Novel integrative methodology for engineering large liver tissue equivalents based on three-dimensional scaffold fabrication and cellular aggregate assembly.

A novel engineering methodology for organizing a large liver tissue equivalent was established by intergrating both 'top down' and 'bottom up' approaches. A three-dimensional (3D) scaffold was engineered comprising 43 culture chambers (volume: 11.63 cm(3)) assembled in a symmetrical pattern on 3 layers, a design which enables further scaling up of the device to a clinically significant size (volume: 500 cm(3)). In addition, an inter-connected flow channel network was designed and proved to homogenously deliver culture medium to each chamber with the same pressure drop. After fabrication using nylon-12 and a selective laser sintering process, co-cultured cellular aggregates of human hepatoma Hep G2 and TMNK-1 cells were loosely packed into the culture chambers with biodegradable poly-L-lactic acid fibre pieces for 9 days of perfusion culture. The device enabled increased hepatic function and well-maintained cell viability, demonstrating the importance of an independent medium flow supply for cell growth and function provided by the current 3D scaffold. This integrative methodology from the macro- to the micro-scale provides an efficient way of arranging engineered liver tissue with improved mass transfer, making it possible to further scale up to a construct with clinically relevant size while maintaining high per-volume-based physiological function in the near future.

The influence of reduced glutathione in fertilization medium on the fertility of in vitro-matured C57BL/6 mouse oocytes.

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo-matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.

Bacillus subtilis induces morphological changes in Fonsecaea pedrosoi in vitro resulting in more resistant fungal forms in vivo

Interactions among microorganisms may be the cause of morphological modifications, particularly in fungal cells. The aim of this work was to examine the changes that occur in cells of the fungus Fonsecaea pedrosoi after in vitro co-culturing with Bacillus subtilis and to explore the results of this interaction in vivo in an experimental murine infection. B. subtilis strain was inoculated into a 15-day pure culture of F. pedrosoi. In vitro, after 48 hours of co-culturing, the fungal cells were roundish. The secretion of fungal dark pigments and production of terminal chlamydoconidia were observed in hyphae after one week. In the in vivo study, two animal groups of 30 BALB/c mice each were employed. One group was inoculated intraperitoneally with hyphal fragments from the co-culture of bacteria and fungi; the other group was infected only with F. pedrosoi hyphae. After seven days of infection, both animal groups developed neutrophilic abscesses. Phagocytosis of bacilli by macrophages occurred at three days. At later periods, generally after 25 days, only roundish cells similar to sclerotic bodies remained in the tissues while hyphae were eliminated by 15 to 20 days. These fungal forms originated mainly from terminal chlamydoconidia. The co-culturing between bacteria and fungi may constitute a mechanism to rapidly obtain resistant fungal forms for host defenses, especially for chromoblastomycosis (CBM) experimental infections.

Stereoselective synthesis of 2-alkenylaziridines and 2-alkenylazetidines by palladium-catalyzed intramolecular amination of alpha- and beta-amino allenes.

Whereas palladium-catalyzed reaction of N-arylsulfonyl-alpha-amino allenes with an aryl iodide (4 equiv) in the presence of potassium carbonate (4 equiv) in DMF at around 70 degrees C affords the corresponding 3-pyrroline derivatives, the reaction in refluxing 1,4-dioxane under otherwise identical conditions yields exclusively or most predominantly the corresponding 2-alkenylaziridines bearing an aryl group on the double bond. Similarly, N-arylsulfonyl-beta-amino allenes can be also cyclized into the corresponding alkenylazetidines bearing a 2,4-cis-configuration under palladium-catalyzed cyclization conditions in DMF.

Trichoblastoma of the skin occurring in the breast. A case report.

BACKGROUND: Trichoblastoma is a rare benign skin appendage tumor constituted mostly of follicular germinative cells. It can arise on any part of the body except the palms, soles, nail units and mucosal membranes. No case of it in breast skin has been reported before. Furthermore, fine needle aspiration cytology findings on this lesion have not been described before. CASE: A 76-year-old female presented with a firm nodule in her left breast. The tumor was well demarcated, about 1.5 cm in diameter. Fine needle aspiration cytology revealed clusters composed of relatively uniform cells with a high nuclear/cytoplasmic ratio. In the midst of some clusters, the tumor cells had a more abundant cytoplasm. Fibrocellular interstitium or dense cyanophilic acellular material occasionally was attached to them. The tumor cells had oval or fusiform nuclei that had fine, evenly dispersed chromatin. To exclude a diagnosis of breast cancer, it is important to recognize that the clusters are composed of basaloid cells with focal squamous eddies and that there is at least focally peripheral palisading. The histopathologic diagnosis was trichoblastoma. CONCLUSION: Fine needle aspiration cytology can distinguish trichoblastoma from malignant diseases of the breast and may be used to diagnose the lesion in conjunction with clinical findings.

