A model for predicting sediment-water partition of toxic chemicals in aquatic environments.

In order to investigate the characteristics of sediment-water partition of chemicals in aquatic environments using published data, we developed a model for predicting the sediment-water partition coefficient (Kp) as the sum of sorption to sediment organic matter and sorption to sediment inorganic matter. This model is so successful that the differences between Kp (median for a variety of Japanese water bodies) and pre-Kp (predicted K) are within one order of magnitude in 24 out of 28 chemicals.

Tumour invasion and metastasis are promoted in mice deficient in tenascin-X.

BACKGROUND: Tenascin-X (TNX) is a member of the tenascin family of large oligomeric glycoproteins of the extracellular matrix (ECM). To determine whether TNX plays a part in tumour invasion and metastasis and to disclose its normal physiological role, we disrupted its gene in mouse embryonic stem cells by homologous recombination and created mice deficient in TNX. RESULTS: TNX-null mutant (TNX-/-) mice arose at normal frequency and showed no obvious defects during their adult life. However, when TNX-/- mice were subcutaneously inoculated in foot-pads with a highly invasive and metastatic cell line, B16-BL6 melanoma cells, the primary tumour size at 30 days after inoculation in the TNX-/- mice had increased by 1.2-fold compared with that in wild-type mice, and the invasion to the ankle and pulmonary metastasis in TNX-/- mice were also augmented by 2.2-fold and 6.8-fold, respectively, compared to those in wild-type mice. To disclose the molecular mechanism(s) of the promotion of tumour invasion and metastasis in TNX-/- mice, we measured the protein levels of matrix metalloproteinases (MMPs), which are recognized as playing a key role in these events, in the foot-pad homogenates of TNX-/- mice prior to the inoculation of melanoma cells. Gelatin zymography showed that the activities of proMMP-2, active MMP-2 and proMMP-9 were significantly higher in TNX-/- mice than in wild-type mice. Furthermore, a Northern blot analysis demonstrated that this increased activity of MMP-2 in TNX-/- mice was due to the induced expression of MMP-2 at the transcriptional level. The elevated expression of MMP-2 and MMP-9 resulted in decreased laminin levels, to less than half that of wild-type mice in the homogenates of TNX-/- mice. CONCLUSIONS: TNX deficiency led to an increase in the production of MMPs, and the increased activity of MMPs may result in the degradation of laminin. Consequently, the melanoma cells inoculated in TNX-/- mice might facilitate invasion and metastasis. These results imply that TNX is required for impeding the invasion and metastasis of tumour cells.

Cloning and characterization of a rat ortholog of MMP-23 (matrix metalloproteinase-23), a unique type of membrane-anchored matrix metalloproteinase and conditioned switching of its expression during the ovarian follicular development.

In our attempt to study the role of matrix metalloproteinases (MMPs) in the process of mammalian ovulation, we isolated a rat ortholog of the recently reported human MMP-23 from gonadotropin-primed immature rat ovaries. Transient expression of epitope-tagged rat and human MMP-23 in COS-1 cells revealed that they were synthesized as a membrane-anchored glycoprotein with type II topology. Indirect immunofluorescent analysis showed that subcellular localization of MMP-23 was predominantly in the perinuclear regions. The transfected human MMP-23 protein was processed endogenously to the soluble form in COS-1 cells. However, cotransfection of MMP-23 with the mouse furin cDNA did not enhance this processing, indicating that furin may not be involved in this event. Notably, in situ hybridization analysis revealed a dramatic switching of MMP-23 mRNA localization from granulosa cells to theca-externa/fibroblasts and ovarian surface epithelium during the follicular development. In serum-free primary culture of rat granulosa cells, a drastic diminution of MMP-23 mRNA expression was observed in response to FSH action between 24 h and 48 h of culture. The observed effect of FSH on MMP-23 expression was mimicked by treatment of granulosa cells with forskolin or 8-bromo (Br)-cAMP. In contrast, MMP-23 mRNA levels increased in theca-interstitial cells regardless of the presence of LH in the culture. However, treatment of theca-interstitial cells with forskolin or 8-Br-cAMP markedly reduced the expression of MMP-23 with a concomitant increase in progesterone production. These results indicate that the MMP-23 gene is spatially and temporally regulated in a cell type-specific manner in ovary via the cAMP signaling pathway.

