The limitations of studies of clarification of response elements of whey acidic protein (WAP) gene to hormones using mammary cell lines has been shown. We studied the response of the upstream region (2.6 kb) of WAP to various steroid hormones using gonadectomized mWAP/hGH transgenic mice. Ovariectomy or castration for transgenic mice was performed at 10 days or 30 days post partum. Various steroid hormones were administered daily for 10 days to the gonadectomized transgenic mice after they reached 2 months of age. Prior to the hormonal administration and 24 hr after the final administration, blood was collected and the hGH levels in the plasma was measured by RIA. Daily doses of estradiol-17β were significantly more effective at increasing hGH levels in transgenic females ovariectomized at 10 days post partum than progesterone of an equal dose. A combined dose of progesterone and of estradiol-17β significantly amplified the increase of hGH levels accompanied by the great development of mammary glands, compared to a dose of progesterone alone. Corticosterone induced only a slight increase of hGH, while testosterone had no effect. The doses of gonadal steroid hormones did not induce an increase in hGH levels and development of mammary glands in the castrated transgenic males. The results showed that the response of 5' region of WAP requires at least some extended development of the mammary gland and that the 2.6 kb upstream region of the exogenous WAP gene contained the element responsive to ovarian hormones.

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The changes in levels of peripheral major lymphocyte subsets were monitored with 10 adult cynomolgus monkeys (5 females and 5 males) during the 9 weeks after immunization with chick type-II collagen in Freund's complete adjuvant. Three females and 3 males developed overt arthritis determined by swelling of small joints and increase of plasma alkaline phosphatase as well as C-reactive protein. An increase of CD16⁺ NK cells was observed in four non-arthritis-developed monkeys (two females and two males). There was no significant difference in the fluctuation pattern of CD4⁺ T cell, CD8⁺ T cell and CD20⁺ B cell levels between arthritis-developed monkeys and non-developed ones. In addition, the percentages of CD45RA ⁺ CD4⁺ T cells to total CD4⁺ T cells, CD28^- CD8⁺ T cells to total CD8⁺ T cells, and IgD^- B cells to total B cells did not significantly differ between them. On the other hand, a significant increase was demonstrated in CD14-positive cells at 3 weeks after immunization in only arthritis-developed monkeys regardless of sex. The expression of CD14 antigen on the surface of increased cells was low in comparison with those appearing in blood obtained before immunization. In addition, increased CD14^low cells showed no response to LPS stimulation. However, there was no significant difference in antibody titer to both chick type-II and monkey type-II collagen between arthritis-developed monkeys and non-developed ones. These results suggest that an increase in number of CD14^low monocytes with immature function might be a part of the autoimmune response, and that the appearance of these cells is of pathogenic importance in the arthritic process in cynomolgus monkeys regardless of the production of autoantibody.

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In the present study, we investigated the characteristics of the postrest contraction (PRC) in chronic diabetic ventricular muscle. We used WBN/Kob rats of 7-8 weeks as the spontaneously diabetic animal and Wistar rats of 7-8 weeks as the control. We found: (1) No significant differences were seen in the amplitude, the contracting speed, and the relaxing speed of electrically stimulated twitch tension between control and WBN/Kob rats. In addition, the relationship between amplitude of twitch tension and stimulus cycle lengths (0.2-5 sec) was very similar in both animals. (2) The ratios of the first twitch tension (T₁) of PRC with various rest intervals (5-600 sec) to the steady-state tension (T_ss) were significantly smaller in the diabetic rats than in the controls. (3) When the preparation was stimulated at shorter cycle lengths, the recovery process of PRC was separated into at least two components (fast and slow components). In the diabetic rats, the time constant (τ) of both components was significantly longer than in controls. (4) After caffeine (10^-3 M) treatment, τ of the fast component in the control rats became longer, whereas it remained unchanged in diabetic rats. These findings suggest a dysfunction of the intracellular calcium handling system in spontaneously diabetic heart that is likely to include impaired calcium sequestration and/or extrusion.

