Purification of swine carbonic anhydrase isoenzyme III and measurement of its levels in tissues and plasma.

The changes in the levels of carbonic anhydrase isozyme III (CA-III) in swine plasma and urine have not been previously determined or reported. CA-III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA-III from swine muscle tissues and determined CA-III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA-III in the tissues of female swine (age, 3 months) and plasma of young swine (age, 1-5 months) and adult female pigs (age, 2-3 years) were determined using the ELISA system for swine CA-III. The mean (± SD) levels of CA-III in the skeletal muscles were 3.8 ± 3.2 mg/g (wet tissue), and in the plasma, 230 ± 193 ng/ml at 1 month, 189 ± 208 ng/ml at 2 months, 141 ± 148 ng/ml at 3 months, 78 ± 142 ng/ml at 4 months and 53 ± 99 ng/ml at 5 months. The mean level of CA-III in the plasma samples from 2- to 3-year-old pigs was 18 ± 60 ng/ml. CA-III in the plasma samples was found to decrease from 1 month until 3 years of age (p < 0.01). We performed far-western blotting to clarify the cause of the observed decrease in CA-III in plasma. Our results demonstrated that CA-III is bound to the transferrin and albumin. In addition, we determined that the levels of CA-III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.

The renal structure in an Asian elephant (Elephas maximus).

The renal structure of a female Asian elephant (Elephas maximus) was observed in both macroscopic and light microscopic levels. The left kidney was elongated-ellipse in shape, whereas the right appeared round. The left kidney was 31 cm in cranio-caudal length, 21 cm in medio-lateral length, and 2950 g in weight. The right kidney was 34 cm in cranio-caudal length, 22 cm in medio-lateral length, and 3250 g in weight. The external appearance showed the six separated renal lobes in both sides of the kidney. The four pairs of the lobes were fused in the deepest region in both sides of kidney, so we considered it as an incompletely lobated kidney in this species. We observed the proximal and distal urinary tubules in histological sections. Many renal corpuscles consisted of the glomerulus and Bowman's capsule. Many mesangial cells and some podocytes were confirmed in each glomerulus; however, Bowman's capsules were larger than those in other mammalian species.

Comparative functional morphology of the masticatory apparatus in the long-snouted crocodiles.

The masticatory muscles and their related structures of the skull were observed in the Indian gavial (Gavialis gangeticus), the false gavial (Tomistoma schlegelii), and the African slender-snouted crocodile (Mecistops cataphractus) to detail some morphological differences in comparison with the other crocodile species, and to compare and elucidate the functional strategy of themasticatory apparatus in these long-snouted species. The Musculus pterygoideus posterior was relatively smaller in the three species compared with many short-snouted crocodiles. It suggests that the masticatory power in fish-eating long-snouted species is not so high as in the short-snouted crocodiles, while the masticatory muscles were morphologically different among the three long-snouted species as follows. The M. pterygoideus posterior of the false gavial was extended in the lateral side of the lower jaw unlike the Indian gavial. The M. pseudotemporalis and the Fenestra supratemporalis were largely developed in the Indian gavial, however we suggest that the other two species possess the weak bundles in this muscle. The false gavial and the African slender-snouted crocodile have the pterygoid bone well-developed extending dorso-ventrally and it is suggested that the M. adductor mandibulae posterior attached to the pterygoid bone may be much larger than the Indian gavial. These data morphologically clarify the masticatory mechanism in the long-snouted crocodiles different from the short-snouted species, and demonstrate that the evolutional strategy to share the functional role in the masticatory muscles have been differently established between the Indian gavial and the other two species. We also obtained the morphological data in the fossil skull of the Machikane crocodile (Toyotamaphymeia machikanense) and concluded from the fossil characters that the considerable developments of the M.pterygoideus posterior and the M.pseudotemporalis in this species had not morphologically been consistent with both the Indian and false gavials.

The structure of the masseter muscle in the giraffe (Giraffa camelopardalis).

