Impaired blood-brain barrier function in angiotensinogen-deficient mice.

Astrocytes in the central nervous system have physiologically important roles in the response to brain injury. Brain damage results in disruption of the blood-brain barrier (BBB), producing detachment of astrocyte endfeet from endothelial cells. The resultant leakage of serum proteins from loosened tight junctions between endothelial cells produces brain edema. At the same time, reactive astrocytes migrate to the injured area, where they proliferate and produce extracellular matrix, thereby reconstituting the BBB. As astrocytes are known to express angiotensinogen, which is the precursor of angiotensins (AI to AIV), we have investigated a possible functional contribution of angiotensinogen or one of its metabolites to BBB reconstitution. The astrocytes of angiotensinogen knockout mice had very attenuated expression of glial fibrially acidic protein and decreased laminin production in response to cold injury, and ultimately incomplete reconstitution of impaired BBB function. Although these abnormalities were rescued by administration of AII or AIV, the restoration of BBB function was not inhibited by AII type 1 and 2 receptor antagonists. These findings provide evidence that astrocytes with angiotensins are required for functional maintenance of the BBB.

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity.

Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.

EVALUATION OF PATIENT EXPOSURE IN FAST kVp SWITCHING DUAL ENERGY COMPUTED TOMOGRAPHY: PHANTOM STUDY.

The aim of this study was to evaluate the suitability of size specific dose estimates (SSDE) to estimate patient dose in Fast kVp switching dual energy CT. An anthropomorphic phantom (RAN-110) was repeatedly scanned (chest, abdomen and the pelvis) using a 64 detector row MDCT (Discovery CT750 HD, GE Healthcare, Milwaukee, WI, USA) with various CT parameters, including Fast kVp switching. Dosimetry was performed using thermo-luminescent dosimeters, positioned both superficially and within the phantom. SSDE was calculated for all slices of the anthropomorphic phantom using both the localiser and axial images. In Fast kVp switching, SSDE underestimated the measured absorbed dose for the chest/abdomen region ~35% at the maximum, but were in closer agreement for the pelvic region about within 10%. In single energy techniques, SSDE could not be applied in the estimation of organ doses, but in Fast kVp switching dual energy techniques, SSDE could be applied for anatomical regions with larger thicknesses.

[Primary cardiac angiosarcoma in the left atrium with adrenal metastasis; report of a case].

We encountered a 61-year-old woman with primary cardiac angiosarcoma in the left atrium. On echocardiography, the tumor extended into the atrial septum and mitral valve, and mitral valve stenosis and regurgitation were significant. We resected the tumor protruding into left atrium, and affecting mitral valve. The surgical procedure was not radical, but on postoperative echocardiography, function of the mitral valve was improved. Three months later, elevation of her right diaphragma was observed on chest X-ray and a giant adrenal tumor was detected by magnetic resonance imaging. Tumor biopsy indicated that this tumor was adrenal metastasis from cardiac angiosarcoma. In addition, echocardiography showed the recurrence of angiosarcoma in the left atrium and the presence of mitral stenosis and regurgitation. She died of heart failure 185 days postoperatively.

Activation of the nuclear oncogenes N-myc and c-jun in carcinoid [correction of cartinoid] tumors of transgenic mice carrying the human adenovirus type 12 E1 region gene.

The adenovirus (Ad) E1 region genes, E1A and E1B, are well known cooperatively to transform primary rodent cells and activate a number of cellular promoters, including nuclear oncogenes such as N-myc and c-jun, in transfected cell lines. However, there is still less information available on the in vivo mechanism(s) by which the E1 region gene, when chromosomally integrated in the living animals, exerts its effect on nuclear oncogene activation coupled with transformation. To investigate such in vivo activity of E1A we have used a series of microinjection experiments into fertilized eggs to generate three transgenic mice carrying the Ad12-type E1A/E1B genes under the control of the human renin gene. This transgene caused an early onset of bowel cartinoid tumors that express neural cell adhesion molecules, but do not metastasize to any region. Northern blot analysis revealed that the transgenes were considerably expressed in the tumors, but not in other tissues at detectable levels. Interestingly, the levels of N-myc and c-jun mRNAs in the cartinoid tumors were elevated 19- and 8-fold, respectively, as compared with those found in the control intestine. In contrast, the major histocompatibility complex (MHC) class I mRNA level was not altered between the tumor and control intestines, suggesting that this unchanged expression may reflect the loss of tumor metastasis. These findings provide the first in vivo evidence that the expression of the Ad12 E1 region gene induces cartinoid tumors associated with the activation of the nuclear oncogenes N-myc and c-jun.

