Production of immunoreactive 2',5'-oligoadenylate synthetase in p48-deficient mice.

2',5'-Oligoadenylate synthetase (2'5'OAS), an enzyme induced by interferon (IFN), is physiologically produced in IFN-untreated normal healthy mice. The enzyme is localized mainly in the epithelium of the digestive tract, reproductive organs, and the choroid plexus in the brain. 2'5'OAS is also detected in oocytes in the ovary and in neurons and glial cells of both the telencephalon and cerebellum. Here, we examined the role of p48 (ISGF3gamma), a component of IFN-stimulated gene factor 3 (ISGF3), in the physiologic production of 2'5'OAS using p48-deficient mice generated by gene targeting. In the p48-deficient mice, the physiologic production of 2'5'OAS localized in the following cells was severely impaired: hepatocytes, Kupffer cells, splenocytes, epithelium of the large intestine, oviduct, and uterus, and neurons and glial cells in both the telencephalon and cerebellum. The results show that 2'5'OAS in these cells is induced physiologically through a pathway including p48. However, the production of 2'5'OAS in oocytes was not affected in the p48-deficient mice, indicating that oocyte 2'5'OAS is produced through a p48-independent pathway. A possible function of the GAS sequence found in the promoter region of the 2'5'OAS gene to which Stat6 may bind also is discussed.

The target cells of injected type I interferons in mouse liver.

Type I interferons (IFNs) have been used for the treatment of viral hepatitis, but it is unclear which cells in the liver are affected by injected IFN. The effects of IFN have been studied by the production of 2',5'-oligoadenylate synthetase (2'5'OAS), an IFN-inducible enzyme. Here, we studied the distribution of 2'5'OAS in mouse liver after injection of natural mouse IFN-alpha/beta by Western blotting and immunohistochemistry using a monoclonal antibody specific to mouse 42-kDa 2'5'OAS. Injection of IFN-alpha/beta increased the levels of liver 2'5'OAS and enhanced the intensities of immunohistochemical staining for this enzyme in both hepatocytes and Kupffer cells. In IFN-untreated normal mice, hepatocytes were lightly stained, but some of the Kupffer cells showed rather strong staining. The 2'5'OAS-positive Kupffer cells comprised approximately 60% of those in normal liver, whereas this increased to approximately 90% following IFN-alpha/beta injection. Thus, hepatocytes and Kupffer cells were the targets of injected IFN.

Localization of 2',5'-oligoadenylate synthetase and the enhancement of its activity with recombinant interferon-alpha A/D in the mouse brain.

Although the expression of 2',5'-oligoadenylate synthetase (2-5AS) is induced by interferon (IFN), low constitutive levels can be detected in animals that have not been treated with IFN. In order to clarify which cells express 2-5AS in the mouse brain, the distribution of this enzyme in the brains of both normal healthy mice and mice treated with recombinant human IFN-alpha A/D was studied by Western blotting and immunohistochemistry, using a specific monoclonal antibody. On the Western blots, the antibody to 42-kD 2-5AS reacted slightly with extracts from the telencephalon, cerebellum, diencephalon, and medulla oblongata of normal mouse brain. 42-kD 2-5AS was predominantly found in the ependymal cells and epithelium of the choroid plexus, and to a lesser degree in neurons and glial cells. Injection of recombinant human IFN-alpha A/D into the left lateral ventricle enhanced the activity of the enzyme in the telencephalon, cerebellum, diencephalon, and medulla oblongata, but did not change the immunohistochemical localization of the enzyme. Direct injection of the IFN into the cortex of the telencephalon enhanced the activity of 2-5AS in all parts of the brain and immunoreactivity was observed in the neurons and glial cells surrounding the injection site. These data indicate that 42-kD 2-5AS activity in the mouse brain is enhanced by the injection of recombinant human IFN-alpha A/D either into the left lateral ventricle or cortex of the telencephalon. Expression of 42-kD 2-5AS in ependymal cells and epithelium of the choroid plexus may prevent viral infections in the brain.

Distribution of immunoreactive 2',5'-oligoadenylate synthetase in mouse reproductive organs.

