Prevention of neural tube defects by loss of function of inducible nitric oxide synthase in fetuses of a mouse model of streptozotocin-induced diabetes.

AIMS/HYPOTHESIS: Maternal diabetes during pregnancy increases the risk of congenital malformations such as neural tube defects (NTDs). Although the mechanism of this effect is uncertain, it is known that levels of nitric oxide synthase (NOS) and nitric oxide are elevated in embryos of a mouse model of diabetes. We postulated that overproduction of nitric oxide causes diabetes-induced congenital malformations and that inhibition of inducible NOS (iNOS) might prevent diabetic embryopathy. METHODS: Mice were rendered hyperglycaemic by intraperitoneal injection of streptozotocin. The incidence of congenital malformations including NTDs was evaluated on gestational day 18.5. We assessed the involvement of iNOS in diabetes-induced malformation by administering ONO-1714, a specific inhibitor of iNOS, to pregnant mice with streptozotocin-induced diabetic mice and by screening mice with iNOS deficiency due to genetic knockout (iNos(-/-)). RESULTS: ONO-1714 markedly reduced the incidence of congenital anomalies, including NTDs, in fetuses of a mouse model of diabetes. It also prevented apoptosis in the head region of fetuses, indicating that iNOS is involved in diabetes-related congenital malformations. Indeed, no NTDs were observed in fetuses of diabetic iNos(-/-) mice and the incidence of other malformations was also markedly reduced. CONCLUSIONS/INTERPRETATION: We conclude that increased iNOS activity during organogenesis plays a crucial role in the pathogenesis of diabetes-induced malformations and suggest that inhibitors of iNOS might help prevent malformations, especially NTDs, in diabetic pregnancy.

Local calcium release in dendritic spines required for long-term synaptic depression.

We have used rats and mice with mutations in myosin-Va to evaluate the range and function of IP3-mediated Ca2+ signaling in dendritic spines. In these mutants, the endoplasmic reticulum and its attendant IP3 receptors do not enter the postsynaptic spines of parallel fiber synapses on cerebellar Purkinje cells. Long-term synaptic depression (LTD) is absent at the parallel fiber synapses of the mutants, even though the structure and function of these synapses otherwise appear normal. This loss of LTD is associated with selective changes in IP3-mediated Ca2+ signaling in spines and can be rescued by photolysis of a caged Ca2+ compound. Our results reveal that IP3 must release Ca2+ locally in the dendritic spines to produce LTD and indicate that one function of dendritic spines is to target IP3-mediated Ca2+ release to the proper subcellular domain.

Computer three-dimensional reconstruction of the sinoatrial node.

BACKGROUND: There is an effort to build an anatomically and biophysically detailed virtual heart, and, although there are models for the atria and ventricles, there is no model for the sinoatrial node (SAN). For the SAN to show pacemaking and drive atrial muscle, theoretically, there should be a gradient in electrical coupling from the center to the periphery of the SAN and an interdigitation of SAN and atrial cells at the periphery. Any model should include such features. METHODS AND RESULTS: Staining of rabbit SAN preparations for histology, middle neurofilament, atrial natriuretic peptide, and connexin (Cx) 43 revealed multiple cell types within and around the SAN (SAN and atrial cells, fibroblasts, and adipocytes). In contrast to atrial cells, all SAN cells expressed middle neurofilament (but not atrial natriuretic peptide) mRNA and protein. However, 2 distinct SAN cell types were observed: cells in the center (leading pacemaker site) were small, were organized in a mesh, and did not express Cx43. In contrast, cells in the periphery (exit pathway from the SAN) were large, were arranged predominantly in parallel, often expressed Cx43, and were mixed with atrial cells. An approximately 2.5-million-element array model of the SAN and surrounding atrium, incorporating all cell types, was constructed. CONCLUSIONS: For the first time, a 3D anatomically detailed mathematical model of the SAN has been constructed, and this shows the presence of a specialized interface between the SAN and atrial muscle.

Elevated serum alpha-fetoprotein level of nude mice bearing hepatoma cells by treatment with monoclonal antibodies to alpha-fetoprotein.