Retinoic acid is a major regulator of chondrocyte maturation and matrix mineralization.

During the process of endochondral bone formation, chondrocytes undergo a series of complex maturational changes. Our recent studies indicate that this maturational process is influenced by the vitamin A derivative retinoic acid (RA). To learn how this agent regulates chondrocyte development, we characterized matrix gene expression during maturation of cartilage cells in chick sternum. RNAs were isolated from the cephalic portion of day 13, 14, 16, 18, and 20 chick embryo sternum and analyzed via northern blots. Type II collagen RNA levels remained fairly constant during this developmental period. In contrast, expression of type X collagen and alkaline phosphatase (APase) genes was first detected at day 16, followed by that of osteonectin (ON) and osteopontin (OP). To explore the mechanisms triggering these changes, chondrocytes were isolated from the cephalic portion of day 17-18 sternum (US cells) and grown in monolayer in standard serum-containing medium. After 3 weeks in culture, most of the cells enlarged and became type X collagen-positive, but they exhibited low APase activity and contained only trace amounts of ON and OP mRNAs. Treatment of parallel 3-week-old cultures with RA (10-100 nM) rapidly increased expression of the APase, ON, and OP genes severalfold. In concert with a significant increase in APase activity, there was abundant calcium accumulation in the RA-treated cultures. Electron microscopy confirmed the formation of large matrix-associated mineral crystals and the presence of numerous matrix vesicles. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum (LS cells).(ABSTRACT TRUNCATED AT 250 WORDS)

Neuron-specific expression of reporter gene in transgenic mice carrying the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha 1A subunit gene fused to E. coli lacZ reporter gene.

To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q type calcium channel alpha 1A subunit gene, transgenic mice carrying a 0.5-kb, 1.5-kb, 3.0-kb or 6.3-kb 5'-upstream region of the gene fused to Escherichia coli lacZ reporter gene were produced. In transgenic mice carrying the 1.5-kb, 3.0-kb or 6.3-kb 5'-upstream region, the reporter gene was exclusively expressed in the nervous system, although those with the 0.5-kb 5'-upstream region failed to show reporter expression. Histological examinations showed that the three 5'-upstream regions induced distinct expression patterns of the reporter gene in the CNS and adrenal medulla. The 1.5-kb 5'-upstream region drove reporter gene expression in the olfactory bulb, dorsal cortex and hippocampus, while the regulatory element for the expression in the amygdaloid nucleus, septum, habenula medial nucleus, choroid plexus, substantia nigra, inferior colliculus, pontine nucleus and cerebellum was located in the 5'-upstream sequence between 1.5 kb and 6.3 kb. In the cerebellum, the expression of the reporter gene was induced by the 3.0-kb region in granule cells, whereas it was induced by the 6.3-kb region in Purkinje cells. The expression of the reporter gene in chromaffin cells in the adrenal medulla was induced only by the 6.3-kb 5'-upstream region. These results suggest that the expression of the mouse P/Q-type Ca2+ channel alpha 1A subunit gene is regulated in a complex fashion by both positive and negative cis-regulatory elements.

Expression analysis of the 5'-upstream region of mouse P/Q-type Ca(2+) channel alpha( lA) subunit gene fused to Escherichia coli lacZ reporter gene in the spinal cord using transgenic mice.

The P/Q-type Ca(2+) channel alpha(1A) subunit is expressed in spinal cord including ventral motor neurons and interneurons and dorsal horn. To identify the transcriptional mechanisms of the mouse alpha(IA) subunit gene in spinal cord, transgenic mice carrying a 0.5, 1.5, 3.0 or 6.3-kb 5'-upstream region fused to the Escherichia coli lacZ reporter gene were examined. Transgenic mice carrying the 3.0-kb region expressed the reporter gene in dorsal horn and interneurons of ventral horn, although those with the 0.5-kb, 1.5-kb or 6.3-kb region did not. No transgenic mice expressed the reporter gene in motor neurons of ventral horn. These results suggest that in spinal cord, the expression mechanisms of the alpha(1A) subunit gene are complex, involving both positive and negative cis-regulatory elements, and the 6.3-kb 5'-upstream region alone is not sufficient for the expression.

Age-dependent paraparesis in WKA rats: evaluation of MHC k-haplotype and HTLV-1 infection.

