CEA (24-27 mol). In contrast to CEA, which contains approximately 8% high-mannose-type sugar chains, all sugar chains of NFA-2 are mono- to tetra-antennary complex-type chains having four types of tri-mannosyl cores, with or without bisecting N-acetylglucosamine and fucose residues. The structures of their outer chain moieties comprise Gal beta 1-->3(HSO(3-)-->6)GlcNAc, Neu5Ac alpha 2-->3Gal beta 1-->3GlcNAc, Type 1, repeating chain, Type 2, Type 2H, Type 1H, Lex, Lea and Leb antigenic determinants. Approximately 50% of the outer chain moieties of the oligosaccharides of NFA-2 contain Type 1 chain, in contrast to those of CEAs produced by the liver metastases of colon tumours in which only a trace amount of Type 1 chain was detected.
The complete amino acid sequence of a Ca(2+)-dependent lectin, CEL-IV, from the marine invertebrate Cucumaria echinata was analyzed. The established sequence showed that CEL-IV comprises 157 amino acid residues with a molecular mass of 17,098 Da (without disulfide bonds). From comparison with other proteins, CEL-IV was apparently homologous with the C-type lectin family. The identity was relatively high with a sea cucumber (Stichopus japonicus) lectin SJL-I (40.0%) and a sea urchin (Anthocidaris crassispina) lectin echinoidin (32.6%). In CEL-IV, one interchain and two intrachain disulfide bonds were identified. Interestingly, one of the two intrachain disulfide bonds that were highly conserved among the other C-type lectins was missing, suggesting that this might be a characteristic feature of C-type lectins in the Holothuroidea.
Subcellular localization and antigenic properties of eight carcinoembryonic antigen (CEA)-related proteins (nonspecific cross-reacting antigens, NCAs) in neutrophils, including CD66 antigens, were examined with a panel of CD66 monoclonal antibodies (MAbs), whose specificities were characterized with CHO cells expressing CEA gene family recombinant proteins. Intracellular granules of neutrophils were isolated by cell lysis followed by centrifugation on a sucrose gradient. SDS-PAGE analysis of immunoprecipitates from the granules revealed that NCA-95 (CD66c, NCA-50/90) exists predominantly in the azurophil granule-enriched fraction. NCA-90 was identified in the same fraction and was suggested to be a molecule closely-related to NCA-95. NCA-26 was identified in the specific granule fraction together with NCA-160 (CD66a, BGP), and was likely a splicing variant of NCA-160. NCA-100 (CD66b, CGM6) was also identified in this fraction. NCA-80, -58 and -30, which are detectable in culture medium of neutrophils with polyclonal anti-NCA-50/90, were not recognized by any of the CD66 MAbs tested. These findings indicate that NCA-160, -100, -95, -90 and -26 are the products of the CEA gene family and stored separately in azurophil and specific granules. It remains to be clarified whether or not the other three secretory antigens, NCA-80, -58 and -30, are still unknown members of the CEA family.
To reduce the high immunogenicity of mouse monoclonal antibodies (MAbs) in man, mouse/human chimeric antibodies have been generated in two ways. The more simple method is to construct chimeric antibodies in which the variable regions from the mouse MAbs are linked to human constant regions. The second is to graft the complementarity determining regions from the variable regions of mouse MAbs into human variable regions. So far, more than a hundred of chimeric antibodies have been reported and about twenty-five have been tried for immunotherapy of various human diseases. In the near future, many mouse/human chimeric antibodies will be commercially available.
The levels of 23 protein components of the sera of 84 patients with uterine cervical cancer undergoing radiotherapy were determined by a single radial immunodiffusion method, before radiation therapy, after whole pelvic irradiation, and after intracavitary irradiation using a remote after roading system (RALS). We examined the correlations between changes in serum protein fractions and the prognosis of the patients. The patients with uterine cervical cancer were treated with combined external irradiation of 60Co gamma-rays or 10 MV X-rays, and RALS therapy. The levels of the same protein components were also measured in 21 normal adult women as a control. All patients were observed between 4 years and 8 years after radiation therapy. In the pretreatment sera obtained from patients with uterine cervical cancer, the concentrations of prealbumin (Prealb), alpha 2HS glycoprotein (alpha 2 HS), alpha 2-plasmin inhibitor (alpha 2PI), transferrin (Tf), plasminogen (Pmg), albumin (Alb), IgM and hemopexin (Hx) were significantly lower than those normal controls. However the serum concentrations of: alpha 1-antichymotrypsin (alpha 1 X), haptoglobin (Hp), C9, fibrinogen (Fib), ceruloplasmin (Cp), alpha 1-acid glycoprotein (alpha 1AG), alpha 1-antitrypsin (alpha 1AT) and C4 were elevated. At the completion of whole pelvic irradiation and RALS therapy, the Hp, C4, and Fib levels were significantly lower than those before radiation therapy, whereas Prealb, alpha 2HS and alpha 2PI were elevated. In patients who had lived between 4 years and 8 years after radiation therapy, the Cp, alpha 1 AG, Hp and C9 levels measured before radiation therapy were reduced significantly, while Tf was elevated compared with patients who died within 4 years. In those who had lived more than 4 years, the alpha 1 AT, Hp, alpha 1X, Cp and C9 levels measured after whole pelvic irradiation were reduced significantly; while the C4 level was elevated. In cases of uterine cervical cancer followed for a period of 4 to 8 years after radiation therapy, multiple regression analysis was used to determine whether increased concentrations of serum protein fractions were associated with good prognosis for the original disease. Tf, Pmg, and alpha 1AT before radiation therapy were positively correlated with survival, whereas AT III, Cp, C1Inh, IgA, alpha 1 AG and C9 showed negative correlations. After whole pelvic irradiation,Pmg, C4 Prealb, Alb,alpha 2M and Hp were found to be positively associated with survival, whereas Tf, alpha 2PI, AT III, alpha 1 AT,C1Inh, C9 and IgA were negative factors.
Recent studies have shown that, even with a minimal content of carcinoembryonic antigen (CEA), normal human colonic epithelial cells express substantial amounts of CEA mRNA and colonic mucosal fragments cultured in vitro produce CEA quite actively, indicating that CEA should no longer be considered to be of an oncofetal nature. To understand the basis of the usefulness of CEA as a tumor marker, we analyzed the release of CEA, a glycosyl-phosphatidylinositol (GPI)-anchored protein, from colonic epithelial cells, by culturing isolated colonic crypts in collagen gel. The crypts appeared to preserve their morphological and biochemical integrity in the gel for at least 16 hr, and released CEA spontaneously. Three forms of CEA--spontaneously released CEA, CEA liberated with phosphatidylinositol-specific phospholipase C (PI-PLC) and CEA in cell lysates--were indistinguishable on SDS-PAGE. This is in contrast to recombinant CEA spontaneously released from CHO transfectants, which showed a smaller molecular mass than that of PI-PLC-cleaved recombinant CEA. By phase separation using Triton X-114, CEA in the cell lysates of crypts was separated mostly into the detergent phase, while the spontaneously released and the PI-PLC-cleaved CEA were separated into the aqueous phase. When the cells were metabolically labeled with the precursors of the GPI-anchor, 3H-ethanolamine but not 3H-palmitic acid was found in the spontaneously released CEA. These findings suggest that, in contrast to the proteolysis-like release of the recombinant CEA from CHO cells, CEA in normal colonic epithelial cells is released by a non-proteolytic cleavage, which probably occurs through the action of some endogenous phospholipase.