The Bisurface total knee replacement: a unique design for flexion. Four-to-nine-year follow-up study.

BACKGROUND: The Bisurface knee prosthesis was designed in 1989 to improve knee flexion without affecting the durability of the prosthesis. The prosthesis has a unique ball-and-socket joint in the midposterior portion of the femoral and tibial components, which functions as a posterior stabilizing cam mechanism and causes femoral rollback. The femoral component was made of alumina ceramic. The purpose of this study was to review the clinical results of the first 223 arthroplasties performed with this prosthesis in order to assess whether this new implant had achieved its design objectives. METHODS: From December 1989 to May 1994, all patients who were scheduled for primary total knee arthroplasty were enrolled in a prospective study of the Bisurface knee. The patients were evaluated clinically according to The Hospital for Special Surgery knee-rating system and with a self-administered questionnaire, and they were evaluated radiographically according to the system of the Knee Society. Kaplan-Meier survivorship analysis was performed with revision of the knee or recommendation for revision as the end point. RESULTS: One hundred and sixty-six patients treated with a total of 223 consecutive primary total knee arthroplasties were enrolled in the study, and 182 knees were followed for 3.9 to 9.0 years (mean, 5.8 years). Preoperatively, the mean Hospital for Special Surgery knee score was 44.5 points. At the time of latest follow-up, the mean knee score was 86.3 points. The mean preoperative and postoperative ranges of flexion were 119 and 124 degrees, respectively. The patients, even those with a good preoperative range of motion, rarely lost deep flexion of the knee after the procedure. A revision operation was performed in eight knees (because of infection in five, instability in two, and breakage of the peg of the patellar component in one). Two knees had recurrent medial-lateral subluxations of the femorotibial articulation, which were treated nonoperatively. No prosthesis had loosened aseptically and no alumina ceramic femoral component had broken by the time of latest follow-up. The rate of survival of the implant was 94 percent (95 percent confidence interval, 90 to 98 percent) at six years. According to the patient questionnaires, 20 percent of the knees sometimes felt loose in daily living activities, which prompted us to improve the intrinsic stability of the prosthesis by improving the congruity of the ball-and-socket joint. CONCLUSIONS: Total knee arthroplasty with the Bisurface prosthesis resulted in an excellent range of motion and a high level of satisfaction with the operation; the durability of the prosthesis is promising.

Inhibition of cell growth and Epstein-Barr virus reactivation by CD40 stimulation in Epstein-Barr virus-transformed B cells.

The CD40 molecule plays important roles in B cell activation, proliferation, and immunoglobulin (Ig) class switching. In Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), CD40 mediates growth inhibition and EBV reactivation via the CD40 signaling pathways. CD40 cross-linking with a monoclonal antibody arrests cell growth in G1 and induces expression of p27kip1 cyclin-dependent kinase inhibitor. CD40 cross-linking also induces EBV reactivation, as detected by the induction of EBV-specific early antigen, immediate early BZLF1 RNA, and its protein product ZEBRA. These results support hypotheses that the proliferation of EBV-infected B cells in vivo can be inhibited by interactions with the CD40 ligand on activated helper T cells, and latent EBV is reactivated via the signaling pathways controlled by CD40 interactions.

Induction of interleukin-10 on activation of Epstein-Barr virus in EBV-infected B-cell lines.

Human (h) interleukin-10 (IL-10) exhibits a strong DNA and amino acid sequence homology to the Epstein-Barr virus (EBV) BCRF1 genome, viral (v) IL-10. We analyzed the production of IL-10 for EBV activation in B-cell lines. The latent EBV in Akata cells was activated by the cross-linking of surface immunoglobulin G (IgG) with anti-human IgG. The levels of IL-10(h+v) and vIL-10 in the culture fluids were measured by a specific enzyme-linked immunosorbent assay (ELISA). IL-10(h+v) was detected at the same time for EBV immediate early gene BZLF1 product ZEBRA and early gene BMRF1 product EA-D. This was more than 4 hours prior to the appearance of vIL-10, and late gene products gp 350/220 and viral capsid antigen. The induction of hIL-10 and vIL-10 mRNAs were detected in anti-IgG-treated Akata cells by reverse transcription-polymerase chain reaction. The induction of IL-10(h+v) and vIL-10 was inhibited with a tyrosine kinase inhibitor, herbimycin, or with an inhibitor of herpesvirus DNA polymerase, phosphonoacetic acid, or acyclovir. IL-10(h+v) and vIL-10 were also detected in the supernatants of Akata and Daudi but not Ramos cells infected with P3HR-1 EBV. These results show the IL-10 induction on EBV activation in EBV-carrying B-cell lines.

