Biomedical subjects

K Fujimoto

Publications and source records attributed to K Fujimoto.

At least 19 recordsLinked to original sources

PubMed1999

Cell membrane dynamics and the induction of apoptosis by lipid compounds.

To investigate the induction of apoptosis by some lipid compounds which are a potent inducer of apoptosis, the plasma membrane fluidity of U937 cells was measured using the fluorescent probe, pyrene. The increase of the membrane fluidity was observed immediately after the treatment of cells with lipid inducers. We also found that the trigger of apoptosis was pulled within 30 min after treatment. Data from the dynamic light scattering experiment indicated that lipid inducers were dissolved to form the emulsion. At the very early stage of apoptosis, possibly, the well-controlled transfer of lipid inducers from the emulsion to the lipid layer of cells can bring about the increase of membrane dynamics which might lead to the induction of apoptosis.

Apoptosis↗

PubMed1999

Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands.

Two related integral membrane proteins, claudin-1 and -2, recently were identified as novel components of tight junction (TJ) strands. Here, we report six more claudin-like proteins, indicating the existence of a claudin gene family. Three of these were reported previously as RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF, but their physiological functions were not determined. Through similarity searches followed by PCR, we isolated full length cDNAs of mouse RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF as well as three mouse claudin-like proteins and designated them as claudin-3 to -8, respectively. All of these claudin family members showed similar patterns on hydrophilicity plots, which predicted four transmembrane domains in each molecule. Northern blotting showed that the tissue distribution pattern varied significantly, depending on claudin species. Similarly to claudin-1 and -2, when these claudins were HA-tagged and introduced into cultured Madin-Darby canine kidney cells, all showed a tendency to concentrate at TJs. Immunofluorescence and immunoelectron microscopy with polyclonal antibodies specific for claudin-3, -4, or -8 revealed that these molecules were exclusively concentrated at TJs in the liver and/or kidney. These findings indicated that multiple claudin family members are involved in the formation of TJ strands in various tissues.

Amino Acid Sequence↗

PubMed1999

Parasite infection and Japanese cedar pollinosis in monkeys.

To evaluate the relationship between helminthic parasite infection and sensitization to Japanese cedar (Cryptomeria japonica; CJ) pollen allergens in Japanese monkeys (Macaca fuscata), we examined the parasite infection, presence of anti-pollen allergen IgE and development of pollinosis. Serum samples and fecal specimens were taken from 169 monkeys in five troops, and the presence of IgE antibody for CJ pollen allergen and of helminthic parasite eggs in their feces was examined. Of the 169 monkeys, helminthic parasite eggs from 138 monkeys were detected. The frequency of anti-CJ IgE (19%) and the level of total IgE did not differ significantly between the monkeys with and without parasite eggs. We examined the presence of anti-CJ IgE and pollinosis symptoms in 31 monkeys of a troop; six of the monkeys demonstrated anti-CJ IgE and pollinosis symptoms. Five of these six monkeys had parasite eggs. We found that the monkeys that demonstrated anti-CJ IgE and pollinosis symptoms had helminthic parasite infections. These data suggest that helminthic parasite infection does not reduce the development of clinical signs of CJ pollinosis in Japanese monkeys.

Allergens↗

PubMed1999

Expiratory and inspiratory chest computed tomography and pulmonary function tests in cigarette smokers.

This study evaluated small airway dysfunction and emphysematous destruction of lung parenchyma in cigarette smokers, using chest expiratory high-resolution computed tomography (HRCT) and pulmonary function tests (PFT). The degree of emphysematous destruction was classified by visual scoring (VS) and the average HRCT number at full expiration/full inspiration (E/I ratio) calculated in 63 male smokers and 10 male nonsmokers (group A). The Brinkman smoking index (BI), defined as cigarettes x day(-1) x yrs, was estimated. Sixty-three smokers were divided into three groups by PFT: group B1 (n=7), with normal PFT; group B2 (n=21), with diffusing capacity of the lung for carbon monoxide (DL,CO) > or = 80% predicted, forced expiratory volume in one second (FEV1) < 80% pred and/or residual volume (RV) > 120% pred; and group B3 (n=35), with DL,CO < 80% pred, FEV1 < 80% pred and/or RV > 120% pred. Heavy smokers (BI > or = 600) (n=48) showed a significant increase in emphysema by both VS and E/I. E/I was significantly elevated in both group B2 (mean+/-SD 0.95+/-0.05) and B3 (0.96+/-0.06) compared with group B1 (0.89+/-0.03). VS could not differentiate group B2 (3.9+/-5.0) from B1 (1.1+/-1.6). These findings suggest that the expiration/inspiration ratio reflects hyperinflation and airway obstruction, regardless of the functional characteristics of emphysema, in cigarette smokers.

