Impairment of Blood-Cerebrospinal Fluid Barrier in Multiple Sclerosis

The blood-CSF barrier (BCB) function in active multiple sclerosis (MS) was studied by means of CSF proteins analysis using disc electrophoresis and immunofixation. Forty-five CSF samples were obtained by repeat lumbar punctures at various intervals, from four autopsy-proven cases and three male and nine female patients with clinically definite MS. When total protein content was increased, the percentages of prealbumin and tau fraction were decreased significantly in association with the presence of haptoglobin (Hp) polymers in nearly all the samples, as a result of increased permeability of the BCB. Even when the total protein content was normal, Hp polymers were detected in 56% of the samples, and the tau fraction tended to be decreased. Monoclonal immunoglobulin and Hp polymers were both recognized in some cases. The results suggested a more frequent occurrence of BCB impairment in MS than had formerly been revealed by CSF albumin analysis, and accorded with the recent reports of contrast-enhancing lesions of MS brain in computerized tomography.     

Evidence for Coadaptation: Negative Correlation between Lethal Genes and Polymorphic Inversions in DROSOPHILA MELANOGASTER

Through examination of all available data on lethal and inversion frequencies on the second chromosome in natural populations of Drosophila melanogaster, we have discovered that there is a clear negative correlation between the two quantities. Lethal genes are located more densely on the regions of standard gene arrangement than the inverted regions, and this accounts for the negative correlation. To reveal the underlying mechanism of the phenomena, we have carried out an experiment and found that effect of EMS-induced mutations on the inversion-carrying chromosome is more severe than that on the standard chromosome. We interpret these results as evidence for coadaptation or position-effect within the inversion chromosomes. New mutations within the coadapted gene complex are quickly eliminated from the population and polymorphic inversions are kept free of mutants through selective elimination.     

Immunological regression of metastatic cancer in the liver as a result of “in vivo xenogenization”

We attempted to induce the regression of liver metastatic tumor cellsin vivo by the administration to rats of Friend leukemia virus (FV) (in vivo xenogenization). The virus which was used in this experiment, FV, is highly immunogenic and does not normally cause disease in an adult rat. At first, we induced a FV viremia in tumor bearing rats in order to deliver the virus to the site of the tumor cells. FV viremia was induced by injecting 60 mg/kg cyclophosphamide (CY) i.v. after the administration of FV, and by transferring syngeneic bone marrow cells so that FV would be able to infect them and then replicate.In order that the tumor cells which were infected with virus should regress, it was necessary to break down their tolerance to FV antigens. As adoptive immunotherapy we therefore, transferred syngeneic spleen cells from rats which had been immunized with FV to tumor bearing rats. The result of this experiment was that these tumor bearing rats infected with FV which had received either normal syngeneic spleen cells or no spleen cells as controls died from liver metastasis (8 out of 9 rats (89%) and 15 out of 17 (88%) respectively). On the other hand, only 4 out of the 15 (27%) tumor bearing rats which were infected with FV and which received FV-immune spleen cells died from liver metastasis.These sets of data indicate that thein vivo xenogenization of tumor cells are indeed able to induce the regression of metastic tumor cells.     

The gene for glycogen-storage disease type 1b maps to chromosome 11q23.

Glycogen-storage disease type 1 (GSD-1), also known as "von Gierke disease," is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase) activity. There are four distinct subgroups of this autosomal recessive disorder: 1a, 1b, 1c, and 1d. All share the same clinical manifestations, which are caused by abnormalities in the metabolism of glucose-6-phosphate (G6P). However, only GSD-1b patients suffer infectious complications, which are due to both the heritable neutropenia and the functional deficiencies of neutrophils and monocytes. Whereas G6Pase deficiency in GSD-1a patients arises from mutations in the G6Pase gene, this gene is normal in GSD-1b patients, indicating a separate locus for the disorder in the 1b subgroup. We now report the linkage of the GSD-1b locus to genetic markers spanning a 3-cM region on chromosome 11q23. Eventual molecular characterization of this disease will provide new insights into the genetic bases of G6P metabolism and neutrophil-monocyte dysfunction.     