Unilateral intrauterine horn insemination of fresh semen in cats.

The sperm count required were investigated to obtain a conception rate of 80% by unilateral intrauterine insemination (UIUI) of fresh semen in cats. The conception rates obtained by insemination before and after ovulation were also examined. Thirty-six female cats aged 1-7 years were used in the experiments, and the number of experimental cases was 44. Seven male cats aged 2-12 years from which semen could be collected by the artificial vagina method were used. In artificial insemination, 100 iu x 2 or 250 iu of hCG was administered on days 2-4 of estrus, and sperm were introduced into the uterine horn with a greater number of ovulations (or mature follicles) 15, 20 and 30 hr after hCG administration by laparotomy. The inseminated sperm counts were 2 x 10(6) (Exp. 1). 4 x 10(6) (Exp. 2), and 8 x 10(6) (Exp. 3). As a result, ovulation was induced in 42 of 44 cases (induction rate: 95.5%) regardless of the dosage of hCG. Conception was obtained by UIUI in two of 16 animals (conception rate: 12.5%) in the Exp. 1, five of 16 animals (31.3%) in Exp. 2, and eight of 10 animals (80.0%) in Exp. 3. Regarding the relationship between the ovulation state at insemination and conception, the conception rate obtained by insemination before ovulation was clearly higher than that obtained by insemination after ovulation (p<0.05). Regarding the number of kits compared to the number of ovulations on the inseminated side, the percentages of cases in which the number of kits exceeded the number of ovulations on the inseminated side were similar in all groups inseminated with a different number of sperm. It is therefore necessary to investigate conception rates obtained by bilateral insemination to increase the fertility rate. Based on the above findings, it was shown that the sperm count required for fertilization by UIUI is 8 x 10(6).

Unilateral intrauterine horn insemination of frozen semen in cats.

Frozen feline semen was prepared using two types of extenders, egg yolk Tris-fructose citric acid (EYT-FC) and egg yolk sodium citrate solution (EYC), and the semen qualities after thawing and the conception rates obtained by unilateral intrauterine horn insemination (UIUI) were investigated. Cats used in the experiment were six males and 11 females aged 2-12 years (the number of experimental cases was 17). For preparation of frozen semen, semen collected by the artificial vagina method was adjusted to I x 10(8) sperm/m/ and 7% glycerol, put in 250 microl straws, and then frozen using a cell freezer. The mean sperm motility after thawing was 30.0+/-9.7 (SE) % in the semen prepared with EYT-FC and 30.0+/-3.3% in the semen prepared with EYC. Four of seven animals were fertilized by UIUI using two straws in both extenders, and the conception rate was 57.1%. The mean ratios of number of kits to the number of ovulations in the inseminated side were 61.1+/-24.5% and 30.5+/-3.4% for EYT-FC and EYC, respectively, showing that the ratio tended to be higher in the semen prepared with EYT-FC. The above findings, comparing the two extenders for preparation of frozen feline semen, showed that EYT-FC is slightly superior to EYC. To increase conception and fertility rates, it may be important to increase the sperm count for insemination and to inseminate both uterine horns.

Localization of the ATP-sensitive potassium channel subunit (Kir6. 1/uK(ATP)-1) in rat brain.

The Kir6.1/uK(ATP)-1, subunit of ATP-sensitive K(+) channels (K(ATP)), was localized in adult rat brain by in situ hybridization and immunohistochemistry. The mRNA of this subunit was ubiquitously expressed in various neurons and nuclei of the adult rat brain. Interestingly, Kir6.1/uK(ATP)-1 mRNA was also expressed in glial cells. Kir6.1/uK(ATP)-1 protein staining gave a dispersed array of fine dots throughout all neurons and glial cells examined. Under electron microscope, the immunoreactive products were specifically restricted to the mitochondria. The present study indicates that this K(ATP) subunit is localized in the mitochondria and may play a fundamental role in vital brain function.