Forced Expiratory Volume↗

PubMed1999

Immunohistochemical analysis of Bcl-2, Bax, Bcl-X, and Mcl-1 expression in pancreatic cancers.

Expression of several members of the Bcl-2 family proteins was investigated by means of both immunohistochemical analysis in 30 invasive ductal adenocarcinomas and 23 intraductal papillary-mucinous tumors (IPMTs) and immunoblot analysis in 6 cancer tissues and 7 pancreatic cancer cell lines. We found that Bcl-2 was expressed in 23%, Bax in 53%, Bcl-X in 90%, and Mcl-1 in 90% of the invasive ductal adenocarcinomas. In intraductal papillary-mucinous adenocarcinomas, the expression rate of Bax was 44% and those of Bcl-XL and Mcl-1 were 88%; these values were higher than those for intraductal papillary-mucinous adenomas. Immunoblot analysis identified Bcl-XL as the predominant form of the Bcl-X protein in both pancreatic cancer tissues and cell lines, and demonstrated that both Bcl-XL and Mcl-1 protein levels were uniformly high in all cell lines. These results suggest that an imbalance between antiapoptosis proteins (such as Bcl-2, Bcl-XL, and Mcl-1) and proapoptotic proteins (such as Bax and Bcl-Xs) is involved in the distinctive biologic features of adenocarcinomas of the pancreas. Furthermore, predominantly high expressions of Bcl-XL and Mcl-1 in intraductal papillary-mucinous adenocarcinomas might be involved in the carcinogenesis in IPMT of the pancreas.

Adenocarcinoma↗

PubMed1999

A new parameter in decision making for transurethral electroresection of benign prostate hyperplasia.

OBJECTIVE: In this study, the clinical usefulness of transition zone (TZ) volume (TZV) measured by transrectal ultrasonography (TRUS) was investigated as a new parameter for the preoperative prediction of the treatment efficacy of transurethral resection of the prostate (TURP). METHODS: Fifty-six men with symptomatic benign prostatic hyperplasia (BPH; age 68.6 +/- 9.7 years) underwent TURP and were evaluated based on ordinary BPH parameters such as the international prostatic symptom score (I-PSS), quality of life (QOL) score, peak urine flow and entire prostate volume (PV), as well as the new TZV parameters and calculation of the TZ index. Relative risks were adjusted simultaneously for potentially confounding variables by multiple logistic regression analysis after adjustment for age, QOL, I-PSS, Qmax and residual urine. RESULTS: The adjusted relative risk for TURP at a TZ index of 0.1 increased to 4.5 (95% confidence interval 2.3-8.78). In general, poor responses were observed in patients with less symptomatic scores or lower values prior to operation, but there was a weak correlation between treatment outcome and preoperative scores or values of ordinary parameters. The volume parameters of BPH and PV did not predict treatment efficacy preoperatively, but TZV and the TZ index correlated with the treatment efficacy of TURP. CONCLUSION: TZV and the TZ index seem to be useful new parameters in preoperative decision-making with regard to TURP.

Aged↗

PubMed1999

Eosinophilic inflammation in the airway is related to glucocorticoid reversibility in patients with pulmonary emphysema.

STUDY OBJECTIVES: We examined the effects of glucocorticoids on airway inflammation and its association with the reversibility of airflow obstruction in response to oral glucocorticoid administration in patients with COPD. PATIENTS: Twenty-four male patients with smoking-related stable pulmonary emphysema without alpha1-antitrypsin deficiency and nine normal healthy volunteers. MEASUREMENTS: We measured the pulmonary function, the inflammatory cells, and the levels of eosinophil cationic protein (ECP), immunoreactive neutrophil elastase-alpha1-protease inhibitor (NE-alpha1-PI) complex, and interleukin (IL)-8 in sputum induced from patients with pulmonary emphysema in its stable phase before and after treatment with 20 mg oral prednisolone per day for 2 weeks. RESULTS: The eosinophil and neutrophil counts and the concentrations of ECP, NE-alpha1-PI complex, and IL-8 in the sputum were significantly increased at baseline. The eosinophil count at baseline was significantly correlated with the reversibility of airflow obstruction following treatment, and the treatment also significantly reduced the eosinophil numbers and ECP level in the sputum. In contrast, the increased neutrophil number and the concentrations of NE-alpha1-PI complex and IL-8 at baseline did not correlate with the reversibility and were not affected by treatment. CONCLUSIONS: These findings suggest that the eosinophilic inflammation, not neutrophilic inflammation, in the airway is involved in the reversible part of the airflow obstruction in response to glucocorticoids in patients with pulmonary emphysema.