This study was carried out to clarify the reason for elevation of serum alpha-fetoprotein (AFP) level of nude mice bearing hepatoma cells after treatment with monoclonal antibodies (MoAbs) to AFP. MoAbs to AFP showed no effect on the cumulative amounts of AFP secreted from human hepatoma cell line, HuH-7, in vitro. However, the treatment of nude mice bearing HuH-7N cells (HuH-7 xenograft) with MoAbs to AFP led to elevation of the serum AFP level in spite of the fact that the growth curve of HuH-7N cells was similar to that for PBS treatment. This apparent elevation of the serum AFP level is thought to be due to the slow elimination of AFP-MoAb immune complexes with little lattice structure from circulation, but not the enhancement of AFP secretion of HuH-7N cells. Thus, when using a MoAb alone or MoAb-drug conjugate, the serum AFP level should only be cautiously used as a tumor marker for evaluating the targeting immunotherapy.

Follow-up study on histogenesis of microcephaly associated with ectopic gray matter induced by prenatal gamma-irradiation in the mouse.

Brain malformation with ectopic gray matter was visualized with magnetic resonance imaging in small-sized heads of prenatally exposed atomic bomb survivors. The identical brain malformation was reproduced in mice and its histogenesis was studied in the present experiment. Pregnant mice were exposed to 60Co gamma-irradiation at a single dose of 1.5 Gy on embryonic day 13 (E13), and then injected intraperitoneally with 30 mg/kg BrdU on E15. The extensive dead cells appeared throughout the brain mantle at 6 hours (h) after exposure. On E16 cell aggregations formed rosettes. On E18 a high proportion of BrdU-labeled cells reached the superficial layers of the cortical plate with the remaining cells located in the ectopic neuronal masses. The quantitative study showed that labeled cells in layers II to III were fewer and those in layers IV to VI more numerous in the prenatally irradiated adult mice than in controls. The anti-GFAP immunostaining revealed that the glial fibers in the irradiated mice were preserved, but disorganized. These findings suggested that the majority of migrating neurons were able to arrive at their normal layers, but some neurons remained due to the interrupted migratory pathway and eventually formed ectopic neuronal masses beneath the subcortical white matter.

Derangement of Purkinje cells in the rat cerebellum following prenatal exposure to X-irradiation: decreased Reelin level is a possible cause.

It has been reported that prenatal X-irradiation of rats during the late gestation period causes heterotopic Purkinje cells in the internal granular layer (IGL) of the abnormally foliated cerebellum. The present study was designed to demonstrate the process of X-ray-induced derangement of Purkinje cells and their surrounding cells. In addition, the expression of some morphoregulatory molecules was examined to determine which molecules are involved in the abnormal pattern of Purkinje cells. Pregnant rats (n = 22) were exposed to 2.5 Gy X-radiation on gestation day 21 and the cerebellum of progeny was examined histologically and by immunohistochemistry to identify Purkinje and Bergmann cells. At 12 h after exposure, extensive cell death was observed in the external granular layer (EGL). By postnatal day (P) 9, while Purkinje cells with well-developed dendrites aligned underneath the EGL in the control cerebellum, Purkinje cells with shorter and abnormally oriented dendrites failed to align and remained in the heterotopic location in the IGL. Bergmann cells and their fibers were also disoriented but later recovered in their proper position. Abnormal folia developed in the irradiated rats. Using immunohistochemistry, we next examined the levels of the neural cell adhesion molecule (NCAM), fibronectin, tenascin, and Reelin. Among them, only the level of Reelin was affected significantly. Reelin decreased strikingly in the premigratory zone of the EGL and IGL in the irradiated cerebellum on P1, and the decrease continued until P9. Decreased Reelin expression was demonstrated quantitatively by Northern blot analysis and the correlation between the mRNA and protein levels was well presented. The expression of reelin mRNA decreased significantly by irradiation from P0, being almost one third of the level in controls on P4, and tended to recover up to P9. It is thus indicated that X-irradiation causes a marked decrease in the level of Reelin at the critical stage for the alignment of Purkinje cells. Since Reelin has been shown to play an important role in the migration of neural cells, it is suggested that the decrease in Reelin by X-irradiation is an important factor for the derangement of Purkinje cells.

