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The genome size (C-value) of a species indicates the total amount of DNA included in its haploid chromosomes. It is one of the fundamental characteristics that relate closely to the evolution of organisms. Information on molluskan C-values is limited despite the abundance of morphological diversity. Therefore, to better understand molluskan genome evolution, we conducted a comprehensive survey of the C-values and AT content of 146 species in four molluskan classes by flow cytometry. The difference in AT content was found to be related to an increase in C-value in gastropods and cephalopods. For cephalopods, a positive correlation of AT content with the increase in chromosome number and C-value was observed from Nautilus pompilius and N. macromphalus (2n = 52) to the specialized groups of octopus (2n > 60) and squid (Sepiida and Teuthida; 2n = 92–112). In contrast, a negative correlation between C-value and AT content in gastropods was demonstrated from the limpet group Patellogastropoda (2n = 16–18) to the specialized group Neogastropoda (2n = 26–72). Our data suggest at a likely positive correlation between AT content and C-value in the cephalopods lineages but a negative correlation between them in the gastropod lineages. Furthermore, the reduction of the C-value in the Bivalvia lineage could not have been caused by the selective deletion of AT-rich regions. Our study adds valuable data to the currently limited knowledge base of molluskan C-values, which will provide important information for future genome projects in mollusks.

In Hiroshima Bay, parasitic isopods of the genus

This paper introduces an ultra-low power 1.7-2.7-GHz fractional-N sub-sampling digital PLL (SS-DPLL) for Internet-of-Things (IoT) applications targeting compliance with Bluetooth Low Energy (BLE) and IEEE802.15.4 standards. A snapshot time-to-digital converter (TDC) acts as a digital sub-sampler featuring an increased out-of-range gain and without any assistance from the traditional counting of DCO edges, thus further reducing power consumption. With a proposed DCO-divider phase rotation in the feedback path, the impact of the digital-to-time converter's (DTC's) non-linearity on the PLL is reduced and improves fractional spurs by at least 8 dB across BLE channels. Moreover, a "variable-preconditioned LMS" calibration algorithm is introduced to dynamically correct the DTC gain error with fractional frequency control word (FCW) down to 1/16384. Fabricated in 40 nm CMOS, the SS-DPLL achieves phase noise performance of -109 dBc/Hz at 1 MHz offset, while consuming a record-low power of 1.19 mW.

Cnidarians are considered ancestral metazoans and, therefore, are important taxa for studying animal evolution. However, little is known about the group’s genome size (C value), which is an important parameter in whole-genome sequencing. To address this issue, we measured the C values of 27 cnidarian species from Japan, using flow cytometry, and found that they ranged from 0.26 to 3.56 pg. Excluding the results for Agalma elegans and Physalia physalis (order Siphonophorae), which had the highest C values among the species included in the present study, the C values for the cnidarians were 0.26–1.49 pg. In particular, we found that hydrozoans possessed relatively large and wide-ranging C values, indicating that evolution within the group involved considerable gains or losses of genomic content. Overall, the C values reported in the present study could be valuable for whole-genome sequencing, using next-generation sequencers, and for future research in cytogenetics.

It is said that ‘(t)he making of a constitution normally follows a fundamental political event’.11 AW Bradley, KD Ewing and CJS Knight, Constitutional and Administrative Law (Pearson, 16th edn 2015...

The Internet-of-Things (IoT) is gaining momentum, and the ultra-low-power (ULP) RF transceiver is one of the key enablers. Generation of the local oscillator (LO) consumes a significant share of the total energy of these ULP transceivers which are typically powered by small batteries. Therefore, a fractional-N PLL needs to perform LO frequency synthesis and modulation with a very stringent power budget, i.e., below 1mW [1]. A digital PLL is favored in these applications because of the benefit of small area, which is also a critical cost consideration in IoT. On the other hand, the LO quality generated by these ULP PLLs cannot be compromised, and it needs to fulfil the RF requirements defined in the IoT standards, e.g., Bluetooth Low Energy (BLE). Although the integrated phase error is less stringent in IoT standards, the spectral purity requirements remain critical in order to fulfill the regional spectrum regulations, e.g., FCC. A high fractional spur level due to the non-linearity in PLLs (e.g., from a TDC) introduces the unwanted emission. In this work, we present a ULP dividerless digital PLL with a power-efficient spur-mitigation technique. Furthermore, one of the critical issues of the dividerless (or sub-sampling) PLLs, the lack of frequency capture capability without the assistance of an extra power-hungry frequency-locked-loop (FLL), is addressed and mitigated by the proposed digital phase unwrap technique.