Interleukin-4 production in Epstein-Barr virus-transformed B cell lines from peripheral mononuclear cells of patients with atopic dermatitis.

Interleukin-4 (IL-4) is known as an immunomodulatory cytokine secreted by relatively few cell types, for example, activated T lymphocytes, basophils, and mast cells, but not by B cells. It plays an important role in promoting the production of the IgE antibody. We established Epstein-Barr virus (EBV)-positive B cell lines from peripheral blood mononuclear cells of patients with atopic dermatitis (AD) and tested the production of several cytokines in the cell lines. We found that IL-4 was produced in a cell line, OB, by an IL-4-specific enzyme-linked immunosorbent assay. IL-4 mRNA was detected in OB and two other AD-derived cell lines by IL-4-specific reverse transcription-polymerase chain reaction. IgE was also produced by the OB cells. The production of IL-4 and IgE was enhanced in the cells treated with 12-O-tetradecanoyl phorbol-13-acetate. This is the first evidence that IL-4 is produced by an EBV-transformed B cell line.

Mild mitral regurgitation reduces exercise capacity in patients with idiopathic dilated cardiomyopathy.

We evaluated 30 patients with dilated cardiomyopathy (New York Heart Association functional Class II or III with medical treatment) to assess the effect of mild mitral regurgitation (MR) on exercise capacity in patients with congestive heart failure. They were classified into two groups based on results of left ventriculography: MR present (n = 10) and MR absent (n = 20). The severity of the MR by left ventriculography was grade I (mild) in all patients with MR. Steady-state hemodynamic data and angiographic data did not differ significantly between the two groups. Heart rate and systolic blood pressure at rest and in response to symptom-limited exercise testing did not differ between the groups. However, the peak work load was significantly lower in the group with MR than that in the group without MR (101 +/- 32 vs. 142 +/- 29 W, respectively; p < 0.005). Peak oxygen uptake and peak oxygen pulse were also significantly lower in the group with MR than in that without MR (peak oxygen uptake: 18 +/- 23 +/- 5 ml/min/kg; p < 0.05, peak oxygen pulse: 6.6 +/- 2.6 vs. 9.5 +/- 2.7 ml/min/beat: p < 0.01, respectively). Thus, mild MR had a detrimental effect on the exercise capacity in patients with dilated cardiomyopathy.

Activation of prophenoloxidase A1 by an activating enzyme in Drosophila melanogaster.

An activating enzyme for prophenoloxidase A1 was isolated from pupae of Drosophila melanogaster, and the activation of purified prophenoloxidase A1 with this enzyme was analyzed. The purification included ammonium sulfate fractionation, DEAE-cellulose, Superdex 75, arginine-Sepharose and hydroxyapatite column chromatography. The prophenoloxidase activating enzyme was determined to be a 28.5-kDa protein consisting of a single polypeptide. The kinetics of the activation reactions was unusual in that the final levels of phenoloxidase activity varied depending on the initial concentrations of the activating enzyme, not those of the prophenoloxidase. The activation was effectively suppressed by the inhibitors of trypsin-type serine protease. The protein has amidolytic activity, and Boc-Val-Pro-Arg-MCA was the best substrate among the synthetic substrates examined. The molecular mass of the activated phenoloxidase was smaller than that of the prophenoloxidase, indicating that a 5-kDa peptide was released from the prophenoloxidase by limited proteolysis with the activating enzyme. The cleavage site of prophenoloxidase A1 was shown to be between Arg and Phe at positions 52 and 53.

Nucleotide sequence of the cDNA encoding the proenzyme of phenol oxidase A1 of Drosophila melanogaster.