Expression of ZAKI-4 messenger ribonucleic acid in the brain during rat development and the effect of hypothyroidism.

We identified ZAKI-4 (also designated as DSCR1L1) as a thyroid hormone responsive gene in cultured human skin fibroblasts. Recently it has been reported that ZAKI-4 belongs to an evolutionary conserved family of proteins that function as calcineurin inhibitor. In human, ZAKI-4 and calcineurin are highly expressed in brain, where thyroid hormones play essential roles in the development during fetal and neonatal periods. In the present study, we examined the temporal and spatial expression patterns of ZAKI-4 messenger RNA (mRNA) in control and hypothyroid rat brains. Northern blot analysis revealed that ZAKI-4 mRNA was detected in both cerebral cortex and cerebellum as early as embryonic day (E)18. In the cerebral cortex, the expression level gradually increased with age, reaching a plateau at postnatal day (P)7 and remained constant thereafter until P30. A similar pattern of increase with age was also observed in hypothyroid rats; however, the magnitude of the increase was significantly reduced. In control rats, the fold increase in ZAKI-4 mRNA level from E18 to P17 was 10.8; whereas in hypothyroid rats, it was 7.4. In cerebellum the expression level did not change with age or by thyroid status. In situ hybridization revealed that ZAKI-4 mRNA is widely expressed in neurons throughout the brain. It is noteworthy that the expression in the neurons of layer VI of the cerebral cortex was more evident in control rats than that in hypothyroid rats from P17 to P30. Though not influenced by hypothyroidism, there were several regions of the brain in which ZAKI-4 mRNA was strongly expressed. These regions were the mitral cell layer of the olfactory bulb, the substantia nigra, and the hippocampus, where calcineurin is also abundantly expressed. Therefore, it may be hypothesized that ZAKI-4 plays an important role in the development and function of the brain by modulating calcineurin function; and decrease in ZAKI-4 mRNA expression in the specific brain areas may explain, in some parts, the mechanism of abnormal brain development by hypothyroidism.

Distribution of the muscarinic K+ channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart.

The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.

Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border.

The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.

Presence of the Kv1.5 K(+) channel in the sinoatrial node.

The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K(+) channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, approximately 64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions.

Purkinje cell abnormalities and synaptogenesis in genetically jaundiced rats (Gunn rats).

The course of cytological abnormalities and synaptogenesis of Purkinje cells were investigated in the culmen of cerebella from homozygous Gunn rats with hereditary hyperbilirubinemia from postnatal day 7 to adulthood (5-10 months old). The affected Purkinje cells were abundant at day 7. A large number of Purkinje cells reached the fully advanced stage of degeneration during the ensuring 16 days and disappeared between days 12 and 30. The Purkinje cells remaining at day 30 were less affected and recovered by the adult stage. Various abnormalities in Purkinje cell synaptogenesis with the parallel fibers, climbing fibers, and basket and stellate cell axons were observed. Primitive junctions between parallel fibers and Purkinje dendritic shafts were often found in adulthood. The parallel fiber boutons lacking postsynaptic partners and facing astrocytic processes were often noted from day 18 to adulthood. The persistence of such presynaptic elements suggests some mechanisms for stabilizing the synaptic elements once they have been formed. Many of the parallel fiber synaptic boutons with or without their postsynaptic partners were enlarged and were assumed to be transsynaptically affected by Purkinje cell damage. A number of climbing fiber synapses with perisomatic process of Purkinje cells, which are transient in normal synaptogenesis, were present at day 30 and a few of them were still found even in adulthood. Basket and stellate cell synapses were often found in abundance on the remaining Purkinje cells in adulthood, though they were not frequently encountered during the development period.

Endoplasmic reticulum is missing in dendritic spines of Purkinje cells of the ataxic mutant rat.

Dilute-opisthotonus (dop) is a spontaneous ataxic mutation in the rat, regulated by an autosomal recessive gene. Immunohistochemical staining with anti-inositol 1,4,5-trisphosphate receptor antibody and electron microscopic examinations revealed that the endoplasmic reticulum in dendritic spines of Purkinje cell was missing in the ataxic rat. This could impair the intracellular signal transduction in the parallel fiber-Purkinje cell synapse, and be a cause of the severe ataxic movement.