Human T-cell leukemia virus type 1 (HTLV-1) infection is shown to be closely associated with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the occurrence of HAM/TSP was reported to be associated with MHC class II, the mechanism is still unclear. The WKA(RT1k) strain of rats was reported to develop HAM/TSP-like paraparesis after HTLV-1 infection, and was suggested to be an animal model of HAM/TSP. We asked whether MHC k-haplotype is specifically involved in the pathogenesis of paraparesis of WKA(RT1k) rats. We injected the HTLV-1 producing human T cells (MT-2 cells) intravenously into WKA(RT1k) rats and MHC congenic WKA.1L(RT1l) rats which have MHC l-haplotype of LEW rats on the WKA background. Positive antibody response to HTLV-1 antigens and presence of provirus in peripheral blood mononuclear cells confirmed that MT-2 cell-injected rats were infected with HTLV-1. Two of 13 MT-2 cell-injected WKA(RT1k) rats and five of 13 MT-2 cell-injected WKA.1L(RT1l) rats developed HAM/TSP-like hindlimb paraparesis between 16 and 26 months old. Interestingly, three of 14 MT-2 cell-uninjected WKA(RT1k) rats and four of 13 MT-2 cell-uninjected WKA.1L(RT1l) rats showed similar paraparesis between 15 and 26 months old. MHC k-haplotype is not specific to the development of paraparesis in WKA(RT1k) rats. The role of aging, genetic background, HTLV-1 infection and other factors on the development of HAM/TSP-like paraparesis in rats are discussed.

A neuropathological study of paraparetic rats injected with HTLV-I-producing T cells.

In order to clarify the pathogenesis of HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP), we injected HTLV-I-producing rabbit or human T cells intravenously into WKA and F344 rats. Infection was confirmed from increase in the anti-HTLV-I antibody titer and from the presence of HTLV-I proviral DNA. Only WKA rats developed hindlimb paraparesis 78-124 weeks after the injection. Neuropathological examination of 5 rats showed degeneration of the anterolateral and posterior funiculi as well as the peripheral nerves, and this degeneration was characterized by prominent vacuolation and macrophage infiltration. The myelopathy and neuropathy were grossly similar to those in human HAM/TSP. Although pathological changes of the spinal cord were very mild in 2 paretic rats, and similar lesions were found in the spinal cords and peripheral nerves of 2 control WKA rats, the myelopathy, radiculoneuropathy, or both in the paretic rats showed greater severity than in the controls. The contribution of the aging process to the lesions of the spinal cord and peripheral nerve is discussed. It appears possible that HTLV-I may accelerate the aging process and give rise to paraparesis. The precise role of HTLV-I in the pathogenesis of rat paraparesis remains to be elucidated taking the role of the aging process of the spinal cord and peripheral nerve into account.

Expression analysis of Escherichia coli lacZ reporter gene in transgenic mice.

To define a gene expression mechanism, it is often advantageous to use a reporter gene and transgenic mouse. The lacZ reporter gene is particularly useful for studies of the cis-regulatory element for tissue-specific expression in transgenic mice because of the ease of the enzyme assay and visualization on sections. In this report, we describe our method for examining the cis-regulatory element in transgenic mice, including choice of the lacZ gene, generation of transgenic mice, and analysis of beta-galactosidase activity.

Effects of interaction between parvovirus minute virus of mice NS1 and coactivator CBP on NS1- and p53-transactivation.

The non-structural protein NS1, encoded by the parvovirus minute virus of mice (MVM), is a potent regulator of viral gene expression in addition to prominent roles in viral replication and cytopathic effects associated with parvoviral infection. Although NS1 involves the modulation of viral and cellular transcription, the primary activation mechanism of MVM NS1 remains unclear. In the present study, we show here that the coactivator CREB binding protein, CBP, could potentiate NS1-mediated transcription as measured on the P38 promoter, which drives expression of the MVM capsid genes. NS1 bound to the two related cysteine-histidine-rich regions of CBP, referred to as C/H1 and C/H3, the former of which has an antagonistic function to CBP upon the NS1-transactivation. Furthermore, NS1 inhibited the synergistic transactivation by CBP and p53. These findings suggested that CBP as a transcriptional coactivator is required for NS1-mediated viral and cellular transcription in parvovirus-infected cells, resulting in cell proliferation and differentiation to achieve its lytic cycle.

Pathologic characterization of hypotensive C57BL/6J-agt: angiotensinogen-deficient C57BL/6J mice.

a fpreviously produced angiotensinogen-deficient mice, i.e. mice with deleted renin-angiotensin system (RAS), with a genetic background on C57BL/6J - C57BL/6J-agt (-/-) -, but no C57BL/6J-agt (-/-) which survived long enough to be weaned. In the present study, we attempted to prevent neonatal death and analyzed pathological development in C57BL/6J-agt (-/-). We indicate that mortality in C57BL/6J-agt (-/-) derived from C57BL/6J-agt (+/-) can be reduced by hypodermic saline injection in the 7 days following birth, that hydronephrosis developed by day 14 in association with polydiplasia and polyuria by day 30, and that chronic hypotension occurs. Hydronephrosis is less damaging to electrolyte resorption in younger mice, but not in adults. We also observed that C57BL/6J-agt (-/-) derived from C57BL/6J-agt (-/-) frequently develop fetal hydronephrosis and die of respiratory failure at birth. These results suggest that maternal RAS is associated with structural maturation of kidney and lung in late fetus and that postnatal RAS plays important roles in structural and functional maintenance of the kidneys.