In the giraffe (Giraffa camelopardalis), the masseter muscle was divided into several layers. The superficial and more medial (second) tendinous sheets of the masseter muscle fused with each other at the dorso-caudal part and a fleshy portion was located between these tendinous sheets. In the rostral part, the most superficial tendinous sheet turned around as a compact tendon and attached to the facial crest (Crista facialis). The turned tendinous sheet, however, never fused with the second tendinous sheet and this layer of the masseter muscle, that originated from the facial crest with the turned sheet, was inserted into the mandible with its fleshy portion. In the cattle, goat, sheep and Sika deer, the rostral layer of the masseter muscle arises from the facial crest with its fleshy portion and is inserted into the tubercle on the mandible through the strong tendinous sheet. In this study, the takin also showed the same structure of the masseter muscle. In the giraffe, however, the rostral layer inserted into the mandible through the strong tendinous sheet could not be distinguished, thus, there was no conspicuous tubercle on the mandible. Moreover in the masseteric region of the skull.,the giraffe was similar to the Sika deer in several ways. However, it is suggested that the giraffe exerts smaller forces on the cheek teeth than does the Sika deer because of its longer Margo interalveolaris.

Radial sesamoid bone as a part of the manipulation system in the lesser panda (Ailurus fulgens).

The well-developed radial sesamoid bone presented a rod-like shape in the lesser panda. It could be separated into two components: (1) an ulnar cartilaginous, (2) a radial osseous part. The radial sesamoid bone was connected with four elements as follows: (1) the tendon of the M. abductor pollicis longus, (2) M. abductor pollicis brevis and M. opponens pollicis, (3) Aponeurosis palmaris, and (4) Flexor retinaculum. The bone made no articulation with the first metacarpal. The movement of the radial sesamoid bone may be controlled by the connecting muscles and muscle-related structures. It is suggested that the bone acts as a supporting ridge in the gripping action in the lesser panda. However, we suggest that the grasping mechanism is obviously different from that of the giant panda, in which the radial sesamoid bone is connected strongly with the first metacarpal.

Musculoskeletal system of the neck of the polar bear (Ursus maritimus) and the Malayan bear (Helarctos malayanus).

The gross anatomical study was undertaken in the musculoskeletal system of the neck of the polar bear, and the findings were compared with those of the Malayan bear. The Musculus splenius and the M. trapezius were well-developed in the polar bear. The long neck of the polar bear consisted mainly of the M. splenius with the M. biventer cervicis and the M. complexus lying tightly underneath. The cervical vertebrae possessed huge ventral tubercle in the ventral part of the transverse process in the polar bear. These morphological characteristics suggest that the polar bear may rotate and bend the skull and the long cervical vertebrae. We postulate that the polar bear has evolved the high-mobility long neck to adapt for swimming. Unlike the polar bear, the Malayan bear has not specialized in the neck structure.

Role of nitric oxide in regulating the ductus arteriosus caliber in fetal rats.

The role of nitric oxide (NO) on the ductus arteriosus (DA) patency was examined in fetal rats at various stages of gestation. N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg, i.p.), an NO synthase (NOS) inhibitor, or indomethacin (3 mg/kg, p.o.), a cyclooxygenase inhibitor, was administered at 3 hr before cesarean section to pregnant rats ranging from day 17 to day 21 of gestation. Dams were decapitated and the fetuses were obtained by cesarean section. The fetuses were rapidly frozen in an acetone-dry ice mixture. Using rapid-freezing and shaving methods, the calibers of the DA and pulmonary artery were measured. The constrictive effect of L-NAME on the fetal DA caliber was stronger than that of indomethacin in 19-day-old and immature fetuses. In near-term fetuses, the constrictive effects of L-NAME were reduced, while indomethacin caused marked DA constriction. We conclude that endogenous NO may play a major role in regulating the patency of the DA in earlier fetal stages, while dilator prostaglandins may play a greater role in regulating the ductal patency in the near-term fetus.

Effect of follicle-stimulating hormone on sertoli cell division in cultures of fetal rat testes.