Octopus (Amphioctopus) areolatus is an important marine cephalopod in Japan. We examined its diploid chromosome number, karyotype, and genome size (C value), and performed fluorescence in situ hybridization (FISH) using a vertebrate telomeric probe. The diploid chromosome number was 60 in embryonic cells, with 24 pairs of metacentric chromosomes, four pairs of metacentric/submetacentric chromosomes, and two pairs of submetacentric chromosomes. The mean C value was estimated to be 5.47 pg. From these findings, and those reported previously for other octopus species, we suggest that the factor causing the quantum change of C value in O. (A.) areolatus was genome duplication, and not polyploidy. In telomeric FISH analysis, hybridization signals were clearly observed in the telomeric regions of the chromosomes. This is the first report of FISH analysis of cephalopod chromosomes, and our findings suggest that the telomere sequence of O. (A.) areolatus is (TTAGGG) n , which may allow gene mapping in the future.

The reproductive cycle of female Tylos granuliferus was investigated in laboratory conditions. We focused our experiments on female copulative timing for effective insemination and on female sexual receptivity for reproduction. We used females who were isolated from males along with delayed copulation. The female reproductive cycle of T. granuliferus is related to the parturial moult cycle. Females copulate soon after shedding the posterior half of their body cuticle through anterior shedding with a shedding interval of 2 days. Copulation ends just before oviposition. The optimum timing for copulation is within a few days after completion of the parturial moult. Oviposition immediately follows insemination. Without copulation, females do not oviposit their eggs. Pre-copulatory mate guarding by males and sperm storage by females for future broods do not appear to occur in this species. Isolated females have a sexual receptive period of 12 days during the early stages of the reproductive cycle and cannot pr...

Deep seawater facilities inadvertently collect organisms from suction inlets and, thus, may serve as unique biological platforms. Organisms collected from a depth of 612 m offshore from Okinawa, Japan, were archived from 2000 to 2006. Of the total of 633 individuals collected, 550 specimens were examined and taxonomically identified; the remaining 83 samples were too seriously damaged to examine. As a result, a total of 63 species were identified, and taxa comprised 34 fishes, 23 crustaceans, 5 mollusks and 1 echinoderm. Although a weak tendency of year-to-year increase in the catch number was apparent, it should be confirmed by monitoring for a longer term. No clear seasonality for the catch number and species composition was observed. A significant decline in the number of the pleurobranchid Pleurobranchella nicobarica and an increase in the benthopelagic holothurian Enypniastes eximia were recorded during the study period; these variations may imply that unspecified environmental changes have occurred.

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP fluorescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.

Colonies of the ‘big mouth’ tunicate, Megalodicopia hians, were first discovered in Toyama Bay, Japan Sea. The suspension-feeding habit of this organism is inferred from the diets in the oral aperture and feces. The colonies were mainly found on the steep slope (water depth > 1 km), particularly at the sites characterized by exposed rocks and/or thin sediment. Most of the individuals faced ‘mouth’ downward, suggesting that they trap suspended matters adrift or re-suspended sediment in the upward current. We have analyzed the composition of total fatty acids of Megalodicopia hians in comparison with those of other bathyal benthic animals having various feeding habits and phylogenies. A dendrogram was constructed by the cluster analysis of the fatty acid compositions. Primary grouping in the fatty acid-based dendrogram reflected the feeding habits rather than the phylogenic relationships of the studied benthic animals. An inference from the dendrogram suggested that suspension-feeding is usual but occasionally this organism engages in the scavenging habit, in general agreement with the inference from the diet analysis.