Although 2', 5'-oligoadenylate synthetase (2-5AS) is an enzyme induced by inferferon (IFN) or viral infections and mediates one of the principal antiviral pathways turned on by IFN, low constitutive levels of the enzyme can be detected in various "normal" animals that have not been treated with IFN or virus. The distribution of this enzyme in the female and male reproductive organs of normal healthy mice was studied by Western blotting and by an immunohistochemical method, using a specific monoclonal antibody. On Western blotting, an antibody to 42-kD 2-5AS reacted with extracts from the ovary, oviduct, uterus, vagina, and placenta among the female reproductive organs, and testis, epididymis, and ductus deferens in the male. Immunohistochemically, the 2-5AS was localized on the following cells in the female reproductive organs: oocytes in the ovary; epithelium in the oviduct, uterus, and vagina; and trophoblasts in the placenta. Furthermore, the 2-5AS was localized on the epithelium and muscular layer in the ductus deferens and epithelium in the penis of the male mice, whereas the epithelium of the testis, epididymis, and seminal vesicle were stained faintly. It is well known that IFN is produced continuously in normal mice, so the 2-5AS in the tissues of normal mice is considered to be induced by such IFN produced under physiological conditions. Expression of the 2-5AS on the epithelium and trophoblasts in the reproductive organs may be responsible for the prevention of viral infections. However, the enzyme in oocytes may have some functions other than as an antiviral agent, since the enzyme was not detectable in embryos during early development.

Distribution of immunoreactive 2',5'-oligoadenylate synthetase in mouse digestive tract.

2',5'-Oligoadenylate synthetase (2-5OAS), an enzyme induced by interferon (IFN), has also been found in various "normal" animals that had not been treated with IFN. The distribution of this enzyme in the digestive tracts of normal healthy mice was studied by western blotting and by an immunohistochemical method using a specific monoclonal antibody. On western blotting, the antibody to 42 kD 2-5OA synthetase reacted with extracts from the stomach and intestines (small and large intestine), but not with extracts from the esophagus. Immunohistochemically, the 42 kD 2-5OAS was localized on the following cells: surface mucous and parietal cells of fundic glands in the stomach; surface epithelial cells in the intestines; and some enteric nervous cells in the esophagus, stomach, and intestines. Treatment with IFN- alpha/beta did not essentially change the distribution of the enzyme in the tissues, although a small amount of the enzyme was detected in the esophagus by western blotting. Expression of the 42 kD 2-5OAS in the digestive tract may be responsible for the prevention of viral infections.

Inhibition of hair growth by subcutaneous injection of a sympathetic neurotoxin, 6-hydroxydopamine in neonatal mice.

The effects of a sympathetic neurotoxin, 6-hydroxydopamine (6-OHDA), on hair growth in neonatal mice were examined. Newborn mice were injected once subcutaneously in the mid-dorsal region with 6-OHDA (0.3 mg/g body weight) and bovine serum albumin (BSA) or with BSA only (controls) on day 0. By day 10, distinct areas of hairless skin were observed surrounding the sites treated with 6-OHDA. The areas of hairless skin were smaller at 15 days of age and were covered with hair by 20 days of age. Control sites injected with BSA only were indistinguishable from the surrounding skin at all ages examined. Microscopic and morphometrical analyses of skin obtained from the neonatal mice at various ages showed that the hairless skin in 6-OHDA-treated mice was thinner than the skin of control animals. The thinning of the skin and delay in hair growth induced by 6-OHDA treatment showed a significant difference from the skin of control animals injected with BSA only. Immunohistochemical experiments demonstrated that the administration of 6-OHDA had caused the complete depletion of tyrosine hydroxylase-immunoreactive fibers (sympathetic fibers) around blood vessels and piloerector muscles and in nerve bundles throughout the dermis and subcutaneous tissue. These findings indicate that the sympathetic neurons are associated with skin thickness and hair growth in neonatal mice.

Culture of marrow stromal cells derived from bone marrow specimens formed at fracture site of human long bone.