Clones encoding pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] A1 were isolated from a lambda gt10 library that originated from Drosophila melanogaster strain Oregon-R male adults. The 2294 bp of the cDNA included a 13-bp 5'-noncoding region, a 2070-bp encoding open reading frame of 690 amino acids, and a 211-bp 3'-noncoding region. A hydrophobic NH2-terminal sequence for a signal peptide is absent in the protein. Furthermore, there are six potential N-glycosylation sites in the sequence, but no amino sugar was detected in the purified protein by amino acid analysis, indicating the lack of an N-linked sugar chain. The potential copper-binding sites, amino acids 200-248 and 359-414, are highly homologous to the corresponding sites of hemocyanin of the tarantula Eurypelma californicum, the horseshoe crab Limulus polyphemus, and the spiny lobster Panulirus interruptus. On the basis of the phylogenetic tree constructed by the neighbor-joining method, vertebrate tyrosinases and molluscan hemocyanins constitute one family, whereas pro-POs and arthropod hemocyanins group with another family. It seems, therefore, likely that pro-PO originates from a common ancestor with arthropod hemocyanins, independently to the vertebrate and microbial tyrosinases.

Delayed improvement in exercise capacity with restoration of sinoatrial node response in patients after combined treatment with surgical repair for organic heart disease and the Maze procedure for atrial fibrillation.

BACKGROUND: Although the Maze procedure successfully restores sinus rhythm in patients with heart disease and atrial fibrillation, it is still uncertain whether an addition of the Maze procedure in cardiac surgery is beneficial for exercise performance of the patients after surgery. METHODS AND RESULTS: The Maze procedure was performed in 25 patients (age, 37 to 70 years) during valve surgery (18 patients) or closure of atrial septal defect (7 patients). A cardiopulmonary exercise test using ramp incremental protocol (15 W/min) was performed before and 1 month, 6 months, and 1 year after surgery. Sinus conversion was obtained in 23 of 25 patients 1 month after surgery. However, sinoatrial (SA) node response to exercise was attenuated by surgery: Mean heart rate (HR) was 83 +/- 13/min at rest, 94 +/- 13/min at 60 W, and 107 +/- 17/min at peak exercise. Peak oxygen uptake (PVO2) was unchanged at this period (before, 17.6 +/- 4.5 mL.min-1.kg-1; 1 month after, 17.5 +/- 4.2 mL.min-1.kg-1). Thereafter, SA node response was restored 6 months after surgery: Mean HR was 84 +/- 13/min at rest, 104 +/- 16/min at 60 W, and 130 +/- 20/min at peak exercise (P < .01 versus 1 month). PVO2 was also improved at this period (20.7 +/- 4.0 mL.min-1.kg-1, P < .01). The increase in PVO2 from 1 month to 6 months after surgery was correlated with the increase in peak HR (y = 0.73x +/- 3.6, r = .79). There were no further changes in heart rate response or PVO2 from 6 months to 1 year after surgery. CONCLUSIONS: Atrial fibrillation was successfully treated by combined treatment with surgical repair for organic heart disease and the Maze procedure. However, SA node response to exercise was attenuated early after surgery. Thus, exercise capacity was improved at the late phase after surgery, which was related to the extent of restoration in SA node response.

Quercetin potentiates TNF-induced antiviral activity.

Tumor necrosis factor (TNF) produces a dose-dependent inhibition of vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), and herpes simplex virus type 1 (HSV-1) replication in WISH cells. The antiviral activity of TNF against VSV and EMCV is greatly enhanced by combination with quercetin. Induction of 2',5'-oligo-adenylate (2-5A) synthetase by TNF is also enhanced by quercetin. Addition of polyclonal antibodies to human interferon (IFN)-beta completely blocked both enhancement of antiviral activity and 2-5A synthetase induction.

Genetic polymorphism of prophenoloxidase A1 in Drosophila melanogaster.

Electrophoretic variations of prophenoloxidase were obtained from natural populations of Drosophila melanogaster. A1, one of the 2 isoforms of the prophenoloxidases, is polymorphic: 10 fast-migrating and 2 slow-migrating types were found in polyacrylamide gel electrophoresis among 271 wild strains. The majority were the intermediate-migrating type. By use of these variants, A1 gene was mapped at 79.6 on the right arm of the second chromosome. By deletion mapping, its cytological position was located at 55A-B. In the electropherograms, hybrids between the types differing in mobility exhibited 3 bands, indicating that A1 is a dimeric protein. The gene for A1 is expressed through larval, pupal and adult stages.

Purification and characterization of prophenoloxidases from pupae of Drosophila melanogaster.