Genome size (C value) is a fundamental characteristic of every species and is very important for the progress of cytogenetic, genomic, and phylogenic studies. However, information on the C value of phylum Brachiopoda is scarce. In this study, we collected 8 brachiopod species from Japan, including 5 from class Articulata, and determined their C values by flow cytometry. The mean C values for these 8 species-namely, Lingula anatina, Lingula reevii, Discradisca stella, Terebratulina crossei, Laqueus rubellus, Laqueus blanfordi, Terebratalia coreanica, and Platidia japonica—were 0.41 pg, 0.41 pg, 0.46 pg, 0.33 pg, 0.44 pg, 0.42 pg, 0.37 pg, and 0.31 pg, respectively. Although the C values were examined across the various taxa within Brachiopoda, we detected very little variation (approximately 1.5-fold, from 0.31 pg in P. japonica to 0.46 pg in D. stella). This low C value variation suggests that the occurrences of evolutionary events such as changing the number of transposable elements and intron s...

We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-dayold green fluorescent protein (GFP)-transgenic mice (1/1). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P , 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 6 0.3 for the experimental group versus 2.0 6 0.7 for the control group, mean 6 SEM), the number of collected oocytes by superovulation per mouse (7.0 6 1.7 for the experimental group versus 22.7 6 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft. in vitro fertilization, ovary, ovulation, ovum pick-up/transport, pregnancy

A member of the mitogen-activated protein kinase superfamily, MAK, has been proposed to have an important role in spermatogenesis, since Mak gene expression is highly restricted to testicular germ cells. To assess the biological function of MAK, we have established MAK-deficient (Mak(-/-)) mice. Mak(-/-) mice developed normally, and no gross abnormalities were observed. Spermatogenesis of the Mak(-/-) mice was also intact, and most of the mice were fertile. However, Mak(-/-) male-derived litter sizes and their sperm motility in vitro were mildly reduced. These data show that function of MAK is not essential for spermatogenesis and male fertility.

The N141I mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease in the Volga German families. We previously reported that mouse brains harboring mutant PS2 contained increased levels of amyloid β protein (Aβ) 42 in the Tris-saline-soluble fraction (Oyama, F., Sawamura, N., Kobayashi, K., Morishima-Kawashima, M., Kuramochi, T., Ito, M., Tomita, T., Maruyama, K., Saido, T. C., Iwatsubo, T., Capell, A., Walter, J., Grunberg, J., Ueyama, Y., Haass, C. and Ihara, Y. (1998) J. Neurochem. 71, 313–322). Here, using a new extraction protocol, we quantitated the Aβ40 and Aβ42 levels in the Tris-saline-insoluble fraction. The insoluble Aβ levels were found to be higher than the soluble Aβ levels, and the insoluble Aβ42 levels were markedly increased in mutant PS2 transgenic mice. To investigate the origin of the insoluble Aβ42, we prepared the detergent-insoluble, low density membrane fraction. This fraction from two independent lines of mutantPS2 transgenic mice contained remarkably increased levels of Aβ42 and significantly low levels of glycerophospholipids and sphingomyelin. This unexpected finding suggests that a large increase in the levels of Aβ42 in mutant PS2 mice is presumably induced through alterations of the lipid composition in the low density membrane domain in the brain.

The prophylactic effect of FK463, a new water-soluble echinocandin-like lipopeptide with inhibitory activity against 1,3-β- d -glucan synthase, against Pneumocystis carinii infection was investigated with the severe combined immunodeficient (SCID) mouse model. Treatment with FK463, pentamidine, and saline only was performed for 6 weeks from the day after the SCID mice were inoculated intranasally with infected lung homogenates. FK463 at 0.2 or 1.0 mg/kg of body weight, pentamidine at 4 mg/kg, or saline was subcutaneously administered daily into the backs of the SCID mice. The effects of the drugs were evaluated by detection of P. carinii cysts in mouse lung homogenates by toluidine blue O staining, lung histology, and PCR amplification of a P. carinii -specific DNA fragment from the lungs. P. carinii cysts were detected in the lungs of all mice administered saline. In contrast, no cysts were detected in mice administered both doses of FK463 and pentamidine. A specific DNA fragment was amplified from all mice administered saline and at least half or more of the mice administered FK463 and pentamidine. These results indicate that FK463 acts on cyst wall formation but not on trophozoite proliferation and is extremely effective in preventing P. carinii -associated pneumonia. These results suggest that FK463 is potentially useful as a prophylactic agent against P. carinii infection.