Pathogenesis of haemagglutinating encephalomyelitis virus (HEV) in mice experimentally infected by different routes.

Three-day-old suckling mice inoculated intracerebrally (i.c.) with the 67N strain of haemagglutinating encephalomyelitis virus (HEV) showed nervous signs and died. The virus was passaged 10 times in suckling mice and was designated the MB-67N strain. The pathogenesis of MB-67N was studied with various ages of mice and inoculation routes. All mice inoculated i.c. with a large dose of virus died regardless of age, although a smaller dose caused fatal infection only in suckling mice. By intranasal, intraperitoneal and subcutaneous inoculation, the virus also killed suckling mice under 16 days old, but not older mice, even with a large dose. The susceptibility of mice for the MB-67N strain was influenced by age and inoculation routes. High titres of virus were re-isolated from the brain of diseased mice after inoculation by any route, but not from other organs. Histologically, numerous areas of severe focal necrosis were produced in the cerebral cortex. Specific immuno-fluorescence and numerous viral particles were found in the cytoplasm of nerve cells by immuno-fluorescence staining and electron microscopy. These findings indicate that the MB-67N propagates mainly in the central nervous system and nerve cells serve as a main target of virus replication.

Neurotropism of mouse-adapted haemagglutinating encephalomyelitis virus.

The propagation of a mouse-adapted strain (67N) of haemagglutinating encephalomyelitis virus in infected mice and murine cells was examined by viral re-isolation and immunostaining. Viral propagation was strictly limited to the neurons and to an established line of neuroblastoma cells in in-vivo and in-vitro experiments. These results provide adequate evidence that this virus is neurotropic.

Chromosomal mapping of human angiotensinogen gene and human renin gene by fluorescence in situ hybridization (FISH) in transgenic mice.

We attempted to apply FISH to chromosomal mapping of the human angiotensinogen (hAG) and human renin (hRN) transgenes which are carried by the parental strains of the Tsukuba hypertensive mouse. We report here that the hAG gene is mapped in the C2 region of Chr 19 and the hRN gene in the A1 region of Chr 6.

Vertical transmission to embryo and fetus in maternal infection with rat virus (RV).

The influence of maternal rat virus (RV) infection on rat embryogenesis and fetus was examined by viral reisolation, immunostaining and PCR analysis. Vertical transmission caused by the UT-1 strain of RV depended on the stage of gestation when maternal infection occurred. When females were infected at the pre-mating point, the number of fetuses was smaller than that normally obtained, possibly due to infection at the stage of the hatched blastocyst, but almost all of the fetuses obtained were free from infection and developed normally. The incidence of transplacental infection was the highest when pregnant females were infected in the middle of the gestation stage, and some of the fetuses died. In pregnant females which were infected late in the gestation stage, all fetuses developed normally. Some of them were infected transplacentally and harbored the infectious virus. Much attention should be paid to performing reliable rederivation of RV-infected rat colonies by hysterectomy and embryo transfer.

Prevalence of "orphan" parvovirus infections in mice and rats.

"Orphan" parvovirus (OPV) infection in laboratory mice and rats was serologically surveyed for 465 mouse sera and 271 rat sera collected from 1986 to 1987 and from 1993 to 1996 in Japan. The results suggest that parvovirus infection is rare in mice but common in rats (positive rate: 13-22%) and that most putative viruses were OPVs. OPV is therefore considered to already have been harbored for at least ten years in Japan.

Vascular remodeling in hypertensive transgenic mice.

We physiologically and histopathologically analyzed vascular damage due to hypertension and vascular remodeling in hypertensive transgenic mice (Tsukuba hypertensive mice; THM). Pubertal (6-week-old) THM already had hypertension similar to blood pressure in adult THM due to an enhanced renin angiotensin system (RAS). They progressively developed remarkable vascular hypertrophy composed of dedifferentiation of vascular smooth muscle cells (VSMCs) and extracellular matrix accumulation in the thoracic aorta, and VSMC hyperplasia was predominant in the abdominal aorta. THM are therefore a useful animal model for studying vascular remodeling mediated by enhanced RAS.