The present study was performed to determine when follicle-stimulating hormone (FSH) begins to promote Sertoli cell division in fetal rats, and to determine whether the effect of FSH is mediated by cAMP-dependent protein kinase (PKA). When testes from 15- to 17-day fetuses were cultured with or without FSH for 48 h, FSH did not promote Sertoli cell division in 15-day testes, but did in 16- and 17-day testes. Anti-rat FSH was injected into 16-day fetuses in utero. Twenty-four hours later, the testes of the injected fetuses and those of their intact littermates were cultured with or without FSH for 48 h. Without FSH, the Sertoli cell division index was significantly lower in anti-FSH-treated fetuses than in intact fetuses. With FSH, however, the index increased. When PKA inhibitor was added to cultures of 16-day testes with FSH, the promotion of Sertoli cell division by FSH was inhibited. We conclude that between 16 and 17 days of gestation, fetal pituitary FSH stimulates the division of Sertoli cells by activating the PKA activity.

Effects of TAK-044, a nonselective endothelin receptor antagonist, on the spontaneous and indomethacin- or methylene blue-induced constriction of the ductus arteriosus in rats.

We studied the effects of TAK-044, a nonselective endothelin (ET) receptor antagonist, on the indomethacin- or methylene blue-induced constriction of the ductus arteriosus (DA) in rats and compared them with the effects on spontaneous DA constriction. Injection of TAK-044 into 21-day-old fetuses in utero was performed through the uterine wall of laparotomized mother rats under light ether anesthesia. The fetuses were autopsied 3 hr after treatment with TAK-044 (10 mg/kg) in utero and simultaneous administration to the laparotomized mother rats of indomethacin (3 mg/kg, p.o.) or methylene blue (100 mg/kg, i.p.). In the second experiment, pregnant rats were decapitated on day 21 of gestation to obtain newborn rats by cesarean delivery. Newborn rats which were given TAK-044 (2, 10 mg/kg) immediately after or 1 hr before cesarean delivery were autopsied at various times after birth. In both experiments, pups were rapidly frozen in an acetone-dry ice mixture at autopsy to evaluate the DA constriction by the whole-body freezing and shaving method. TAK-044 injection into the fetus 3 hr before autopsy completely inhibited the DA constriction induced by maternal treatment with indomethacin or methylene blue. TAK-044 caused dose-dependent inhibition of the spontaneous closure of the DA after birth. The inhibitory effect was more pronounced in pups which were given TAK-044 in utero 1 hr before birth; however, the inhibitory effect was incomplete in newborn pups. These results, together with the previous finding that BQ-123, an ETA-specific receptor antagonist, inhibits the ductal constriction induced by oxygen in vitro [Coceani et al., 1992], indicate that the ETA receptor plays a significant role in the indomethacin- or methylene blue-induced DA constriction as well as in the spontaneous DA constriction after birth, and also indicate that the inhibition of ETA receptor by TAK-044 was more easily achieved in fetuses than in neonates.

Adaptation of the muscles of mastication to the flat skull feature in the polar bear (Ursus maritimus).

The muscles of mastication of the polar bear (Ursus maritimus) and those of the brown bear (U. arctos) were examined by anatomical approach. In addition, the examination of the skull was carried out in the polar bear, brown bear and giant panda (Ailuropoda melanoleuca). In the polar bear, the rostro-ventral part of the superficial layer of the M. masseter possessed the abundant fleshy portion folded in the rostral and lateral directions like an accordion. Moreover, the rostro-medial area of the superficial layer became hollow in the nuchal direction when the mouth was closed. The M. temporalis of the polar bear covered up the anterior border of the coronoid process of the mandible and occupied the almost entire area of the cranial surface. The M. pterygoideus medialis of the polar bear was inserted on the ventral border of the mandible and on the ventral part of the temporal bone more widely than that of the brown bear. As results of our measurements of the mandible, an effect of the leverage in the polar bear was the smallest in three species. In the polar bear, the skull was flat, and the space between zygomatic arch and ventral border of the mandible, occupied by the M. masseter was the narrowest. It is suggested that the muscles of mastication of the polar bear is adapted to the flat skull feature for supplementing the functions.

