Morphological study of disordered myelination and the degeneration of nerve fibers in the spinal cord of mice lacking complex gangliosides

Gangliosides, a family of glycosphingolipids that contain sialic acid, are abundant on the neuronal cell membranes, but their precise functions in the central nervous system remain largely undefined. In a previous study of GalNAc-T(-/-) mice engineered to lack beta1,4-N-acetylgalactos-aminyltransferase (GM2/GD2 synthase) to abolish any, complex gangliosides, we observed the reduction of nerve conduction velocity but did not find any obvious morphological change in the brain. In the present study, we observed morphological changes in the nerve fiber tracts of the spinal cord in these mice. In GalNAc-T(-/-) mice, the number of degenerated axons was markedly increased in the dorsal funiculus, tract of Lissauer, and dorsolateral funiculus of the cervical segment of the spinal cord as well as the dorsal funiculus and tract of Lissauer of the lumbar segment of the spinal cord. There were also increased numbers of unmyelinated fibers in GalNAc-T(-/-) mice. Loosened myelin sheaths and myelin sheaths separated from axons by wide spaces were also observed in GalNAc-T(-/-) mice. These results provide a morphological basis for the previously observed reduction in the nerve conduction velocity and suggest that complex gangliosides are essential for the maintenance of myelin and the integrity of nerve fibers of the spinal cord.     

Genomic structure and eight novel exonic polymorphisms of the human N-cadherin gene

 Analysis of the detailed genomic structure of human N-cadherin revealed that the 16-exon gene is more than 72 kb in length and that it consists of a mosaic of exons. Five repeated cadherin domains, a transmembrane domain, and a cytoplasmic domain are encoded by exons 4 to 13, 13 and 14, and 14 to 16, respectively. A search for molecular variants in the entire coding region in 96 Japanese individuals resulted in the identification of eight sequence polymorphisms including three CCT- or GCC-type trinucleotide repeat polymorphisms adjacent to the initiation codon and five other novel single-nucleoticle polymorphisms (SNPs) in the coding region. Three of the five SNPs accompanied an amino acid substitution: Ala118Thr, Ala826Thr, and Asn845Ser. Knowlege of the fine gene structure and eight novel polymorphisms will be useful for the genetic study of the role of N-cadherin in diseases involving cell adhesion in the brain and in cardiomyocytes. Received: January 23, 2002 / Accepted: March 12, 2002     

Polycondensation-addition. V. Polyurethan-ureas with asymmetric carbon atoms in the main chain from L- and DL-2-alkyl-2-isocyanatoethyl chloroformates and diamines1

L - and DL-2-Alkyl-2-isocyanatoethyl chloroformates were synthesized by the phosgenation of the corresponding amino alcohols. These optically active or racemic difunctional compounds were submitted to polycondensation-addition with various diamines to form high molecular weight polyurethan-ureas having asymmetric carbon atoms along the main chain. The optically active polyurethan-ureas obtained had slightly higher melting points than the corresponding racemic polymers.     

Nature and significance of calcium oxalate crystals in normal human thyroid gland

To elucidate the significance and nature of calcium oxalate crystals in the thyroid, we studied these crystals clinicopathologically and immunohistochemically in 182 normal thyroids from patients autopsied within 5 h of death. Under polarized light, calcium oxalate crystals showed brilliant birefringence and were invariably found within the colloid. The crystals were found in 73.1% of all cases but were more prevalent and denser in older individuals, with the highest prevalence (85.2%) being observed in those over 70 years of age. No crystals were seen in those under 10 years of age. Although underlying diseases seemed to have little influence, post-mortem delay apparently affected the prevalence and density of occurrence since the crystals tended to disappear with hours after death. An immunohistochemical study using anti-thyroid hormone antibodies revealed that the crystals were within negatively or weakly stained colloid and were not common in strongly stained colloid. These findings support the hypothesis that the occurrence of calcium oxalate crystals in normal human thyroid is associated with a low functional state of the thyroid follicles.     