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Transgenic mice hemizygously carrying human c-Ha-ras proto-oncogene, Tg-rasH2 show very sensitive and facilitated carcinogenicity to various carcinogens. In this study, activities of certain enzymes related to drug metabolism and energy metabolism were measured in microsome and cytosol fractions of livers of Tg-rasH2 mice and their wild type littermates with both sexes treated with 3-methylcholanthrene (MC) and phenobarbital (PB). Aminopyrine N-demethylase activities increased significantly in livers of all mice treated with PB. MC and PB treatments induced significant increases in activities of UDP-glucuronosyltransferase and S-adenosyl homocysteinase compared to those in the non-treated groups in microsome fractions from all mice. In cytosol fractions of livers of all mice, glutathione S-transferase activity was significantly induced in the PB treated groups. There were no significant differences in activities of lactate dehydrogenase, glucose 6-phosphate dehydrogenase, pyruvate kinase and glucose 6-phosphatase related to energy metabolism in livers and kidneys among all mice. Tg-rasH2 mice showed stable activities of enzymes related to drug detoxication and energy metabolism similar to those of non-transgenic mice. These results suggest that the human c-Ha-ras transgene may not affect drug metabolism-related enzymes, and the facilitated carcinogenic response in the Tg-rasH2 mouse is not due to these enzymatic disorders.

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A congenic C57BL/6JJcl-Tcrb^tm1MomTrp53^tm1 (Tcrb ^-/-: Trp53 ^-/-) mouse lacking T-cell receptor beta chain (TCRβ) and transformation related protein 53 (p53) has been established at the N8th generation of backcrossing male Tcrb ^-/- :Trp53 ^-/- mice, which had been obtained by mating a Tcrb ^-/- mouse with a Trp53 ^-/- mouse, with female C57BL/6JJcl mice. In the mice deficient for the both genes, occurrence of tumor masses was observed mostly in the cecum with high frequency as examined at 3 months of age. The majority of the masses had histologic features of hyperplasia or dysplasia while occasional lesions were noted to be adenocarcinomas invading the submucosa (invasive adenocarcinoma). As examined at 4 months of age and thereafter, all mice had 4-5 colorectal tumors per animal, the lesions being located mainly in the cecum and, histopathologically, all the obvious neoplastic growths in the regions examined were invasive adenocarcinomas. The Tcrb and Trp53 genes deficient mouse strain which develops spontaneous colorectal carcinoma with fairly high frequency at early age would be useful as an animal model for colorectal cancer.

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The expression balance of M₂ and M₃ muscarinic receptor subtypes on the pathogenesis of airway hyperresponsiveness was investigated by using two congenitally related strains of guinea pigs, bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR). CCh-induced airway responses in vivo and in vitro were investigated by comparing the effects of muscarinic receptor subtype antagonists, and the relative amounts of M₂ and M₃ muscarinic receptor mRNA in tracheal smooth muscle and lung tissue were investigated. After treatment with muscarinic receptor subtype antagonists, the ventilatory mechanics (V_T, R_aw, and C_dyn) of response to CCh aerosol inhalation were measured by the bodyplethysmograph method. The effects of these antagonists on CCh-induced tracheal smooth muscle contraction were also investigated. The effects of M₂ muscarinic receptor blockade were less but the effects of M₃ muscarinic receptors blockade on the airway contractile responses were greater in BHS than in BHR. In M₃ muscarinic receptor blockades, CCh-induced tracheal contractions in BHS were significantly greater than those in BHR. In tracheal smooth muscle from BHS, the relative amount of M₂ muscarinic receptors mRNA was less but that of M₃ muscarinic receptor mRNA was more than those in BHR. These results suggest that the high ACh level as a consequence of dysfunction of M₂ muscarinic autoreceptors and the excessive effect of M₃ muscarinic receptors on the airway smooth muscle may play an important role in the pathogenesis of airway hyperresponsiveness.