Association of cathepsin E deficiency with the increased territorial aggressive response of mice

Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, but physiological functions of this protein in the brain remains unclear. In this study, we investigate the behavioral effect of disrupting the gene encoding cathepsin E in mice. We found that the cathepsin E-deficient ( CatE −/−) mice were behaviorally normal when housed communally, but they became more aggressive compared with the wild-type littermates when housed individually in a single cage. The increased aggressive response of CatE−/− mice was reduced to the level comparable to that seen for CatE+/+ mice by pretreatment with an NK-1-specific antagonist. Consistent with this, the neurotransmitter substance P (SP) level in affective brain areas including amygdala, hypothalamus, and periaqueductal gray was significantly increased in CatE−/− mice compared with CatE+/+ mice, indicating that the increased aggressive behavior of CatE−/− mice by isolation housing followed by territorial challenge is mainly because of the enhanced SP/NK-1 receptor signaling system. Double immunofluorescence microscopy also revealed the co-localization of SP with synaptophysin but not with microtubule-associated protein-2. Our data thus indicate that cathepsin E is associated with the SP/NK-1 receptor signaling system and thereby regulates the aggressive response of the animals to stressors such as territorial challenge.     

Capturing enzyme structure prior to reaction initiation: tropinone reductase-II-substrate complexes

To understand the catalytic mechanism of an enzyme, it is crucial to determine the crystallographic structures corresponding to the individual reaction steps. Here, we report two crystal structures of enzyme-substrate complexes prior to reaction initiation: tropinone reductase-II (TR-II)-NADPH and TR-II-NADPH-tropinone complexes, determined from the identical crystals. A combination of two kinetic crystallographic techniques, a continuous flow of the substrates and Laue diffraction measurements, enabled us to capture the transit structures prior to the reaction proceeding. A structure comparison of the enzyme-substrate complex elucidated in this study with the enzyme-product complex in our previous study indicates that one of the substrates, tropinone, is rotated relative to the product so as to make the spatial organization in the active site favorable for the reaction to proceed. Side chains of the residues in the active site also alter their conformations to keep the complementarity of the space for the substrate or the product and to assist the rotational movement.     

Activation of anchorage-independent growth of HT1080 human fibrosarcoma cells by dexamethasone

Anchorage independence is an important hallmark of the transformation that correlates with tumorigenicity. We have isolated a variant clone of HT1080 human fibrosarcoma cells (cl-2) that is specifically defective in anchorage-independent growth. Interestingly, 10(-7) M dexamethasone (DEX) substantially rescued the anchorage-independent growth of cl-2 cells in semisolid culture. DEX also promoted the anchorage-independent growth of parental HT1080 cells. However, the agent had no effect on the anchorage-dependent growth of cl-2 and parental cells in ordinary liquid culture. Cell cycle analysis demonstrated that the population of G0/G1 cells increased, whereas that of S and G2/M cells decreased in growth-arrested cl-2 cells in suspension culture. However, such an effect of anchorage loss on cell cycle progression was alleviated by adding 10(-7) M DEX. In cl-2 cells in semisolid culture, DEX suppressed the expression of P27Kip1, whereas it stimulated the expression of cyclin A and hyperphosphorylated retinoblastoma (Rb) proteins. On the other hand, DEX had no effect on cyclin D1 and P21Cap1 expression. These effects of DEX, except for the suppression of P27Kip1, were blocked by an antimicrofilament drug, cytochalasin D. Our results suggest that the stimulation of anchorage-independent growth by DEX involves at least two regulatory mechanisms, i.e., one that leads to the suppression of P27Kip1 protein without requiring cytoskeletal integrity, and another that requires cytoskeletal integrity, leading to stimulation of cyclin A and hyperphosphorylation of Rb protein.     

Synthesis of 61-bis(1-adamantylcarbamoyl)-1,2-methano[60]fullerene and its antagonistic effect on haloperidol-induced catalepsy in mice

The 61-bis(1-adamantylcarbamoyl)-1,2-methano[60]fullerene was synthesized from N,N'-di(1-adamantyl)malondiamide and C(60) in the presence of 1,8-diazabicyclo[5,4,0]-7-undecene. The intraperitoneal administration of this fullerene derivative (10mg/kg) caused an antagonistic effect on haloperidol-induced catalepsy in mice.