One of the most characteristic clinical features in cutaneous leishmaniasis is the development of nodules followed by ulcerations at the site of infection.Leishmania amazonensis-infected mice show similar ulcerative lesions.Leishmania-infected severe combined immunodeficiency (SCID) mice, however, have been shown to develop nonulcerative nodules. In the present study, the roles of T cells in ulceration were examined using SCID mice in cell reconstitution experiments. After development of nonulcerative nodules, SCID mice were inoculated with splenocytes from eitherLeishmania-infected or naive immunocompetent mice, resulting in ulceration in all mice. When naive splenocytes were depleted of CD4+, CD8+, or B220+cell populations and the remaining cells were injected intoLeishmania-infected SCID mice after the development of nodules, only SCID mice inoculated with splenocytes depleted of CD4+cells did not show ulceration. The evidence obtained in this study clearly shows that the CD4+cell population is indispensable for ulceration in leishmaniasis lesions of SCID mice.

In order to synthesize MgB 2 films, electrolysis is performed in an electrolyte comprising a fused mixture of B 2 O 3 , KCl and MgCl 2 in a molar ratio of B 2 O 3 :KCl:MgCl 2 = x :3.5:5. The results of the Meissner effects and X-ray diffraction measurements indicate the formation of MgB 2 films on graphite and Si substrates. The superconducting critical temperature depends on both the synthesis temperature of the electrolysis and the amount of B 2 O 3 in the electrolyte.

Five specimens of Ostrea fluctigera Lamy, 1925 were collected from the Sagami Bay, adhering by their left valves to the lowest body whorl (near the aperture) of empty shells of several gastropod species, including those used by hermit crabs. In each case, the curved edge of the left valve precisely matched the curvature of the lowest body whorl of the snail shell, and both the left and right valves grew as an extension of the snail shell's aperture. The possibility of a mutualistic symbiosis between O. fluctigera and hermit crabs is discussed.

Although severe combined immunodeficient (SCID) mice are considered useful as an animal model for human hematopoietic diseases, the complete reconstruction of human hematopoietic cells can not be established even in these mice. This appears to be because human cytokines, adhesion molecules and extracellular matrices which support differentiation and growth of human hematopoietic cells differ from those in animals. To improve this animal model, we attempted to produce transgenic (Tg) mice producing human interleukin 3 (hIL-3) and human granulocyte macrophage colony stimulating factor (hGM-CSF) with the homozygote of the scid gene. We established two Tg mouse lines, one releasing both 0.5-1 ng/ml of hIL-3 and 0.05-0.2 ng/ml of hGM-CSF in their sera and another releasing only high (2-10 ng/ml) levels of hGM-CSF. When human cytokine-dependent myeloid cell line, TF-1, was subcutaneously transplanted into these two Tg-SCID mouse lines, TF-1 could be successfully engrafted and grew in all lines of Tg-SCID mice but not in control mice. We also observed that TF-1 grows in GM-CSF Tg-SCID mice in a dose dependent manner in vivo and IL-3 shows an additive effect on its growth. These results indicated that these Tg-SCID mice were an useful in vivo model for investigating human leukemogenesis, especially the role of IL-3 and GM-CSF in leukemogenesis.

The platelet glycoprotein (GP) Ib/IX/V is a hetero-oligomeric receptor complex for von Willebrand factor (vWF) and mediates platelet adhesion and aggregation under high shear stress conditions. It is composed of alpha and beta chain of GP Ib, GP IX, AND and GP V. To establish transgenic mice carrying human GP Ib alpha, we injected into mouse zygotes a 6 kb DNA fragment containing human GP Ib alpha gene that included entire coding sequence and putative promoter region. One hundred and thirteen offsprings were screened, and only one was found to express human GP Ib alpha protein and has passed the human GP Ib alpha gene as well as the expression of the gene to next generation. The expression of human GP Ib alpha in transgenic mice was limited to platelets and megakaryocytes. Glycocalicin, a proteolytic fragment of human GP Ib alpha found in normal human plasma, was not detected in transgenic mouse plasma. Human vWF in the presence of ristocetin supported agglutination of transgenic mouse platelets, but not of control mouse platelets.