[Accuracy of participation rate data for health examination research].

Each local government conducts health examinations based on the Health and Medical Law for the Aged. However, since some residents are able to take health examinations at their own work places, for example, and the local government are allowed to exclude such people from taking the law-mandated health examination, it is difficult to obtain an accurate picture of the examination rate in each area. We investigated the actual participation status for health examination services of all of the 6,080 persons 20 years and over in age in Sakuragawa-mura, Ibaraki Prefecture. A comparative investigation was made on 3,655 non-bedridden/non-hospitalized persons of 40 years and over to ascertain the reliability of responses to questions about participation in lung cancer and gastric cancer examination services given by the village. The rate of valid responses was extremely low in those who had not participated in health examinations (male 54%, female 55% for the lung cancer, and male 53%, female 56% for the gastric cancer). These discrepancies are assumed to be the result of confusing the current health examinations with: (1) the health examination given in the previous year, (2) other kinds of health examinations, or (3) the health examination given in the work place or the like. A comparative investigation through logistic regression analysis, between the responses to the questions in this investigation and the actual health examination participation records, for persons who had not yet taken either lung cancer examinations or gastric cancer examinations (908 males and 938 females for the former, and 1,038 males and 1,187 females for the latter). Results showed that the influence of (1) and (2) were more or less detected in every kind of cancer examination, and the influence of (1) on the gastric cancer examination was particularly clear. No definite result was obtained about (3), because the actual record of the health examination service at the work place, etc. was unavailable. The results of this study suggests the necessity of a careful examination of methods when conducting a comprehensive service investigation for the health examinations.

Vasodilation by intrathecal lipopolysaccharide of the cerebral arteries after subarachnoid haemorrhage in dogs.

To investigate the influence of inducible nitric oxide synthase on cerebral arteries after subarachnoid haemorrhage (SAH) in vivo, lipopolysaccharide (LPS), a major inducer of inducible nitric oxide synthase, was injected intracisternally into control and SAH model dogs. Intracisternal injection of LPS (0.5 mg) produced a long-lasting, submaximal vasodilation of the basilar artery of control dogs on angiography. This effect became significant at 4 hours after LPS injection and plateaued after 6 hours. This vasodilation was reduced by N(G)-monomethyl-L-arginine. Vasopressin slightly suppressed the vasodilation, while bradykinin increased it. The concentration of L-arginine in CSF decreased after LPS injection, while that of L-citrulline increased. In cytokines, the concentration of tumour necrosis factor-alpha; (TNF-alpha;) in CSF increased transiently at 4 hours after LPS injection, while interleukin-1 beta, interleukin-6, interferon-gamma, did not change. These data suggest that vasodilation by LPS is mainly due to nitric oxide predominantly synthesized by an inducible nitric oxide synthase, proximally induced by TNF-alpha. Our data make it unlikely that SAH itself induces the inducible nitric oxide synthase in vascular tissue, since isolated endothelium-denuded basilar artery from SAH model dogs did not respond to L-arginine. In SAH model dogs, the degree of vasodilation by LPS differed with the severity of vasospasm. Vasodilation was much greater in mild than in severe vasospasm in dogs, and was increased by superoxide dismutase. These findings suggest that the induction of inducible nitric oxide synthase or its activity may be less effective in severe vasospasm.

[Improved quality of life (QOL) in terminal renal carcinoma patients through home nurse visitation: some considerations].

Ours is a general hospital administered directly by the government. There are 585 beds and some 1,200 outpatients on average per day. Visits by home nurses began in September of 1990, and beginning in April of 1994 a home nursing department has been in place at the Community Medical Center. There are 5 persons in charge of the visits. Home visits are paid to 40 patients at present on an ongoing basis. In recent years, malignant diseases have been increasing, and there has been much discussion devoted to the quality of life (QOL) of the patients. In 1994, 15 of the 60 patients visited had malignant diseases. Presently, a 63-year-old female patient with terminal kidney cancer is receiving HPN. Thanks to home nursing visits, she has been able to be discharged. This patient had been receiving home nursing visits from December 21, 1993 until June 20 of 1994 at the rate of 2 visits per month. The person in charge gave guidance for HPN management, and the patient began to regain her pre-hospitalization life and a sense of her role in her family and society. As a result, though she had not actively fought the disease while in the hospital, she gained confidence with the HPN, and with her husband's help, she became a Culture School teacher, resumed her role in society and found her life meaningful. Since there were limits to her husband's ability to care for her, she agreed to enter the hospital again, but was able to continue meaningful life at home for 6 months with assistance from a caregiver, and thus achieved a high QOL.

