Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi.

Transferrin was isolated from plasma of the ascidian Halocynthia roretzi by ion-exchange chromatography. The molecular weight of the plasma transferrin was determined to be 52K by SDS-polyacrylamide gel electrophoresis and gel filtration. Ascidian plasma transferrin was found to bind one mole of iron ion per mole of protein. The reductive S-pyridylethylated transferrin was subjected to Edman degradation analysis for determination of the N-terminal amino acid sequence, and it was also subjected to proteolytic fragmentation to yield peptide fragments, whose amino acid sequences were determined by Edman degradation analysis. Using the above amino acid sequences, a cDNA clone (1880 base pairs) encoding a protein of 372 amino acids containing a signal peptide of 21 amino acids was isolated from an H. roretzi hepatopancreas cDNA library. The reduced amino acid sequence contains the same sequences of the peptide fragments. A comparison of the amino acid sequence of ascidian transferrin with those of other members of the transferrin family revealed that the ascidian transferrin is composed of only the N-terminal lobe of two-lobed vertebrate transferrins. Thus, a one-lobed transferrin is present in the ascidian H. roretzi.

Involvement of beta 2-glycoprotein I and anticardiolipin antibodies in oxidatively modified low-density lipoprotein uptake by macrophages.

Anticardiolipin antibodies (aCL) in the sera of patients with antiphospholipid syndrome (APS) recognize an altered structure of beta 2-glycoprotein I (beta 2-GPI) interacting with solid-phase negatively charged phospholipids. beta 2-GPI bound to Cu2+-oxidized plasma lipoproteins, i.e. oxidized very low-density lipoprotein (oxVLDL), oxidized low-density lipoprotein (oxLDL), or oxidized high-density lipoprotein (oxHDL). beta 2-GPI inhibited in vitro uptake, i.e. cell surface binding, cellular association, and proteolytic degradation of oxLDL by murine macrophage J774A.1 cells. The binding of oxLDL to the macrophages was inhibited by the addition of polyinosinic acid (poly (I)), a competitor of the scavenger receptor, but not by another polyanionic acid, polycytidylic acid (poly (C)). Conversely, the binding of oxLDL was significantly increased by the simultaneous addition of human beta 2-GPI and monoclonal aCL derived from NZW x BXSB F1 (WB F1) mice, an animal model of APS, or anti-beta 2-GPI antibodies from BALB/c mice immunized with human beta 2-GPI. These findings indicate that beta 2-GPI may be an antiatherogenic protein and that the autoimmune response against beta 2-GPI may have a role in the development of atherosclerosis in APS.

Advanced glycation endproducts and diabetic nephropathy.

Diabetic nephropathy is currently the single largest cause of endstage renal disease (ESRD) in the United States and many European countries. The primary cause for the development of diabetic complications (including diabetic nephropathy) is persistent exposure to hyperglycemia, although genetic and other incompletely understood factors also play an important role. Although much consideration has been given to the pathogenesis and genetics of the disease itself, the mechanisms by which persistent exposure to hyperglycemia cause biochemical and metabolic alterations have been very sketchily understood. Recently, a growing body of evidence has linked the accumulation of the late products of glucose-protein interaction to a variety of chronic complications, including diabetic nephropathy. The formation of irreversible advanced glycosylation endproducts (AGEs) resulting from the spontaneous reaction between glucose and proteins occur most noticeably on long-lived structural proteins. Recent studies demonstrate that the pathogenesis of diabetic nephropathy is caused by the hyperglycemia-accelerated formation of AGEs. Also, reactive AGE peptides in the circulation are thought to play a role as a new version of so called middle molecule toxic substances. This evidence is opening a new window for our understanding of the pathogenesis of diabetic nephropathy.

Expression and function of fibronectin binding integrins on rat mast cells.