Modifying effects of 1'-acetoxychavicol acetate (ACA) and the novel synthetic retinoids Re-80, Am-580 and Am-55P in a two-stage carcinogenesis model in female rats

Effects of dietary administration of 1'-acetoxychavicol acetate (ACA) and the novel synthetic retinoids 4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (Re-80); 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (Am-580); and 6-[(3,5-di-tert-butylphenyl) carbamoyl]nicotinic acid (Am-55P) were examined using a two-stage rat carcinogenesis model. A total of 190 female SD rats was treated sequentially with 1,2-dimethylhydrazine (DMH, s.c.); 7,12-dimethylbenz(a)anthracene (DMBA, i.g.); and 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN, in the drinking water) during the first three weeks (DDD-initiation), and an additional 60 rats received the vehicle alone (non-initiation). One week after the completion of the initiation period, they were divided into nine groups and administrated Re-80 (at dose levels of 1.0 or 0.4 ppm), Am-580 (20 or 4 ppm), Am-55P (20 ppm), ACA (100 ppm), all-trans-retinoic acid (10 or 2 ppm) or no supplement in the diet for 33 weeks, until survivors were euthanatized at week 37 weeks. After DDD-initiation, all-trans-retinoic acid at the high dose delayed the development of mammary tumors. The multiplicity of colon tumors in the group fed Am-55P and the incidences of nephroblastomas with ACA or Am-580 were decreased as compared with the control values, but the other chemicals had no modifying effects on tumor development in any organs. Thus, among ACA and the novel synthetic retinoids tested, only Am-55P showed a weak inhibitory effect on a neoplasm of general interest under the present experimental conditions.     

Involvement of cAMP response element-binding protein activation in salivary secretion.

OBJECTIVE: Saliva secretion is mediated by cAMP and the calcium signaling pathway in salivary acinar cells. The PKA signaling pathway plays an important role in protein secretion through the activation of cAMP, in fluid secretion through the elevation of intracellular calcium and in the activation of cAMP response element-binding protein (CREB), which is involved in these signaling cascades. In this study, we investigated whether the activation of CREB plays a part in the salivary secretion in mice. METHODS: We examined CREB activation by assessing phosphorylation at the serine-133 position using Western blotting. RESULTS: Carbachol (a muscarinic acetylcholine agonist) and isoproterenol (a beta-adrenergic agonist) markedly activated CREB in parotid acinar cells. Carbachol and isoproterenol-induced CREB phosphorylation was blocked by atropine (a muscarinic acetylcholine antagonist) and propranolol (a beta-adrenergic antagonist), respectively. The PKA inhibitor H89 inhibited CREB activation, but the PLC inhibitor U73122 did not. Moreover, carbachol- and isoproterenol-stimulated amylase secretion from parotid acinar cells was inhibited by H89 and adenoviral dominant-negative CREB. CONCLUSION: These results indicate that the muscarinic and beta-adrenergic activation of CREB was mediated through the PKA pathway and that CREB is involved in protein secretion from parotid acinar cells.     

Pseudorandom testing technique for the characterization of local hemodynamic control

Summary In order to investigate the low frequency properties of renal and femoral hemodynamic variables, pseudorandom testing techniques were used. The arterial flow of each bed, in separate experiments, was modulated by a low amplitude signal based on a pseudorandom binary sequence (PRBS) generated by digital computer.The cross-correlation functions between input flow and arterial pressure, venous pressure, and venous flow exhibit damped oscillations in all cases. These responses are parameterized in terms of a damping ratio () and an undamped natural frequency _n for a second order model. The parameters of the model are dependent upon the state of the bed as defined by mean arterial and venous pressures, mean flow through the bed, resistance, and oxygen consumption.The results of this study offer further insight into the dynamic low frequency autoregulation phenomenon for the renal and femoral beds of the dog.Supported by NIH Grant HE 11747.     