Two isoforms of prophenoloxidase were isolated from pupae of Oregon-R strain of Drosophila melanogaster. The purification procedure included ammonium sulfate fractionation, Sephacryl S-200 gel chromatography, DEAE-cellulose, and hydroxylapatite column chromatography. The two isoforms, A1 and A3, could be separated by ammonium sulfate fractionation. The isoelectric points of A1 and A3 were determined to be pH 5.8 and 6.7, respectively. The molecular weights of the monomers of A1 and A3 were estimated by SDS-PAGE to be 78 and 77 kDa, respectively. The native states of A1 and A3 are considered to be homodimeric, as judged by gel-filtration chromatography.

Glucosidation of estradiol-17 beta in the cultured ovaries of the silkworm, Bombyx mori.

Metabolic pathways of estradiol-17 beta and other vertebrate steroid hormones of cultured silkworm pupal ovaries were examined using 14C-labeled steroids. The isolated ovaries showed significant uptake and metabolic activity of the 14C-labeled estradiol-17 beta added to the medium. Analysis of the metabolized compounds by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed extensive metabolic conversion of [14C]estradiol-17 beta and estrone; i.e., estrone was reduced to estradiol-17 beta and estradiol-17 beta was metabolized to conjugates, including estradiol-3-beta-D-glucoside and estradiol-17-alpha-D-glucoside. [14C]Testosterone was not transformed appreciably by the ovaries. Metabolic activity and physiological significance of the vertebrate steroid hormones in the silkworm ovaries are discussed.

Does estradiol play a role in ovarian maturation or embryonic development of the silkworm?

Since estradiol has been detected in Bombyx ovaries effects of estradiol and other steroids on the growth and maturation of the silkworm ovary, rate of oviposition, and embryonic development were examined as a part of a study aimed at the clarification of physiological significance of estradiol in insects. These steroids were injected at various doses into the whole pupae and into the isolated pupal abdomens. No significant effect by the injections was observed on the ovarian development, as judged by increase in protein content or wet weight of ovaries and pattern of protein constituents including vitellin. However, rate of oviposition was considerably affected by the injection of estradiol at high doses. No clear effect was observed on embryonic development or determination of diapause by the injection of estradiol into the pupae. Effects of injection of anti-estrogen, nafoxidine, into the isolated abdomens and whole pupae were also examined. No effects were observed on ovarian development by injection. The relationship between physiological significance of the vertebrate steroids and metabolic activity of the ovary is discussed.

Identification of estradiol in the ovaries of the silkworm, Bombyx mori.

Estradiol was extracted and partially purified from the ovaries of the silkworm, Bombyx mori. Identification of estradiol was done by use of radioimmunoassay (RIA) and by gas chromatography-mass spectrometry (GC-MS) after derivatization into the ethyldimethylsilyl derivative. Concentration of estradiol in the ovaries was estimated to be 176 pg/g (RIA) and 63 pg/g (GC-MS).

Uptake and metabolism of [3H]ecdysone in cultured ovaries of the silkworm, Bombyx mori.

Uptake of ecdysone by ovaries of the silkworm, Bombyx mori, was studied by organ culture. [3H]Ecdysone was transported almost linearly into the ovary for up to 3 h of the incubation. The uptake was proportional to the concentration of the labeled ecdysone, unsaturably, at concentrations ranging up to 10(-6) M. Ecdysone which had been transported into the ovary could usually be removed when the ovary was re-transferred to the ecdysone-free medium. Analysis of the transported compounds by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed the marked conversion of ecdysone into 20-hydroxyecdysone, unknown metabolites and conjugated forms. Physiological significance of the metabolic activity of the ovary is discussed with respect to the accumulation of ecdysteroids in the ovary.

2-Deoxy-alpha-ecdysone from ovaries and eggs of the silkworm, Bombyx mori.

From ovaries dissected from developing and emerged adults of the silkworm, Bombyx mori, two substances having high molting hormone activity were isolated. One of these was identified as 2-deoxy-alpha-ecdysone by means of high-pressure liquid chromatography and mass spectrometry. Although this compound had previously been isolated from the fern Blechnum minus and postulated to be the precursor of alpha-ecdysone, it had not been obtained from insect material. The compound is also contained in the form of a conjugate in ovaries as well as in diapausing silkworm embryos.