Adhesion molecules of the integrin family are implicated not only in leukocyte migration but also in leukocyte activation. Here we characterize the expression and function of fibronectin receptor integrins on rat mast cells. A rat basophilic leukemia cell line (RBL-2H3) and phorbol ester-stimulated rat peritoneal mast cells adhered to fibronectin (FN), vitronectin and fibrinogen. These mast cells expressed fibronectin receptor integrins, including very late antigen (VLA)-4, VLA-5 and vitronectin receptor (VNR), as estimated by immunofluorescent staining and inhibition of FN adherence by newly established mAbs reactive with the rat alpha 4 (MR alpha 4-1), alpha 5 (HM alpha 5-1) or beta 3 (HM beta 3-1) chains of the integrin molecules. The beta-hexosaminidase release, a marker for mast cell degranulation, triggered by high affinity IgE receptor (Fc epsilon RI)-mediated stimulation, was enhanced by adhesion of RBL-2H3 cells to either immobilized FN, MR alpha 4-1, HM alpha 5-1 or HM beta 3-1. This FN enhancement of beta-hexosaminidase release was inhibited by soluble MR alpha 4-1, HM alpha 5-1 and HM beta 3-1 as well as by GRGDSP and DELPQLVTLPHPNHLGPEILDVPST peptides which abrogate VLA-5/VNR and VLA-4 binding to FN respectively. In vivo, passive cutaneous anaphylaxis induced by IgE anti-DNP and DNP-BSA was inhibited by concurrent s.c. injection of MR alpha 4-1, HM alpha 5-1 and HM beta 3-1. These results demonstrate that FN receptor integrins expressed on rat mast cells play an important role in regulating mast cell activation both in vitro and in vivo.

[A double blind controlled study on S6472 capsules and granules in complicated urinary tract infection].

The efficacy, safety and usefulness of S6472 capsules (cefaclor, long-acting preparation) were investigated in non-catheterized patients with complicated cystitis using the double blind method with S6472 granules as a control. Each of the patients was orally administrated with 375 mg of the drug twice a day for 7 days, and clinical effectiveness was evaluated according to the Criteria of Drug Efficacy of UTI (the Third Edition). The following results were obtained. 1. There was no significant bias between the capsule-administrated group (A) and the granule-administrated group (B) in background factors. 2. The overall clinical efficacy rates were 78.7% in Group A and 80.5% in Group B, which showed no significant difference. No significant difference was observed also between groups IV and VI in the efficacy for any of the UTI groups. 3. Eradication rates in bacteriological response were 85.9% and 86.0% in Group A and B, respectively. 4. Clinical efficacy rates evaluated according to doctors in charge of patients were 78.7% and 74.7% in Group A and B, respectively, showing no significant difference between the two groups. 5. The incidences of adverse reactions were 3.0% in Group A (3 out of 101 cases) and 7.1% in Group B (7 out of 98), thus no significant difference was found between the two groups. Abnormal test values in laboratory examinations included 5 cases (in 4 among 75 patients) in Group A, but were not detected in any of the 76 patients in Group B. No statistically significant difference were observed between the two groups, however. 6. No significant difference between the two groups was observed in the usefulness evaluation including efficacy and safety profiles on an analog scale by the doctors in charge of patients. These results indicate that S6472 capsules have the same efficacy, safety and usefulness as S6472 granules, suggesting that both drugs are equally excellent for non-catheterized patients with complicated cystitis.

Adjuvant chemotherapy for invasive urothelial cancer: experience with a methotrexate, vincristine, cisplatin, cyclophosphamide, adriamycin and bleomycin (MVP-CAB) regimen: a preliminary report.