To study the process of the internal callus formation, we cultivated marrow stromal cells derived from bone marrow specimens formed at fracture sites of human long bone in alpha-modified Eagle's medium containing 10% fetal calf serum. After 2 weeks of culture, the cells formed two types of colonies; one consisted of spindle cells, and the other comprised of polygonal cells. The two types of colonies were separated and cultured further. The spindle and polygonal cells proliferated to confluence within 3 weeks and after 4 weeks, respectively, after the separation. Both the spindle and polygonal cells showed on the plasma membrane moderate intensity of staining reaction of alkaline phosphatase (ALPase) activity before the period of confluence and strong intensity during the period of confluence. Then, the spindle cells did not produce calcified matrix, but detached from the dish after 6-8 weeks of culture. In the colonies of polygonal cells, however, dense nodules were formed after 9 weeks of culture, which became visible to the naked eye as white aggregates after 11-12 weeks. Electron microscopic studies on the polygonal cells demonstrated matrix vesicles in the intercellular ground substance after 6 weeks of culture, and electron-dense needle-like crystals on the matrix vesicles after 8-10 weeks of culture. On the basis of infrared spectroscopic analysis, the aggregates were composed of hydroxyapatite. Thus, stromal cells derived from bone marrow specimens formed at fracture site of human long bone differentiated to spindle and polygonal cells containing high ALPase activity (a marker for osteogenic capacity) during culture, and the polygonal cells but not spindle cells produced the calcified matrix.

A female sterile mutant, unfertilizable (uf), in Xenopus laevis.

We describe a female sterile mutant, unfertilizable (uf), in Xenopus laevis whose eggs cannot be fertilized by either natural mating or artificial insemination. The uf eggs were able to be activated by pricking with a fine glass needle. Ultraviolet-solubilized jelly from the uf eggs could permit the fertilization of the dejellied wild-type eggs by artificial insemination, while uv-solubilized jelly from the wild-type eggs did not permit the fertilization of the dejellied uf eggs. The uf eggs, whose jelly coats and vitelline envelopes were removed, could be fertilized by insemination in the uv-solubilized jelly from either wild-type or uf eggs. These results show that the eggs and the jelly coats are intact but the vitelline envelopes are abnormal in uf mutant eggs. One- and two-dimensional gel electrophoretic comparisons of vitelline envelopes from wild-type and uf eggs demonstrated that five protein components, having estimated molecular masses of about 26, 64, 69, 78, and 250 kDa, were present in the vitelline envelopes of uf eggs but were absent in those of wild-type eggs. All of these components were polymorphic in terms of isoelectric points. Thus the cause of unfertilizability of uf eggs resides in the vitelline envelopes whose protein components exceed those of the wild-type eggs.

Furrow-related contractions are inhibited but furrow-unrelated contractions are not affected in af mutant eggs of Xenopus laevis.

In embryos from af mutant females of Xenopus laevis, the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions continued (Kubota et al., 1991). To gain insights into the roles of the normal product of af on early development, contractile events which have been observed in the period from fertilization until first cleavage in wild-type eggs were examined in af mutant eggs. Activation waves, activation contraction, and surface contraction waves which were identical to those in wild-type eggs were observed in af eggs by time-lapse video recording. However, second polar body elimination was inhibited in af eggs, although a sign of the polar body formation was indicated by the cytoplasmic bulge of the egg surface as seen by light and electron microscopy. These results indicate that the normal product of af regulates furrow-related contractile events which involve formation of the contractile ring, but exerts no effects on furrow-unrelated contractions in early Xenopus eggs.

Cytological and biochemical analyses of the maternal-effect mutant embryos with abnormal cleavage furrow formation in Xenopus laevis.

We describe an embryonic lethal mutation in Xenopus laevis that provokes regression of cleavage furrow formation. The mutant females (designated as af) were obtained by the back-cross of a female with one of her sons. All the fertilized eggs laid by the mutant females, regardless of the wild-type male used in the mating, failed to cleave although each furrow ran at a proper position superficially. Light and electron microscopic observations of the embryos revealed that the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions did. Two-dimensional gel-electrophoretic comparisons of af and wild-type embryos demonstrated that two proteins, having estimated molecular masses of about 38 kDa (pI 6.6) and 78 kDa (pI 7.6), were missing in af embryos. Microinjection of clear cytoplasm from a wild-type egg into fertilized af eggs provoked partial surface contraction and cleavage furrow formation in recipient af eggs. The results showed that the af females carry a lethal maternal-effect mutation which causes cleavage furrow regression by being deficient in a few proteins, and that cytoplasm of wild-type eggs can partially rescue the cleavage furrow formation of af eggs by furnishing the corrective material, presumably a product of the normal allele of af.

Studies on the expression of intracellular and surface polarity in animal pole cells of Xenopus embryos cultured on various substrata.