The inactivation efficacy of eight disinfectants commonly used in laboratories and animal rooms to inactivate Pneumocystis carinii cysts was estimated by experimental infection in C.B-17-scid mice. The disinfectants examined in this study were 70% ethyl alcohol, 10% iodoform, 0.5% hypochlorous acid, two 1% quanternary ammonium salts, 3% hydrogen peroxide, sodium chlorite and 1% cresol soap. The lung homogenates from P. carinii infected C.B-17-scid mice were treated with each disinfectant for 15 min at room temperature, washed with saline, and inoculated into C.B-17-scid mice. Eight weeks after inoculation, lungs from these mice were examined by staining with toluidine blue O to detect P. carinii cysts. PCR amplifying 346 bp of P. carinii specific mitochondrial ribosomal RNA large segments was also performed using DNA extracted from the lungs of the mice. As a result, seven disinfectants, excepting for 0.5% hypochlorous acid, were effective in the inactivation of P. carinii cysts. These results suggest that P. carinii cysts were sensitive to chemical disinfectants even though they have been commonly considered as insensitive.

We investigated whether refined follicle stimulating hormone (FSH) with only a little contaminating LH can promote the responsiveness of rabbits to multiple-ovulation treatment. One group of female rabbits was stimulated with refined porcine FSH (pFSH), an FSH source with low LH activity, and another group was treated with pFSH. The mean number of eggs recovered from donors stimulated with refined pFSH (27 +/- 3) was significantly greater (P0.05) than that with pFSH (20 +/- 2). Furthermore, the mean number of remaining follicles of donors stimulated with refined pFSH (19 +/- 4) was significantly greater (P0.05) than that with pFSH (12 +/- 1). To decrease the number of remaining follicles in donors treated with refined pFSH, the dose of human chorionic gonadotropin (hCG) was increased from 75 to 150. However, there were no differences in the numbers of eggs and remaining follicles. The results of the present study suggest that refined pFSH with little contaminating LH promotes the responsiveness of rabbits to multiple-ovulation treatment compared with pFSH.

第32回極域生物シンポジウムポスター発表

Aluminum hydroxide gel (Al-gel), which is used as an adjuvant, can absorb macromolecules. We investigated the applicability of Al-gel to the sustained release of follicle-stimulating hormone (FSH) as a simplified method of superovulation (SOV) in rabbits. The responsiveness of rabbits to SOV by a single injection of FSH dissolved in Al-gel suspension (3.2 mg Al/ml) and in 10% (w/v) polyvinylpyrrolidone (PVP), and by multiple injections of FSH in saline was examined. The numbers of total and fertilized eggs recovered from rabbits treated with FSH in Al-gel (40.5 and 26.3, respectively) were similar to multiple injections (47.4 and 28.6, respectively) and were significantly greater (P

We developed a rat line showing small eye from transgenic rats that were obtained by microinjection of a DNA segment containing the human (h)tau cDNA (GenBank: BC000558: 31-677,774-1180) expressed under control of CAG promoter, which is related to Alzheimer disease, into the pronuclei rat embryos. The rat line was established by selective brother-sister mating of rats showing small eyes. Of 11 offspring in the 11th generation, there were eight animals with microphthalmia and the transgene. The remaining three rats without transgene did not show the small eyes phenotype. The globes of affected rats were 1.2 mm in length compared with normal globes (3.5 mm), and all other ocular structures were normal. The expression of hTau protein was evident immunohistochemically in the ciliary body, extraocular muscle, lens epithelium, and pigment epithelium. Cytogenetic analysis suggested that the chromosome location of the transgene was chromosome 1 (1p12). This region may include genes related to lens development, such as Cat5.