Aged↗

PubMed1999

[Severe stenosis of the internal carotid artery and intracerebral hematoma associated with neurofibromatosis type 1: a case report].

Neurofibromatosis Type 1 produces a broad spectrum of clinical manifestations as a result of widespread dysplasia of mesodermal and neuroectodermal tissues. One of the most serious aspects of the disease relates to the arterial involvement that may occur. We report a case of severe stenosis of the internal carotid artery and intracerebral hemorrhage associated with neurofibromatosis Type 1. A 49-year-old female was admitted to our hospital after she had suddenly become comatose. On admission, she demonstrated a decerebrate posture in response to painful stimuli, and was assessed as grade 200 according to the Japan Coma Scale. Physical examination disclosed widespread cutaneous neurofibromas and cafe-au-lait spots. Computed tomography of the head revealed a right putaminal extensive hematoma, with a maximum diameter of 7 cm. The hematoma was removed. After this surgical treatment, cerebral angiography was performed. It showed severe stenosis of the terminal portion of the right internal carotid artery associated with a fine telangiectatic network, indicating the presence of moyamoya vessels in the basal ganglia. Although intracranial hemorrhage associated with neurofibromatosis type 1 is a rare condition, fine telangiectatic collateral vessels caused by occlusive cerebrovascular disease, intracranial aneurysms, brain tumors, or hypertension caused by pheochromocytoma or stenosis of the renal artery should be considered as the cause of hemorrhage.

Carotid Artery Diseases↗

PubMed1999

Identification of protein kinase C phosphorylation sites involved in phorbol ester-induced desensitization of the histamine H1 receptor.

The histamine H1 receptor (H1R)-mediated signaling cascade is inhibited by phorbol ester-induced protein kinase C (PKC) activation. Cloning studies of the H1Rs have shown that several potential PKC phosphorylation sites are located in the third intracellular loop of H1R. To elucidate the molecular mechanism of PKC-mediated desensitization, we identified amino acid residues that are involved in the desensitization of the H1R. Two amino acid residues (Ser396, Ser398) were determined to be PKC phosphorylation sites by in vitro phosphorylation studies using a series of synthetic peptides. Treatment with phorbol ester decreased histamine-induced accumulation of inositol phosphates in Chinese hamster ovary cells expressing the H1R with a rightward shift in the EC50 value, which implies the uncoupling of the receptor from the G protein. Site-directed mutagenesis studies showed that substitution of alanine for Ser398 but not for Ser396 markedly attenuated the effect of phorbol ester, which suggests that the Ser398 residue was primarily involved in PKC-mediated desensitization.

Amino Acid Sequence↗

PubMed1998

A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts.

Three integral membrane proteins, clau- din-1, -2, and occludin, are known to be components of tight junction (TJ) strands. To examine their ability to form TJ strands, their cDNAs were introduced into mouse L fibroblasts lacking TJs. Immunofluorescence microscopy revealed that both FLAG-tagged claudin-1 and -2 were highly concentrated at cell contact sites as planes through a homophilic interaction. In freeze-fracture replicas of these contact sites, well-developed networks of strands were identified that were similar to TJ strand networks in situ and were specifically labeled with anti-FLAG mAb. In glutaraldehyde-fixed samples, claudin-1-induced strands were largely associated with the protoplasmic (P) face as mostly continuous structures, whereas claudin-2-induced strands were discontinuous at the P face with complementary grooves at the extracellular (E) face which were occupied by chains of particles. Although occludin was also concentrated at cell contact sites as dots through its homophilic interaction, freeze-fracture replicas identified only a small number of short strands that were labeled with anti-occludin mAb. However, when occludin was cotransfected with claudin-1, it was concentrated at cell contact sites as planes to be incorporated into well- developed claudin-1-based strands. These findings suggested that claudin-1 and -2 are mainly responsible for TJ strand formation, and that occludin is an accessory protein in some function of TJ strands.