The expression of intracellular and surface polarity in animal pole cells of Xenopus embryos (stage 10) cultured on various substrata was studied by electron microscopy. When animal pole cells of Xenopus embryos were cultured on type I collagen- or gelatin-coated dishes until control embryos reached stage 23, the cells in confluent layers expressed an apical-basal polarity so that the apical surface membrane domain faced the culture medium. However, the cells in confluent layers cultured on naked plastic dishes were suppressed to express intracellular and surface polarity. In addition, single attached cells which were formed by being sparsely plated neither spread on any substrata nor displayed the apical-basal polarity perpendicular to the dishes. These results indicate that the expression of intracellular and surface polarity in cultured animal pole cells of Xenopus embryos requires not only cell-cell contact but also an adhesive substrata such as type I collagen or gelatin.

Cellular polarity in cultured animal pole cells of Xenopus embryos.

The expression of intracellular and surface polarity in cultured animal pole cells of Xenopus embryos (stages 6, 8, and 10) was examined morphologically and immunocytochemically. When control embryos reached stage 23, daughter cells derived from a single or a few animal pole cells formed aggregates. Outer cells of the aggregates displayed intracellular and surface polarity and expressed an epidermis-specific antigen (XEP-1) on the apical surface circumference, while these characteristics had not yet been established in the animal pole cells at the time of isolation. However, inner cells of the aggregates did not display the cellular polarity along an outer-inner axis of the aggregates and displayed the antigen randomly within the aggregates. These results indicate that the expression of cellular polarity in epidermal differentiation of Xenopus embryos in vitro depends on the position within the aggregates formed by daughter cells derived from isolated animal pole cells.

A monoclonal antibody specific for an epidermal cell antigen of Xenopus laevis: electron microscopic observations using a gold-labeling method.

A monoclonal antibody (EPI-1), raised against the supernatant of a homogenate of Xenopus laevis larvae at the tailbud stage (stage 36/37), interacts specifically with a 250 KD epidermal antigen of Xenopus. An immunocytochemical gold-labeling technique was used to investigate changes in antigen distribution during epidermal development of Xenopus laevis. Specific immunolabeling was initially detected over the endoplasmic reticulum in the outer epithelial cells of the late gastrula stage (stage 12.5). After the early neurula stage (stage 13), immunolabeling appeared over moderately electron-dense bodies (these bodies disappear after stage 29), and also over the apical cell surface and adjacent cytoplasm of all the outer epithelial cells. During metamorphosis, labeling decreased and disappeared after stage 62, as the superficial layer had peeled off. These data suggest that the antigen is useful as a marker of general differentiation in studies of epidermal development during the embryonic and larval stages of Xenopus laevis.

Selective binding of colloidal gold-protein conjugates to secretory granules of granular skin glands and myelin sheaths in the skin of Xenopus laevis.

Colloidal gold-protein conjugates are widely used in immunoelectron microscopical studies as general "second antibody" markers. This report demonstrates that colloidal gold conjugated with goat anti-mouse immunoglobulin G (IgG), protein A, and bovine serum albumin (BSA) binds selectively to secretory granules of granular skin glands and myelin sheaths in the skin of Xenopus laevis. The gold labeling pattern was identical whether goat anti-mouse IgG, protein A, or BSA was used to stabilize the colloidal gold sols. Selective labeling over secretory granules of granular skin glands and myelin sheaths with colloidal gold alone was also observed. Such selective labeling could be prevented by increasing the ionic strength (elevation of NaCl content up to 30%) of the washing buffers and the diluent of the antibody. These results demonstrate that secretory granules of granular skin glands and myelin sheaths in the skin of X. laevis have affinity for colloidal gold-protein conjugates and may bind to the charged gold particles electrostatically.

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes.

The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytes in vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell-cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.

Morphometric analysis of fetal rat hepatocytes cultured in monolayer or following gyratory shaking.

The stereological technique was used to quantify glycogen areas and endoplasmic reticulum in fetal rat hepatocytes cultured for 24 hr in monolayer (monolayer cells) or following shaking by gyratory rotation (shaken cells). The volume density and volume per cell of glycogen areas decreased in order of freshly isolated hepatocytes, monolayer cells, and shaken cells. The surface density and area per cell of smooth endoplasmic reticulum increased in order of freshly isolated cells, monolayer cells, and shaken cells. The results show that the decrease of glycogen areas and proliferation of the smooth endoplasmic reticulum are more prominent in shaken cells than in monolayer cells. Prominent proliferation of the smooth endoplasmic reticulum in shaken cells may be due to the consumption of glycogen for energy release as a result of gyratory rotation.