MVP-CAB chemotherapy (methotrexate, vincristine, cisplatin, cyclophosphamide, adriamycin and bleomycin) was administered to 28 patients with high grade, locally-invasive or regional lymph node-involved urothelial cancer as an adjuvant therapy after radical surgery. The median follow-up period of all the evaluated patients was 24 (range, 5-62) months. The median disease-free durations in patients with pT1b+2+3 bladder and upper urothelial cancer were 26 and 22 months, respectively. In contrast, all patients with pT4 disease or lymph node metastases had a recurrence within 24 months of surgery. The median disease-free durations in patients with pure transitional cell carcinoma (grade 3) of the bladder and upper urothelial cancer were 19 and 18 months, respectively. The median disease-free duration in patients with grade 3 pT1b+2+3 pure transitional cell carcinoma was 19 months. In contrast, the median disease-free duration in bladder cancer patients with pT1b+2+3 squamous cell carcinoma components was 35 months. The three-year actuarial survival rates were 79 and 89% for pT1b+2+3 bladder and upper urothelial cancer, respectively, while the three-year actuarial survival rates of patients with pT4 bladder cancer and pT4 upper urothelial cancer were 0 and 100%, respectively. The two-year actuarial survivals in the bladder cancer and upper urothelial cancer patients with lymph node involvement were 0 and 100%, respectively. The three-year actuarial survivals of patients with pure transitional cell carcinoma (grade 3) were 53 and 80% in bladder cancer and upper urothelial cancer patients, respectively. The three-year actuarial survival rate in patients with squamous cell carcinoma or adenocarcinoma components which did not recur was, however, 100%. Although randomized studies comparing MVP-CAB and M-VAC (methotrexate, vinblastin, adriamycin and cisplatin) or other chemotherapeutic regimens will be necessary, we believe our results indicate that MVP-CAB chemotherapy may be useful as an adjuvant therapy for patients with urothelial cancer, including those with squamous cell carcinoma and adenocarcinoma components. More intensive MVP-CAB chemotherapy, i.e., increasing the dose of cisplatin and giving at least five courses, as well as the use of granulocyte colony stimulating factor and a new antiemetic drug (granisetron), will, however, be necessary for patients with pT4 or lymph node-involved disease.

[A case of chronic scrotal hematocele and review of the literature].

A 38-year-old man visited our hospital because of swelling of left scrotal content. He had no history of trauma of scrotum, fever, pain or dysuria. Physical examination revealed a tumor larger than a fist in the left scrotum. Ultrasonography revealed an echogenic mass with echolucent area in the scrotum. Surgical extirpation of the left scrotal tumor was performed under the diagnosis of left testicular tumor. The mass was encapsulated by a white fibrous membrane and was 700 g in weight. The tumor contained 200 ml of dark brown pus-like material. Histological examination revealed deposition of cholesterine crista and infiltration of lymphocyte in tunica vaginalis with extremely atrophic testis, destructive spermatogenesis and atrophic epididymis. Twenty one cases of chronic scrotal hematocele have been reported in the Japanese literature. The age of the patients reported was 38 to 77 years old with a mean age of 65 years. Orchiectomy was done under the diagnosis of testicular tumor in 20 of the 21 cases. Our case was thought to be of an idiopathic chronic scrotal hematocele. The disease should be considered even in the absence of a particular cause such as injury and inflammation of scrotal content.

Requirement for VLA-4 and VLA-5 integrins in lymphoma cells binding to and migration beneath stromal cells in culture.

Physical interaction between human lymphomas and murine bone marrow derived stromal cells were studied. Nalm-6 pre-B cells adhered to BMS2 stromal cells and subsequently migrated beneath them, while Ramos Burkitt lymphoma cells, adhered but did not migrate. Four mAbs were established against Nalm-6 cells, which were able to block initial adhesion of Nalm-6 cells. Two of them were directed against the alpha 4 chain of VLA-4, and other two recognized the beta 1 chain of VLA integrins. Therefore, the initial adhesion of Ramos and Nalm-6 cells to BMS2 was largely mediated by the VLA-4 integrin expressed on lymphocytes. The corresponding ligand on stromal cells appears to be VCAM-1, because antibodies against murine VCAM-1 blocked the adhesion. However, antibodies against the alpha chain of VLA-4 were not capable of blocking subsequent migration beneath stromal cells. In contrast, antibodies against the beta chain of VLA integrins blocked the migration beneath stromal cells as well as the initial adhesion. Because a common beta chain can be shared among integrins, the role of other VLA integrins in Nalm-6 cells migration was investigated. VLA-5 and VLA-6 as well as VLA-4 were expressed on Nalm-6 cells, but not on Ramos cells. Additional blocking experiments revealed that VLA-4 and VLA-5 are likely to work in concert to mediate the migration of Nalm-6 cells beneath stromal cells. Thus, particular VLA integrins appear to be responsible not only for lymphocyte adhesion but also for migration with respect to stromal cells. These findings may have implications for cell-cell interactions and directed migration of lymphocytes in bone marrow and other tissues.