Serological titers to Pneumocystis carinii (Pc) and porcine reproductive and respiratory syndrome virus (PRRSV) were measured on a herd with epidemic Pc pneumonia (case herd) and two comparison herds, by an indirect fluorescent-antibody technique. In the case herd, the geometric mean titer (GMT) for Pc were 1:80 in pigs 1 week old, 1:10 in pigs 5 weeks old, and 1:80 to 1:190 in pigs over 6 weeks old. GMTs for PRRSV were >1:145 in most of age groups over 7 weeks old. In comparison herds, Pc and PRRSV antibody titers were low in weanling pigs. The results clarified the kinetics of antibodies to Pc and concurrent infection of PRRSV in the case herd.

To improve the efficiency of transgenesis, we investigated the effects of a radical scavenger during microinjection on the development to blastocysts or pups of mouse pronuclear embryos, microinjected with the enhanced green fluorescent protein (EGFP) transgene. When embryos were microinjected in medium containing 0-1,000 units/ml catalase, the developmental rate to blastocysts was significantly higher (P

The mammalian fertilization process takes place in a complex microenvironment within the female genital tract. A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), has been identified in oviductal fluid from various mammalian species, including humans. Although OGP is widely believed to be involved in the process of mammalian fertilization, including spermatozoon function and gamete interactions, based on experimental results obtained in vitro, its physiological significance remains controversial. The present study established OGP gene-null (ogp−/−) mice, and primarily characterized their reproductive properties to study the physiological function(s) of OGP. Results obtained from studies using an in vivo or in vitro system showed that the fertility of ogp−/− females was within normal limits. These results indicate that OGP is not essential for the process of in vivo fertilization, at least in mice.

To evaluate the effects of cryopreservation and in vitro fertilization (IVF) on genotypic frequencies in mouse colonies, genotypic frequencies at 15 biochemical, 4 immunological and 20 microsatellite loci were examined in three colonies of MCH (ICR) mice derived from noncryopreserved embryos obtained by natural mating without the induction of superovulation, cryopreserved embryos obtained by natural mating with the induction of superovulation, and cryopreserved embryos obtained by the induction of superovulation and IVF. Three (Pgm-1, Ldr-1 and Hbb) out of the 15 biochemical loci, two (Thy-1 and H2K) out of four immunological loci and five (D5Mit18, D6Mit15, D12Mit5, D13Mit26, and D14Mit7) out of 20 microsatellite loci that showed polymorphisms in every colony were used for detection of genotypic frequencies. The genotypic frequencies of the loci in the three colonies did not differ from the predicted genotypic frequencies (P > 0.05). The results suggested that genetic drift does not occur among colonies established from treated and untreated embryos, and it was clear that the embryo banking by cryopreservation is suitable for preservation of outbred stock without genetic drift.

BALB/cA mice homozygous for both nu and scid mutations (BALB/cA-nu/nu, scid/scid) were developed by mating between BALB/cA-scid and BALB/cA-nu. These mice have greater longevity than C.B-17-scid because no thymic lymphoma occurs in them unlike in the latter. C.B-17-scid is known to show the leaky phenomenon in which a few clones of functional T and B cells develop in aged C.B-17-scid. Unexpectedly, the leaky B cells and T cells were absent or suppressed in BALB/cA-nu, scid mice when cytokine expressions were determined by RT-PCR, lymphocyte phenotypes by flow cytometry and serum immunoglobulin levels by ELISA. These results indicate that B cell leakiness may be induced by leaked T cells. BALB/cA-nu, scid mice may be useful as a recipient in allo- and xeno-transplantation experiments because of the absence of both thymic lymphomas and leakiness, in addition to lack of hair.

Cutaneous leishmaniasis begins as papules or nodules at the site of promastigote inoculation. The next key pathogenic event in this disease is the formation of an ulcer at this site. Leishmania infection in immunodeficient mice, however, showed non-ulcerative cutaneous lesions suggesting the involvement of the immune system in ulcer formation. Severe combined immunodeficient (SCID), recombination-activating gene 2 knockout (RAG-2-/-), and immunocompetent mice were inoculated subcutaneously with cultured L. amazonensis promastigotes. Macroscopic nodules appeared at the inoculation site within 2 weeks of infection in all the mice and gradually extended to the surrounding skin tissue. Although nodules of immunocompetent mice ulcerated within 6 weeks, immunodeficient mice did not form ulcers even after 25 weeks of inoculation. These results strongly suggest the importance of functional T and B cells in ulcer formation of cutaneous leishmaniasis and are consistent with clinical features of non-ulcerative cutaneous leishmaniasis in some AIDS patients. The present study also indicates that the L. amazonensis-infected immunodeficient mouse model might be suitable for studying the mechanisms of ulcer formation in cutaneous leishmaniasis.