Smooth muscle cell proliferation in the ductus arteriosus and the descending aorta, and effects of enalapril on SMC proliferation in perinatal rats.

This study was carried out to determine the proliferation profile of the smooth muscle cells (SMC) in the media of the ductus arteriosus (DA) and the descending aorta (Ao), and to examine the effects of the angiotensin-converting enzyme inhibitor enalapril on the proliferation of these cells in perinatal rats. The proliferating cell nuclear antigen (PCNA) index of the DA peaked in 19-day-old fetuses at 75%, and the index significantly declined in 20-day-old fetuses. The PCNA index of the Ao showed a similar profile until pups reached 1 day of age; however, the index of the Ao then increased in 3-day-old pups. The PCNA indices of the DA and Ao decreased significantly after maternal oral treatment with enalapril (10 mg/kg for 7 days), with a more marked decline in the DA than in the Ao. The PCNA indices of these vessels in 20-day-old fetuses were not altered by maternal treatment with enalapril. These results indicate that the SMC proliferation rate in the DA was similar to that in the Ao until pups reached the age of 1 day, and that the inhibitory effect of enalapril on the SMC proliferation was age-dependent and more prominent in the DA than in the Ao.

MRI examination of trachea and bronchi in the Ganges River dolphin (Platanista gangetica).

The MRI examination was carried out in a formalin-fixed specimen of the Ganges River dolphin (Platanista gangetica), one of the evolutionary primitive species of cetaceans. We could morphologically elucidate the tracheobronchial ramification in the intact whole body. We demonstrated from the MRI sections that the characteristic tracheal bronchus branches from the trachea at the cranial portion. These findings suggest the phylogenetic relationships between cetaceans and artiodactyls. The left bronchus is obviously larger in diameter than the right one. We suggest that the right bronchus has smaller capacity of gas exchange than the left one, because the dolphin possesses the tracheal bronchus in the right lung. The MRI method will be important in the non-invasive study of the anatomy in endangered animal carcass as Ganges River dolphin.

Comparative anatomy of the radial sesamoid bone in the polar bear (Ursus maritimus), the brown bear (Ursus arctos) and the giant panda (Ailuropoda melanoleuca).

Since we have clarified the manipulation mechanism using the radial sesamoid (RS) in the giant panda (Ailuropoda melanoleuca), our aim in this study is to examine the position, shape and function of the RS morphologically, and to observe the attachment to the RS of the M. abductor pollicis longus and the M. opponens pollicis in the other Ursidae species. So, we focused on the carpus and manus of the polar bear (Ursus maritimus) and the brown bear (Ursus arctos) in this study. The RS was tightly articulated to the radial carpal, and could not adduct-abduct independently of the radial carpal. The M. abductor pollicis longus tendon and the M. opponens pollicis belly were attached to the RS, independently. In the polar bear, the deep concave and the flat surface were confirmed in attachment area for these two muscles. The morphological relationship between the RS and the M. abductor pollicis longus and the M. opponens pollicis in the two species of bears were essentially consistent with that in the giant panda. It also demonstrated that the manipulation mechanism of the giant panda has been completely based on the functional relationship between the small RS, and the M. abductor pollicis longus and the M. opponens pollicis in Ursidae species.

Morphological character of the shoulder and leg skeleton in Przewalski's horse (Equus przewalskii).

The skeletal feature was observed in Przewalski's horse (Equus przewalskii) and the domestic horse (E. caballus). As results of the observations, remarkable differences were found in the scapula. The caudal border of the scapula in Przewalski's horse is curved in the caudal direction more than that of the domestic horse. Moreover, in the domestic horse, the sharp caudal border is formed by the shift of the outer muscular line (Linea muscularis) to the border. In Przewalski's horse, however, the caudal border is rounded because the outer muscular line does not shift to the border but lies beside it. We suggest that the morphological difference observed between the scapulae of these horses may lead to changes in the shoulder movement with the various attachments of the long head of the triceps muscle (Caput longum musculi tricipitis). The skeletal measurement and the measurement ratio show that the leg skeleton of Przewalski's horse is shorter and thicker than that of the domestic horse. These results show that the center of gravity of Przewalski's horse is low in position and that it has a short stride.