The dilute-lethal (dl) gene attacks a Ca2+ store in the dendritic spine of Purkinje cells in mice.

The absence of smooth endoplasmic reticulum (SER) in the dendritic spine of Purkinje cells was found in dilute-lethal (dl) mouse cerebella as detected by immunohistochemistry using anti-inositol 1,4,5-triphosphate receptor antibody and electron microscopy. Since SER in the spine has been suggested to play a crucial role for synaptic regulation as an intracellular Ca2+ store (for reviews, see [Miller, R.J., Prog. Neurobiol., 37 (1991) 255-285: Simpson, P.B., Challiss, R.A.J. and Nahorski, S.R., Trends Neurosci., 18 (1995) 299-306]), a neurological defect, characterized by clonic convulsions with opisthotonus and ataxia, in the dilute-lethal mouse with homozygous trait may be attributable to the absence of SER in the dendritic spine of Purkinje cells.

Immunotoxins composed of monoclonal antibody to alpha-fetoprotein and gelonin as a potent hepatoma-targeted drug delivery system.

This study was carried out to evaluate our monoclonal antibody (MoAb) to alpha-fetoprotein (AFP), 80G, as a carrier for targeting AFP-producing hepatoma. Pharmacokinetic analysis showed that the MoAb 80G was actively incorporated into AFP-producing HuH-7N cells (xenograft of human hepatoma cell line, HuH-7) in nude mice. Four conjugates composed of MoAb 80G, and a type 1 ribosome-inactivating protein, gelonin, were prepared. They involve two disulfide-linked and two thioether-linked conjugates. The binding activity of conjugates against AFP remained as high as that of intact 80G according to enzyme-linked immunosorbent assay. The in vitro cytotoxic effects of all the conjugates were specific against AFP-producing HuH-7 cells. Of these conjugates, two containing gelonin modified with 2-iminothiolane were more potent than the others. They showed significant antitumor activity upon AFP-producing HuH-7N cells in nude mice. However, the disulfide conjugate was more toxic to mice than the thioether conjugate judging from the loss in body weight and the liver damage. These results suggest that our MoAb 80G is a suitable carrier for targeting AFP-producing hepatoma cells, and that the noncleavable thioether conjugate is promising as an AFP-producing hepatoma-targeted drug delivery system.

Radioadaptive response in primary mouse spermatocytes revealed by analysis of synaptonemal complexes.

This study was aimed at estimating the damage to the synaptonemal complex (SC) of male mice caused by radiation exposure at the ultimate pre-meiotic interphase. Four experimental groups were formed: group 1 was an untreated control, group 2 received a low dose (50 mGy) of radiation, group 3 a high dose (4 Gy) of radiation, group 4 was first treated with a low dose (50 mGy) and 4 h later with a high dose (4 Gy). Mice were killed 4 days after the treatment. Early pachytene cells were selected for the electron microscopic analysis of SCs. These cells were supposed to be at pre-meiotic interphase at the time of irradiation. Treatment with a low dose of radiation did not produce any substantial increase in the frequency of SC aberrations, while exposure to the high dose resulted in various types of damage. Group 4 demonstrated significantly lower frequencies of axial breaks/fragments and multiaxial configurations than group 3. At the same time, these two groups did not differ in the frequencies of inter- and intrachromosomal exchanges such as translocations, inversions, and deletions. We suppose that the number of breaks produced by a high dose of radiation was the same in the both groups. However, a proportion of the breaks that remained unrepaired until pachytene and were expressed as gaps, fragments, and multiaxial configurations was reduced by pretreatment with the low dose of radiation.

Effect of oily vehicles on absorption of mepitiostane by the lymphatic system in rats.