[Statistics on operations at Hara Genitourinary Hospital (1971-1991)].

The statistical analysis of the inpatients and operations in our department from April, 1971 to March, 1991 revealed a total of 9,170 operations. One of the biggest changes in treatment is a marked increase in closed surgery. This includes endourological surgeries; transurethral resections of prostate and bladder tumor (TUR-P, TUR-Bt), percutaneous nephrolithotripsy (PNL), transurethral uretero-lithotripsy (TUL) and extracorporeal shock-wave lithotripsy (ESWL). From 1976 to 1980 there were 721 open surgical procedures (71.0% of cases) and 295 closed procedures (21.0% of cases). This ratio has now been reversed completely. From 1986-1991, there were 342 open procedures (8.2% of cases) and 3808 closed procedures (91.8% of cases). The main reason for this change was the introduction of the new techniques PNL, TUL and ESWL. Other important observations include: The incidence of emergency operations was higher in this private institution than that seen in non-private hospitals. From 1986, there was an increase in the use of PNL and TUL for the management of urinary calculi, and from 1988, ESWL has overtaken PNL and TUL in the treatment of this condition. Recently the limitations of ESWL have been appreciated and the value of a combined approach to therapy, for selected cases, is being recognized. A total of 238 ileal or ileocecal conduits were created with good results. A low complication rate has been observed with preservation of renal function.

Enhancement of aniline hydroxylation in human liver microsomes.

Effects of cyanide, acetone, 2,2'-bipyridine and paraoxon on aniline hydroxylation by human liver microsomes were studied. The activity of aniline hydroxylation was inhibited by cyanide when low concentrations of substrate were used, whereas the activity was increased by the addition of cyanide when higher concentrations of substrate were used. Although there were large variences among different liver samples in the extent of the activation of aniline hydroxylation, acetone, 2,2'-bipyridine and paraoxon also exerted stimulatory effects on aniline hydroxylation in human liver microsomes. The degree of the stimulation was not related with the activities observed in the absence of these compounds.

[Efficacy of paromomycin sulfate against human cestodiasis and its pharmacological action on tapeworm in vitro].

Recently, it has been reported that paromomycin sulfate has marked anthelmintic efficacy against tapeworm infections in man. In the present study this drug was used in the treatment of 14 cases of diphyllobothriasis latum and 1 case of taeniasis saginata. Also, the actions of paromomycin sulfate on Diphyllobothrium ditremum and D. erinacei were examined pharmacologically using Magnus apparatus and biochemical methods. The results obtained were as follows. For the treatment, a total of 50 mg/kg of paromomycin sulfate divided into 2 doses was given orally at intervals of 30 minutes. Two hours after medication, 20 g of magnesium sulfate dissolved in 200--300 ml of water was given as purgative. One or 2 worms were found in the stools of 11 cases with D. latum and 1 case with T. saginata within 24 hours after medication, but scolex was found in only 2 of them. All cases were negative for the eggs or segments in stool examinations at 1 and 3 months after treatment. Except 1 case complained mild and transient vomiting no side effects were noticed. All cases showed no abnormality in blood examination, liver function test and urinalysis. Both of the proglottids of D. ditremum and D. erinacei showed muscle relaxation in Tyrode solution containing 10(-4) g/ml of paromomycin sulfate. In D. ditremum the recovery of muscle tonus was observed within 10--15 minutes after affection of this drug, while the persistence of muscle relaxation was seen in D. erinacei. The activity of phosphoglucose isomerase was slightly inhibited by 10(-3) M paromomycin sulfate while those of hexokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase were not inhibited. In phosphoenolpyruvate-succinate pathway, the activity of fumarate reductase was slightly inhibited 10(-3) M paromomycin sulfate while those of phosphoenolpyruvate carboxykinase and malate dehydrogenase were not inhibited.