Animals↗

PubMed1998

alpha-Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells.

alphaE-catenin, a cadherin-associated protein, is required for tight junction (TJ) organization, but its role is poorly understood. We transfected an alphaE-catenin-deficient colon carcinoma line with a series of alphaE-catenin mutant constructs. The results showed that the amino acid 326-509 domain of this catenin was required to organize TJs, and its COOH-terminal domain was not essential for this process. The 326-509 internal domain was found to bind vinculin. When an NH2-terminal alphaE-catenin fragment, which is by itself unable to organize the TJ, was fused with the vinculin tail, this chimeric molecule could induce TJ assembly in the alphaE-catenin-deficient cells. In vinculin-null F9 cells, their apical junctional organization was impaired, and this phenotype was rescued by reexpression of vinculin. These results indicate that the alphaE-catenin-vinculin interaction plays a role in the assembly of the apical junctional complex in epithelia.

Binding Sites↗

PubMed1998

Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.

Occludin is the only known integral membrane protein localizing at tight junctions (TJ), but recent targeted disruption analysis of the occludin gene indicated the existence of as yet unidentified integral membrane proteins in TJ. We therefore re-examined the isolated junction fraction from chicken liver, from which occludin was first identified. Among numerous components of this fraction, only a broad silver-stained band approximately 22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of databases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 amino acids, respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as "claudin-1" and "claudin-2", respectively. Although the precise structure/function relationship of the claudins to TJ still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands.

Amino Acid Sequence↗

PubMed1998

Interleukin-15 (IL-15) can replace the IL-2 signal in IL-2-dependent adult T-cell leukemia (ATL) cell lines: expression of IL-15 receptor alpha on ATL cells.

Interleukin-15 receptor (IL-15R) and IL-2R have the same beta and gamma chains, but IL-15R has a specific alpha chain distinct from that of IL-2Ralpha, which is indispensable for the high affinity binding of IL-15. In the present study, we examined four IL-2-dependent adult T-cell leukemia (ATL) cell lines for their IL-15R expression. All cell lines bound IL-15, which was not inhibited by a 100-fold excess amount of IL-2, proliferated in response to IL-15 to the same degree as to the stimulation with IL-2, and were maintained without IL-2. The responses to 1L-15 were inhibited by the antibodies against IL-2R beta or gamma chains but was not by the IL-2R alpha chain antibody. [125I]-IL-15 exhibited a single high-affinity binding with an apparent kd of 0.17 nmol/L. Reverse transcription-coupled polymerase chain reaction (RT-PCR) showed that the cell lines had the mRNA of IL-15R alpha. The cell lines also had IL-15 mRNA. Despite the presence of IL-15 mRNA, the cell lines did not secrete IL-15, and the culture supernatants of fresh ATL cells and plasma from the patients did not contain a detectable amount of IL-15 with a few exceptional cases, although fresh ATL cells also responded to IL-15. These results suggest that ATL cells have the complete form of IL-15R and respond to IL-15. Such an IL-15-dependent cell proliferation mechanism might be used in the development of ATL and for the invasion and proliferation of ATL cells in the visceral organs.

Adult↗

PubMed1998

Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro.

Glial cell line-derived neurotrophic factor (GDNF) is known as a potent neurotrophic factor for dopaminergic neurons. Since adeno-associated virus (AAV) vector is a suitable vehicle for gene transfer into neurons, rat E14 mesencephalic cells were transduced with an AAV vector expressing GDNF. When compared with mock transduction, a larger number of dopaminergic neurons survived in AAV-GDNF-transduced cultures (234% and 325% of controls at 1 and 2 weeks, respectively; P < 0.01). Furthermore, the dopaminergic neurons in the latter cultures grew more prominent neurites than those in the former. These findings suggest that AAV vector-mediated GDNF gene transfer may prevent dopaminergic neuron death, and is therefore a logical approach for the treatment of Parkinson's disease.

Animals↗

PubMed1998

Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions.

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.

Animals↗

PubMed1998

Involvement of matrix metalloproteinase-2 activity in invasion and metastasis of pancreatic carcinoma.