To generate an appropriate model for human acute myeloblastic leukemia (AML), we have successfully established a human hematopoietic growth factor-dependent AML cell line (TF-1 and UT-7/GM)-ascites model using human granulocyte-macrophage colony-stimulating factor (hGM-CSF)- and human interleukin 3 (hIL-3)-releasing transgenic (Tg)-SCID mice. When 1 x 10(7) cells of TF-1, a human erythroleukemia cell line, were transplanted into the peritoneum of irradiated Tg-SCID mice (TF-1 ip/Tg-SCID mice), TF-1 cells grew in both the single cell suspension form (asTF-1) and solid form in ascites and invaded various tissues: lungs, liver, pancreas, and genitals, 3-6 weeks following transplantation. Subsequently, 0.5-1 x 10(7) cells of UT-7/GM, a subline of the UT-7 human megakaryoblastic leukemia cell line, grown in the back of hGM-CSF Tg-SCID mice after subcutaneous inoculation, were transplanted into the peritoneum of other irradiated hGM-CSF Tg-SCID mice. After 4 weeks, UT-7/GM cells (asUT-7/GM) also grew in the same manner as TF-1 cells in hGM-CSF Tg-SCID mice. Analysis of the cells from the peritoneum and tissues by PCR amplifying ALU and human GM-CSF receptor beta sequences and by immunohistochemical staining using anti-human CD45 revealed that they possessed the original characteristics of the parental cells. To confirm the usefulness of this human AML-ascites model, experimental treatment of AML cells grown in these mice was carried out with a differentiation inducer, delta-aminolevulinic acid (deltaALA), which induces hemoglobin synthesis for TF-1 in vitro and is thus regarded as an anti-leukemia drug candidate. Unexpectedly, growth promotion of TF-1 cells was observed in the treated TF-1 ip/hIL-3 Tg-SCID mice without differentiation to erythroid cells after treatment with delta-ALA (5 mM) for 7 days. These results indicate that Tg-SCID mice can support the growth of human hematopoietic growth factor-dependent AML cell lines which are usually rejected by SCID mice, without modification of the parental cell characteristics. In addition, this Tg-SCID leukemia-ascites model may become a useful preclinical tool for estimation of drug efficacy in vivo, since the drug candidate which was promising in vitro did not act in the same manner in vivo.

The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild-type or mutant PS2 under transcriptional control of the chicken beta-actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five- to 15-fold higher than that of endogenous mouse PS2 mRNA. The amyloid beta-protein (Abeta) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild-type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the Abeta42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild-type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N-terminal and C-terminal fragments between wild-type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age-dependent increase of Abeta42 content in the brain.

Previous work has shown the usefulness of severe combined immunodeficient (SCID) mice as in vivo models for the growth of normal human haemopoietic cells and leukaemic cells. Many approaches have been made to improve the engraftment of human haemopoietic cells in SCID mice. We established transgenic mice producing human granulocyte-macrophage colony stimulating factor (hGM-CSF) with the homozygote of the scid gene. Endogenous serum hGM-CSF levels were detected by ELISA [mean 9585 pg/ml (line A, n = 4); mean 1610 pg/ml (line B, n = 4)]. Expression of hGM-CSF was observed in all organs tested including the heart, lung, liver, kidney, spleen, thymus, bone marrow and brain of hGM-CSF transgenic (hGMTg) mice. Morphological analysis of organs and peripheral blood cell counts showed no differences between hGMTg mice and their littermates. Murine Ba/F3 cells expressing functional hGM-CSF alpha beta receptor (BAF/alpha beta cells) could be successfully engrafted in hGMTg SCID mice. The cells invaded multiple organs and caused death within a few weeks of transplantation, although they infiltrated only the spleen of their littermates. These results showed that these hGM-CSF-producing SCID mice are useful as an in vivo assay system for investigating leukaemogenesis.