A functional and structural heterogeneity is formed among fetal rat hepatocytes during culture.

Fetal rat hepatocytes (22-day-old, full-term) in vivo were homogeneous in ultrastructure and glucose 6-phosphatase (G6Pase) distribution throughout the liver acinus. These cells were isolated and cultured for 17 days in Williams medium E containing 10% fetal calf serum, dexamethasone, insulin, and glucagon. Heterogeneity among hepatocytes appeared progressively during culture. There were variations in numbers of binucleated cells, cytoplasmic ultrastructure (e.g., in the distribution of glycogen and the quantity of ultrastructure (e.g., in the distribution of glycogen and the quantity of mitochondria), and intensity of histochemical reactivity for G6Pase. The cells could be classified generally into two types: type I cells had some characteristics of periportal hepatocytes and type II cells those of centrolobular hepatocytes in in vivo adult liver. The results show that a functional and structural heterogeneity can be formed among fetal hepatocytes in monolayer culture without differences in supply of oxygen, nutrients, and hormones. A hidden heterogeneity might already exist among hepatocytes in full-term fetuses even though it cannot be detected ultrastructurally or cytochemically.

Inhibitory effect of colchicine and vinblastine on transport of glucagon receptors to the plasma membrane in cultured rat hepatocytes.

The role of microtubules in the regulation of glucagon receptors on cultured rat hepatocytes was studied. Antimicrotubular reagents, colchicine and vinblastine, did not affect the binding of 125I-labelled glucagon to hepatocytes at 4 degrees C. At 20 and 37 degrees C, however, the reagents reduced the binding after 60 or 90 min of incubation. Scatchard analysis indicated that the reduction in the binding was due to loss of glucagon-receptor populations. If hepatocytes were preincubated with both unlabelled glucagon and the reagents at 37 degrees C, the binding of the ligand to the cells decreased markedly after a certain delay. The reagents did not inhibit the internalization of the ligand in the cells until 30 min of incubation at 37 degrees C. The results suggest that the microtubule system plays a role in the transport of glucagon receptors to the plasma membrane, which is followed by their internalization.

Receptor-mediated endocytosis of glucagon in isolated mouse hepatocytes.

The binding of glucagon to the cell surface and the pathway of intracellular transport of the hormone in isolated mouse hepatocytes were studied by autoradiography, colloidal gold-labeled glucagon (Au-glucagon), and biochemical methods. In cells incubated with 1251-glucagon at 4 degrees C, the label was mainly localized to the plasma membrane even after 60 min of incubation. At 20 degrees C, the labeled ligand was internalized by the cells and the amount of internalized ligand increased with time of incubation. At 37 degrees C, the ligand was rapidly internalized and found to be associated with coated or uncoated vesicles. Au-glucagon experiments revealed clearly the process of internalization of glucagon. Au-glucagon bound to the plasma membrane was transported to coated regions and then internalized into vesicles via coated pits. Biochemical results supported these findings from autoradiography and Au-glucagon experiments. Thus, glucagon is internalized by hepatocytes via receptor-mediated endocytosis.

Quantitative analysis of smooth endoplasmic reticulum proliferation in hepatocytes of early postnatal and adult mice treated with phenobarbital.

The smooth endoplasmic reticulum in hepatocytes of early postnatal and adult ddY male mice injected with phenobarbital was analyzed stereologically. The animals received daily intraperitoneal injections of phenobarbital and were killed about 24 h after the last injection. In early postnatal animals (injected with 35 mg/kg of the drug at days 1 and 2 after birth, or injected with 35 mg/kg at day 2 and with 50 mg/kg at days 3 and 4 after birth), the smooth endoplasmic reticulum proliferated both in perivenular and periportal hepatocytes; there was no significant difference in amount of smooth endoplasmic reticulum between the cells of the two zones. In adult animals, however, proliferation occurred only in perivenular cells after the administration of 35 mg/kg of phenobarbital for 2 days, and predominantly in perivenular cells even after the administration of 150 mg/kg of the drug for 7 days. These results suggest that the conspicuous proliferation of the smooth endoplasmic reticulum in perivenular hepatocytes in response to phenobarbital administration, which is characteristic of adult liver, does not occur in early postnatal mice but becomes manifest during postnatal development.