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We recently demonstrated sexual dimorphism in the S3 segment of the ICR mouse kidney, as differences in periodic acid Schiff (PAS) staining on the brush border and the number and size of PAS-positive granules. However, whether these sex dependent features in the S3 segment of the mouse kidney occur only in the ICR strain or are a general feature also observed in other strains is unclear. In the present study, we examined the renal S3 segment of the ICR, BALB/c, C57BL/6, C3H/He and DBA/2 mice strains, which are commonly used in laboratory experiments. PAS staining of the brush border in females of all strains was more intense than that of males, and PAS-positive granules were detected in all females. In male groups, PAS-positive granules were detected in the DBA/2 strain only, but their number was very few. In addition, PAS-positive giant bodies, larger than the nuclear size, were detected in females except those of the C57BL/6 strain. Histometrical investigation demonstrated apparent strain differences in a number of PAS-positive granules and PAS-positive giant bodies. The ultrastructural and cytochemical investigations suggest that the PAS-positive granules and PAS-positive giant bodies were multilamellar lysosomes. We propose that the present findings are significant for comparative morphology in laboratory animal science.

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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.

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The coronary artery of the black Bengal goat was studied by light microscopy. The wall of the coronary artery consisted of the tunica intima, tunica media and tunica externa. The tunica intima consisted of a single layer of flattened endothelium. The tunica media was well-developed and composed of mainly of smooth muscle cells together with some fine elastic fibers. The tunica externa consisted of predominant collagen fibers, and some elastic fibers and smooth muscle cells. Elastic fibers in the tunica externa formed a circular arrangement around the tunica media. Sex differences were not observed. The media with well-developed smooth muscle cells may be responsible for changes in functional physiological conditions of the heart.

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Senile plaques (SPs) were found in the cerebral cortex of a 44-year-old Western lowland gorilla (Gorilla gorilla gorilla). All the SPs were obtained as dense assemblies consisting of fibrous materials by silver impregnation, but were not detected by Congo red. More SPs were detected by immunostaining for amyloidb protein (Aβ) and a half of Aβ-positive-SPs were also immunoreactive for apolipoprotein E. Moreover, all SPs were immunoreactive for Aβ 42 and Aβ 43, but not for Aβ 40. SPs also did not contain Aβ precursor protein-positive structures. These findings suggested that SPs in this case were diffuse plaques. To our knowledge, this is the first report of SPs in the gorilla.

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In the present study, 2 experiments were carried out. In experiment 1, mouse spermatozoa were frozen and stored in an ultra-low temperature freezer maintained at -79°C, from 1 week to 8 months. In vitro fertilization rates of the frozen-thawed sperm after 1 week and 4 months of storage were high at 71 and 71%, respectively. These values did not differ significantly from the value (73%) of the control stored at -196°C. In contrast, the 8-month storage rate was significantly lower at 51%. In experiment 2, frozen spermatozoa were transported in a Styrofoam box packed in dry ice from Hokkaido to Tokyo. In vitro fertilization rate of frozen-thawed sperm after transport at -79°C was high at 88%, which was not significantly different from that (84%) of the transported control at -190°C. After transferring two-cell embryos derived from frozen spermatozoa to recipients, 37-62% of the embryos developed into offspring in both experiments. These results indicate that mouse spermatozoa can survive cryopreservation in an ultra-low temperature freezer (-79 °C) for up to 4 months and transport at -79°C.

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The purpose of this study was to evaluate the most appropriate rat age for the start of administration, and the effect of weaning, in the immature rat uterotrophic assay using ethynylestradiol (EE). Animals weaned on postnatal day (PND) 20 were administered subcutaneously EE at doses of 0.06-6 μg/kg/day for 3 days beginning on PND 21, 23 or 25. EE at the same doses was also administered to rats weaned on PND 17 or 20 from PND 21 for 3 days. Although uterine weight was significantly increased in the rats given 0.6-6 μg/kg EE in both of the studies, the percentage increase relative to the control in each group given EE from PND 21 and weaned on PND 20 was higher than in those groups given EE from PND 23 or 25, and the group weaned on PND 17.

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To clarify the effect of acetylcholinesterase (AChE) on the pathogenesis of airway hyperresponsiveness, AChE activities in tracheal smooth muscle and lung tissue from congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs were compared. For this purpose, AChE activities were determined by measuring the rate of absorbance of tisssue homogenate. Relative amounts of AChE mRNA were also evaluated by the RT-PCR method. In both tracheal smooth muscle and lung tissue from BHS, the AChE activity and the relative amount of AChE mRNA were less than those in BHR. These results suggest that the reduced AChE activity is at least a candidate for inducing airway hyperresponsiveness.

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