The molecular structures of 1,1-diphenylethyl methacrylate and triphenylmethyl methacrylate

The molecular structures of 1,1-diphenylethyl methacrylate (1,1-DPEMA) and triphenylmethyl methacrylate (TrMA) were determined by means of X-ray diffraction. 1,1-DPEMA: monoclinic, space group P2₁/a,a = 9,666(6), b = 19,94(2), c = 8,132(6) Å, β = 104,49(7)°, and Z = 4; TrMA: monoclinic, space group P2₁/n, a = 17,349(3), b = 9,487(2), c = 11,254(2) Å, β = 102,30(2)°, and Z = 4. Both structures were solved by the direct method and refined by the block-diagonal least-squares procedure to R = 0,175 and 0,056 for non-zero reflections, respectively. In both molecules, conformations about the C(1)? C(2) and C(1)? O(1) bonds are all synperiplanar and one of the two or three phenyl groups attached to the C(5) atom is in trans to the O(2).     

Difference in the expression of three tight junction proteins, barmotin, occludin, and ZO-1, in phenotypically different human colon cancer cell lines

Epithelioid disorganization is a hallmark of gastrointestinal cancers and is believed to be associated with malignant phenotypes such as invasiveness and the potentiality for metastasis. Although tight junctions (TJs) are known to be crucial for the maintenance of polarized organization of the gastrointestinal epithelium, changes in the TJ proteins in human cancers have not yet been fully elucidated. In this report, we investigated the expression and localization of three TJ proteins-barmotin (7H6 antigen), occludin, and ZO-1-in three phenotypically different human colon cancer cell lines exhibiting differnt grades of epithelioid organization. All three proteins were localized at the most apical part of the cell border corresponding to the site of TJs in T₈₄ cells, in which epithelioid organization was well preserved. In contrast, in COLO320DM cells, which showed no epithelioid phenotypes, occludin was not detectable at either the protein or mRNA level, although barmotin and ZO-1 were present in the cytoplasm. In the third cell line, DLD-1, which showed an epithelioid phenotype intermediate between T₈₄ and COLO320DM, aberrant expression of occludin was found in the basolateral cell membrane. On the other hand, barmotin was present in the cytoplasm, whereas ZO-1 was localized at the cell border. These observations showed that changes in the expression of TJ proteins occur in close correlation with epithelioid disorganization in human colon cancers.     

Antitumor immune response by CX3CL1 fractalkine gene transfer depends on both NK and T cells

The CX3C chemokine fractalkine (CX3CL1) exists as both a membrane-bound form promoting firm cell-cell adhesion and a soluble form chemoattracting leukocytes expressing its receptor CX3CR1. When adenoviral vector expressing mouse fractalkine (AdFKN) was transduced to the tumor cells, fractalkine was expressed as both membrane-bound form on the tumor cells and soluble form in the supernatant in vitro. Intratumoral injection of AdFKN (1 x 10(9)PFU/tumor) into C26 and B16F10 tumors resulted in marked reduction of tumor growth compared to control (C26: 86.5%, p<0.001; B16F10: 85.5%, p<0.001). Histological examination of tumor tissues revealed abundant infiltration of NK cells, dendritic cells, and CD8(+) T lymphocytes 3 and/or 6 days after treatment with AdFKN. Splenocytes from mice treated by AdFKN developed tumor-specific cytotoxic T cells, and thereby protected from rechallenging with parental tumor cells. Antitumor effects by AdFKN were completely abrogated in both NK cell-depleted mice and CD8(-/-) mice, and partially blocked in CD4(-/-) mice. These data indicated that fractalkine mediates antitumor effects by both NK cell-dependent and T cell-dependent mechanisms. This study suggests that fractalkine can be a suitable candidate for immunogene therapy of cancer because fractalkine induces both innate and adaptive immunity.