Enhancement of neonatal rat ductal responsiveness to prostaglandin E2 after maternal treatment with enalapril or captopril.

This work was conducted to determine whether the angiotensin-converting enzyme inhibitors (ACEIs) (enalapril and captopril) administered to mother rats prenatally can potentiate a re-opening of the neonatal ductus arteriosus (DA) induced by prostaglandin E2 (PGE2) after postnatal closure. A subcutaneous injection of PGE2 (4 micrograms) was administered to newborn rats 3 hr after a Cesarean delivery from females which had been orally given 0.1, 1 or 10 mg/kg/day of enalapril or 15 or 150 mg/kg/day of captopril from day 14 to day 20 of gestation. The ratio of the DA to the pulmonary artery (PA) was determined at intervals after the injection. The DA/PA ratio was significantly higher in the newborn rats of mothers who were transplacentally administered these agents compared to the controls, except at the low dose (0.1 mg/kg) group of enalapril. We found that the level in the neonatal lungs of 15-hydroxy prostaglandin dehydrogenase, a key enzyme that catalyzes PGE2 to convert it to its inactive metabolite 15-keto-PGE2, was not affected after maternal treatment with enalapril or captopril. These results indicate that the increased ductal responsiveness to PGE2 in newborn rats was a common response after maternal ACEI treatment, but the catabolism of PGE2 in the lungs did not contribute to this response.

Effect of combined fenthion and cimetidine use in rats on lethality, blood cholinesterase activities, and serum cholinesterase isoenzymes.

H2-receptor antagonists inhibit cholinesterase (ChE) activity. We examined perturbations in ChE isoenzyme patterns and ChE activities of rats from the combined effects of fenthion (FEN) and cimetidine (CIM). Sixty-four female Sprague-Dawley rats were divided into 8 groups. Four rat groups were given FEN or gum arabic solution and each group divided into 2 small groups according to the CIM or gum arabic administration. FEN was administered po at 12.3 mg/kg (1/20 LD50) or 24.5 mg/kg (1/10 LD50) for 14 days or 49 mg/kg (1/5 LD50) every 4 days. CIM was given po at 1,500 mg/kg from days 7 to 13. Samples were collected 3 h after CIM administration on days 8 and 13. CIM did not influence ChE isoenzyme patterns or ChE activity. FEN inhibited both the ChE isoenzyme patterns and ChE activities without producing clinical signs. Although 1 rat in the 12.5 mg FEN/kg + CIM group died on day 10, all rats in other FEN (24.5 mg/kg or 49 mg/kg) + CIM groups died on days 8-10. Differences in suppression of ChE isoenzyme patterns were detectable between the FEN-dosed and FEN + CIM-dosed groups. There were no differences in ChE activities between the FEN-dosed and FEN + CIM-dosed groups. The i.p. administration of 500 mg CIM/kg (LD50) did not suppress ChE activities.

Increased ductal responsiveness to PGE2 after maternal treatment with aspirin and ibuprofen.