Vasorelaxant effect of PACAP-27 on canine cerebral arteries and rat intracerebral arterioles.

The vasorelaxant effects of pituitary adenylate cyclase activating polypeptide (PACAP)-27 were examined and compared with those of PACAP-38 and vasoactive intestinal polypeptide (VIP) on isolated canine cerebral arteries and rat intracerebral arterioles in vitro. The addition of PACAP-27, PACAP-38 or VIP resulted in similar concentration-dependent relaxations in both canine basilar arteries and rat intracerebral arterioles. There were regional differences in the PACAP-27-induced relaxations measured in canine cerebral arteries. The maximum relaxation induced by PACAP-27 was significantly lower in the basilar arteries (23.0 +/- 5.6%) than in the rostrally located arteries (proximal middle cerebral arteries: 45.4 +/- 5.7%, anterior cerebral arteries: 55.2 +/- 5.8%). The maximum relaxation induced by PACAP-27 in the basilar arteries was significantly enhanced by mechanical removal of the endothelium (16.4 +/- 4.5% vs. 32.7 +/- 5.8%) as well as by pretreatment with indomethacin or aspirin (12.9 +/- 4.1% vs. 48.7 +/- 6.1% and 46.5 +/- 9.2%, respectively). Incubation of canine cerebral arteries with PACAP-27 in vitro resulted in an increased release of prostaglandin F2 alpha in the buffer from 14.5 +/- 2.1 pg/min/1 mg vessel to 31.1 +/- 4.2 pg/min/1 mg vessel, while other cyclooxygenase cascade metabolites such as prostaglandin E2, thromboxane B2 and 6-keto prostaglandin F1 alpha did not change. These data suggest that the PACAP-27-induced relaxation of canine basilar arteries may be associated with prostaglandin F2 alpha or its precursor, prostaglandin H2.

Effects of adrenomedullin, calcitonin gene-related peptide, and amylin on cerebral circulation in dogs.

The effect of human adrenomedullin on cerebral circulation was investigated in dogs in vivo and in vitro. Bolus administration of adrenomedullin or its homologous peptides, calcitonin gene-related peptide (CGRP) and amylin, into the vertebral artery induced a dose-dependent increase in vertebral blood flow. The potencies of adrenomedullin and CGRP were similar and approximately 100 times more than that of amylin. The effects of adrenomedullin and CGRP were inhibited by CGRP8-37, an antagonist of CGRP. In contrast to substance P, adrenomedullin did not induce an increase in blood flow after prior administration of CGRP. Pretreatment with either NG-nitro-L-arginine methyl ester or indomethacin did not affect the adrenomedullin-induced increase in blood flow. Intracisternal administration of adrenomedullin induced dilation of the basilar and other major cerebral arteries in a dose-dependent manner, accompanied by an increase in the concentration of cyclic AMP in the cerebrospinal fluid. Adrenomedullin also induced relaxation of isolated basilar and middle cerebral arterial rings. These data suggest that adrenomedullin induces vasodilation of cerebral arteries and an increase in vertebral blood by acting at CGRP receptors positively coupled to adenylate cyclase, and that these effects are not dependent on nitric oxide or prostaglandin formation.

Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats.

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T3 injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about approximately 35-40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly approximately 25-32 and approximately 29-41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by approximately 72-81 and approximately 51-70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels.

Onset of paternal gene activation in early mouse embryos fertilized with transgenic mouse sperm.

We investigated the onset of paternal gene expression in the early mouse embryo. We obtained transgenic mouse embryos by fertilizing BD (C57BL/6N x DBA) F1 hybrid female oocytes in vitro, with sperm from homozygous transgenic males carrying integrated chicken beta-actin promoter-driven firefly luciferase cDNA. We then examined the RNA and protein synthesis of the luciferase gene in embryos from the 1- to 2-cell stage. RNA transcripts of the luciferase gene were first detected in the 1-cell stage embryos as early as 13 hr postinsemination, just prior to elongation. By photon-count imaging, functional luciferase was identified at the 2-cell stage 23 hr postinsemination. These findings indicate that the paternal endogenous gene is already transcribed in the late 1-cell embryos, although paternally derived protein is not synthesized until the 2-cell stage. Therefore, these results suggest that the embryonic gene is activated as early as the late 1-cell stage.