[14C]Mepitiostane in various vehicles was administered to the small intestine of anaesthetized rats with cannulated thoracic ducts, and the effect of lipids on lymphatic absorption was examined. The extent of lymphatic absorption was greatest when administered in triolein and sesame oil, which are triglycerides of long-chain fatty acids. Absorption in the presence of other vehicles was in the order of 10% Tween 80 aqueous solution greater than monolein greater than oleic acid approximately oleic acid/monolein (2:1 mol/mol) greater than aqueous suspension. Differences between the extents of lymphatic absorption of mepitiostane in the various formulations were not due to variation in the lymph flow but to the increased secretion of chylomicron and very low density lipoproteins. During absorption of mepitiostane from the small intestine, oil affected not only the penetration into epithelium cells and the metabolism in them, but also the partition between blood and lymph.

Effect of bile on absorption of mepitiostane by the lymphatic system in rats.

The effects of bile and site of gastrointestinal absorption on the lymphatic absorption of the highly lipophilic drug, mepitiostane were examined using thoracic duct-cannulated rats. The lymphatic absorption from the small intestine was very small in the absence of bile compared with that when bile was present. The lymphatic absorption was greatest when drug was administered to the upper small intestine with bile, was smaller for the lower regions of the small intestine, and was negligible for the stomach and the large intestine. A correlation was observed between the extent of lymphatic absorption and the secretion of chylomicron and very low density lipoproteins after administration to various regions with or without bile. The portal absorption data of mepitiostane confirmed that site specificity occurs in the partition of drug between blood and lymph.

Cerebellar hypoplasia in the hyperbilirubinemic Gunn rat: morphological aspects.

Gunn rats, a mutant strain of rats, suffer from autosomal recessive hyperbilirubinemia. The homozygotes (j/j) develop jaundice soon after birth and often exhibit kernicterus and cerebellar hypoplasia that are due to bilirubin. Therefore, j/j Gunn rats have been used as an animal model of bilirubin encephalopathy, as well as of neonatal hyperbilirubinemia. In this review, we discuss morphological aspects of the cerebellar hypoplasia that is due to bilirubin and describe the relationship between plasma bilirubin levels and cerebellar hypoplasia, as well as the pathogenesis of cerebellar hypoplasia, including abnormal histogenesis of the cerebellar cortex, abnormalities associated with Purkinje cells and abnormal synaptogenesis in j/j Gunn rats.

Histological characteristics of the pelage skin of rough fur mice (C3H/HeJ- ruf/ruf).

Pelage skin of C3H/HeJ mice homozygous at an autosomal recessive mutant locus, rough fur (ruf) which is located on chromosome 9, was histologically analyzed. Sebaceous glands synthesizing lipids were larger in the mutant mice than in controls in an examination by Sudan IV staining. Electron microscopic analysis of the sebaceous gland showed that lipid droplets were denser in mutant mice than in control mice, and that they were irregular in shape in ruf mice while those of controls were round. Our results suggested that rough fur (ruf) mice might be an animal model for hyperlipogenesis of the pelage skin.

[Pharmaceutical dosage form design of copoly (lactic/glycolic acid) microspheres. Mechanism of in vitro release of gentamicin].

The sustained release mechanism of gentamicin (GM) from lactic acid/glycolic acid copolymer (PLGA) microspheres was investigated. The terminal free carboxyl group of polymer was proved to be necessary for GM to be highly incorporated into microspheres by comparing interactions with GM and two types of polymers; free (ionized and non ionized) and the terminal esterified carboxyl group of polymer. The weight-average molecular weights (Mws) of component PLGAs of microspheres with an ionizable carboxyl group used here were approximately 4900 and 10000. The release pattern of GM was tested in phosphate buffered saline. The release rate of GM was dependent on the initial Mw and surface form. The GM release continued for 20 and 30 d from PLGA 4900- and PLGA10000-microspheres, respectively. The changes of total weight of microspheres tended to decrease with time, and the molecular weight distribution of PLGA gradually shifted to lower distribution, indicating a decrease in Mw. The changes and the shifts were dependent on the initial Mws of PLGAs but independent of their surface form. The half-times of wight loss of PLGA 4900- and PLGA10000-microspheres were about 10 and 20 d, respectively. From these results, the release profile of GM from PLGA microspheres was explained by the following three steps, i.e., 1) the release from the surface, 2) the relatively slow release caused by the obstruction of channels followed by the degradation of PLGA, 3) the release accompanied by the erosion of microspheres.