This work was conducted to determine whether aspirin and ibuprofen, when administered prenatally may potentiate a reopening of the neonatal ductus arteriosus (DA) induced by PGE2 after postnatal closure. In the first experiment, a subcutaneous injection of PGE2 (4 microgram(s)) was administered to newborn rats 3 hr after a Cesarean delivery from pregnant females which had been orally given 100 or 300 mg/kg/day of aspirin and 10 or 30 mg/kg/day of ibuprofen on days 18, 19 and 20 of gestation. The ratio of the DA to the pulmonary artery (PA) was determined at intervals after the injection. The DA/PA ratio was significantly higher in newborn rats from mothers who were transplacentally administered these agents than the control. We also examined the hypothesis that maternal treatment with nonsteroidal anti-inflammatory drugs (NSAID), such as aspirin and ibuprofen, inhibits the catabolism of PGE2 and that the increased reopening of the DA was partly due to this inhibition. 15-hydroxy prostaglandin dehydrogenase (15-PGDH) in neonatal lungs, the key enzyme involved in catalyzing PGE2 to convert it to its inactive metabolite 15-keto-PGE2, was not affected by maternal treatment with aspirin and ibuprofen. These results suggest that the increased ductal responsiveness to PGE2 in newborn rats was a common response after maternal NSAID treatment, but the catabolism of PGE2 in the lungs did not always contribute to this response.

Effects of follicle-stimulating hormone on intermediate filaments and cell division of Sertoli cells of fetal rat testis in culture.

The present study was aimed to examine the effects of follicle-stimulating hormone (FSH) on cell division of Sertoli cells from rat fetal testes and on the kinetics of 2 kinds of intermediate filaments, cytokeratin and vimentin, which comprise the cytoskeleton of Sertoli cells. Testes from rat fetuses of different ages (from day 15 to day 17 of gestation ) were cultured for 48 hr, with or without added FSH. In 15-day testes, FSH influenced neither cell division of Sertoli cells nor kinetics of intermediate filaments. In 16-day testes, FSH promoted cell division of Sertoli cells and kinetic differentiation of intermediate filaments distributed toward the lumen of the seminiferous tubules. These findings suggest that 16-day testes in culture can respond to FSH in a fashion that cell division of Sertoli cells is promoted and that intermediate filaments increase in number and change in intracellular distribution. It is concluded that FSH influences both proliferation and morphological differentiation of Sertoli cells.

Maternal adrenocortical hormones maintain the early development of pancreatic B cells in the fetal rat.

To investigate the effect of maternal adrenocortical hormones on the development of fetal pancreatic islet cells, pregnant rats were adrenalectomised on d 6 of gestation. On d 12-16 the growth patterns of fetal insulin-producing B cells, glucagon-producing A cells, and somatostatin-producing D cells were observed histometrically. Maternal adrenalectomy resulted in growth retardation of fetal B cells on d 12-15. Maternal corticosterone therapy prevented this retardation. Maternal adrenalectomy, however, did not affect the developmental patterns of A and D cells. By Western blotting and immunohistochemistry, glucocorticoid receptors were demonstrated to be present in the islet cells from d 12 to d 15. These results suggest that maternal adrenocortical hormones, glucocorticoids in particular, maintain the early development of fetal pancreatic B cells through their specific intracellular glucocorticoid receptor.

Immunocytochemical changes in the fetal pancreatic islet following fetal administration of streptozotocin in the rat.

BACKGROUND: Streptozotocin (STZ) is selectively toxic to the B cells in the pancreatic islets. It is well known that in the adult rat, STZ causes the death of B cells, and it eventually induces diabetes mellitus. The present study was conducted to detect what morphological changes could be induced in the fetal B cells following a direct injection of STZ into the fetus in utero during late pregnancy in the rat. METHODS: STZ (400 micrograms/g body weight) was injected into the fetus in utero at 10:00 on day 19 of gestation. Three, 6, 24, and 48 hr after injection, the changes in the B cells (anti-insulin serum positive cells) were examined immunohistochemically. The total volume of the B cells was measured. Electron microscopic observation was made as well. RESULTS: Six hr after STZ injection, some B cells were destroyed so that their granules were distorted and burst. Twenty-four hr after STZ injection, a large majority of the existing B cells were disintegrated, and a number of small isletlike clusters of immature cells appeared among the exocrine acini. The total volume of anti-insulin serum positive cells was strikingly decreased. At 48 hr after injection, however, the volume returned to a level that was comparable to that of their littermate controls. CONCLUSIONS: The regeneration of the B cells may occur because of the high cell proliferative activity of undifferentiated cells following the destruction of the B cells caused by an injection of STZ.