[Production of transgenic mice from in vitro fertilized eggs cryopreserved by ultrarapid freezing].

In vitro fertilized mouse eggs (C57BL/6N), followed by ultrarapid freezing were used for production of transgenic mice by microinjection of the chicken beta-actin promoter-driven the firefly luciferase cDNA (beta act-Luc). Following micromanipulation, the survival rates of the cryopreserved eggs and of the fresh in vitro fertilized eggs (control) were 70.8% (131/185) and 71.9% (159/221), respectively. After transferring them into oviducts of psudopregnant recipients on Day 1, 13.6% (17/125) of the cryopreserved eggs developed to live offspring and 14.1% (21/149) of fresh eggs did so. It was confirmed by Southern blotting analysis that each two transgenic mice were produced from the cryopreserved eggs (12%, 2/17) and the fresh eggs (10%, 2/21). All of transgenic mice produced from both eggs showed the expression of the luciferase gene. These results indicate that the in vitro fertilized eggs cryopreserved by ultrarapid freezing, can be, easily and conveniently, used for generation of transgenic mice.

[Cryopreservation of in vitro fertilized embryos from transgenic rat by ultrarapid freezing].

We cryopreserved pronuclear stage-embryos from the Transgenic rats produced by introducing the rat GH antisense transgene, by ultrarapid freezing. The embryos were obtained by fertilization in vitro with the sperm of homozygous males for the transgene. After thawing of a part of cryopreserved embryos (97 embryos), the 40 embryos (41%) were morphologically normal, and were transferred to oviducts of recipients. All four live young (10%) obtained (one male and three females) preserved the transgene and exhibited dwarfism. We also produced the homozygous transgenic rats after brother-sister mating of those young. These results demonstrated the possibility of the cryopreservation of embryos by ultrarapid freezing for sustaining transgenic rat line.

Behavioral study of visits by aged outpatients to medical facilities in Japan.

In this study we analyzed the changing behavior patterns of aged persons seeking medical care at hospitals and clinics over a recent ten-year period. The results showed a rapid increase in the number of aged outpatients receiving care at hospitals since 1977. In contrast, the number of aged outpatients receiving care at clinics since 1977 fluctuated with an overall downward trend, indicating outpatients' preference to seek treatments in hospital as opposed to clinics. The following are expected to affect future outpatient care rate differences between clinics and hospitals: differences in care rates for new outpatients, and differences between the sexes and among the types of illnesses treated. Having an especially large effect are: hypertension and heart diseases, diseases of the circulatory system also known as "adult diseases"; diseases of the digestive system, and diseases of the respiratory system.

Basic studies of radiopaque resin monomer (III). Physical properties of trial composite resins.

A series of studies has been conducted on the synthesis of radiopaque monomers and the development of a composite resin having these monomers. Using octachlorocyclotetraphosphazene, P4N4Cl8 (4PNC), three kinds of radiopaque cyclophosphazene monomers, 4PN(Br3Ph)1-3-(EMA)7-5, were synthesized by reacting 1-3 mols of tribromophenol (Br3Ph) and 7-5 mols of 2-hydroxyethyl methacrylate (HEMA). As the monomer for an organic composite filler, 70% (wt) synthesized monomer was used with silica (OX-50) treated with silane mixed at 30% and ground after heat-polymerization and then run through a 325-mesh sieve after polymerization. As a base monomer, 25% urethane monomer (U-2TH) was mixed in 25% synthesized monomer and photosensitizer was added. The composite resin was prepared by mixing 50% organic composite filler with 50% base monomer, and polymerized with a Dentacolor XS (Kulzer) visible light-curing apparatus by irradiation for 90 s on each side, 180 s in total. Mechanical properties did not vary with the increase in the number of tribromophenol replacements. Compressive yield strength was more than 120 MPa in all cases. Transverse strength was 60-75 MPa and hardness was HK 26-30. However, the aluminum equivalent increased with the increase in the number of tribromophenol replacements. In the case of 4PN-(Br3Ph)3, the value was 8.8 mm, whereas in the case of commercial composite resin, it was 0.4-10.4 mm.