BACKGROUND: Activation of matrix metalloproteinase-2 (MMP-2) has been implicated in the progression, invasion, and metastasis of various cancers, but little information is available with regard to its role in pancreatic carcinoma with poor prognosis. METHODS: Gelatin zymography was used for the detection of latent and activated forms of MMP-2 and MMP-9 in 13 normal pancreatic tissue specimens, 14 chronic pancreatitis tissue specimens, and 33 pancreatic carcinoma tissue specimens. The gelatinase activity was quantified by densitometer, and the 66-kilodalton (kDa)/(66-kDa + 72-kDa) ratio was calculated as the MMP-2 activation ratio. Western blot analysis was performed to confirm the zymographic profile. RESULTS: Latent forms of MMP-2 and MMP-9 were detected in all samples of pancreatic carcinoma, chronic pancreatitis, and normal pancreatic tissue. The expression rate of the MMP-2 activated form in pancreatic carcinoma tissue specimens was 100% (33 of 33) but that of MMP-9 was 21%. The MMP-2 activation ratio in pancreatic carcinoma tissue specimens was significantly higher than that of chronic pancreatitis and normal pancreatic tissue specimens. The MMP-2 activation ratio in pT3 tumors was significantly higher than that in pT1 tumors. The MMP-2 activation ratio also was significantly higher in pancreatic carcinoma specimens with histologically positive regional lymph node metastasis and distant metastasis than those without metastasis. The MMP-2 activation ratio observed in patients who developed postresection recurrence within 6 months was significantly higher than that in patients without recurrence at 6 months. CONCLUSIONS: The results of the current study indicate that MMP-2 activation plays a significant role in tumor invasion and metastasis in pancreatic carcinoma.

Adenocarcinoma, Mucinous↗

PubMed1998

Expression of interleukin (IL)-12 mRNA in gastric carcinoma specimens: cellular antitumor immune responses.

BACKGROUND AND OBJECTIVES: Several tumor-related antigen peptides that are recognized by autologous cytolytic T cells (CTL) have been reported. However, most human solid tumors, including gastric carcinoma, are only weakly immunogenic. In this study, we focused on interleukin (IL)-12 and interferon-gamma (IFN-gamma) as key cytokines for estimating positive cellular immune responses. METHODS: To estimate the immunogenicity of gastric carcinomas, we examined IL-12 and IFN-gamma at mRNA levels by reverse transcription-polymerase chain reaction assay (RT-PCR) in tumor specimens and adjacent nontumor specimens from 36 gastric carcinoma patients. RESULTS: IL-12 expression was detected in 12 tumor specimens and in only two adjacent nontumor specimens (P = .003). The frequency of IFN-gamma gene expression was higher in the IL-12 mRNA-positive tumor specimens than in the IL-12 mRNA-negative tumor specimens (P = .015). In the IL-12 mRNA-positive tumors, IFN-gamma expression was higher in the tumor specimens than in the adjacent nontumor specimens (P = .007). Conversely, in the IL-12 mRNA-negative tumors, IFN-gamma expression was lower in the tumor specimens than in the nontumor specimens (P = .03). Many tumor-infiltrating mononuclear cells, predominantly T cells, were found in four of the 12 IL-12-mRNA-positive tumor specimens and in none of the 24 IL-12-mRNA-negative tumor specimens (P = .008). CONCLUSIONS: These data suggest that possible immune responses against a tumor may occur at the mRNA level in approximately one-third of gastric carcinomas.

Blotting, Southern↗

PubMed1998

Collagen-induced arthritis in nonhuman primates: multiple epitopes of type II collagen can induce autoimmune-mediated arthritis in outbred cynomolgus monkeys.

OBJECTIVE: To define which regions of the type II collagen (CII) molecule result in anticollagen antibody production and the subsequent development of autoantibodies in a collagen-induced arthritis (CIA) nonhuman primate model. METHODS: Male and female cynomolgus monkeys (2-6 of each sex per group) were immunized with either chicken (Ch), human, or monkey (Mk) CII, or with cyanogen bromide (CB)-generated peptide fragments of ChCII emulsified in Freund's complete adjuvant. Monkeys were observed for the development of arthritis, and sera were collected and analyzed for anticollagen antibody specificity by enzyme-linked immunosorbent assay. RESULTS: Overt arthritis developed in all groups of monkeys immunized with intact CII and with all major CB peptide fragments of ChCII except CB8. Onset and severity of arthritis correlated best with serum anti-MkCII antibody levels. The levels of IgG autoantibody to MkCII were a result of the cross-reactivity rate of anti-heterologous CII antibodies with MkCII, which was based on the genetic background of individual monkeys rather than on sex differences. CONCLUSION: CII from several species and disparate regions of the CII molecule were able to induce autoantibody-mediated arthritis in outbred cynomolgus monkeys. The strong anti-MkCII response suggests that epitope spreading or induction of broad-based CII cross-reactivity occurred in these animals. Autoantibody levels to MkCII were higher in CIA-susceptible monkeys than in resistant monkeys, despite comparable antibody levels in response to the various immunizations of CII. These results closely parallel the type of anticollagen responses found in sera from rheumatoid arthritis patients. Perhaps this can be accounted for by similar major histocompatibility complex heterogenicity associated with an outbred population, or maybe this is a primate-specific pattern of reactivity to CII.

Animals↗