Analysis of genes induced in peripheral nerve after axotomy using cDNA microarrays

One of the most striking features of neurons in the mature peripheral nervous system is their ability to survive and to regenerate their axons following axonal injury. To perform a comprehensive survey of the molecular mechanisms that underlie peripheral nerve regeneration, we analyzed a cDNA library derived from the distal stumps of post-injured sciatic nerve which was enriched in non-myelinating Schwann cells using cDNA microarrays. The number of up- and down-regulated genes in the transected sciatic nerve was 370 and 157, respectively, of the 9596 spotted genes. In the up-regulated group, the number of known genes was 216 and the number of expressed sequence tag (EST) sequences was 154. In the down-regulated group, the number of known genes was 103 and that of EST sequences was 54. We obtained several genes that were previously reported to be involved in regeneration of the injured neurons, such as cathepsin D, ninjurin 1, tenascin C, and co-receptor for glial cell line-derived neurotrophic factor family of trophic factors. In addition to unknown genes, there seemed to be a lot of annotated genes whose role in nerve regeneration remains unknown.     

Role of side-edge site of sphingomyelinase from Bacillus cereus

Bacillus cereus sphingomyelinase (Bc-SMase) belongs to the Mg(2+)-dependent neutral sphingomyelinase (nSMase) which hydrolyzes sphingomyelin (SM) to produce phosphocholine and ceramide, and acts as an extracellular hemolysin. Bc-SMase has two metal ion-binding sites in a long horizontal cleft across the molecule, with one Mg(2+) in the central region of the cleft and one divalent metal ion at the side-edge of the cleft. The role of the Mg(2+) at the side-edge of the long horizontal cleft in Bc-SMase remains unresolved. The replacement of Asn-57, Glu-99, and Asp-100 located in close proximity to Mg(2+) at the side-edge with alanine resulted in a striking reduction in binding to and hydrolysis of sphingomyelin in membranes of sheep erythrocytes or SM-liposomes but that of Phe-55 did not. However, the replacement of these residues had little effect on the enzymatic activity. N57A, E99A, and D100A contained 2 mol of Mg(2+) per mol of protein, and the wild type and F55A contained 3 mol. A crystal analysis showed that N57A with Mg(2+) had no metal ion at the side-edge. These results indicate that the Mg(2+) at the side-edge of Bc-SMase plays an important role in the binding to membranes.     

Establishing interspecific mosaic genome lines between Drosophila ananassae and Drosophila pallidosa by means of parthenogenesis

Strong sexual isolation exists between the closely related species Drosophila ananassae and D. pallidosa, but there is no obvious post-mating isolation; both sexes of the hybrids and their descendants appear to be completely viable and fertile. Strains exhibiting parthenogenesis have been derived from wild populations of both species. We intercrossed such strains and established iso-female lines after the second generation of parthenogenesis. These lines are clones, carrying homozygous chromosomes that are interspecific recombinants. We established 266 such isogenic lines and determined their genetic constitution by using chromosomal and molecular markers. Strong pseudo-linkage was seen between loci on the left arm of chromosome 2 and on the right arm of chromosome 3; the frequency of inheriting the two chromosome regions from the same species was significantly larger than expected. One possible cause of pseudo-linkage is female meiotic bias, so that chromosomes of the same species origin tend to be distributed to the same gamete. But this possibility is ruled out; backcross analysis indicated that the two chromosome regions segregated independently in female hybrids. The remaining possibility is elimination of low-fitness flies carrying the two chromosome regions from different species. Thus, genetic incompatibility was detected in the species pair for which no hybrid breakdown had previously been indicated. The 'interspecific mosaic genome' lines reported here will be useful for future research to identify genes involved in speciation and phenotypic evolution.     

5'-,3'-inverted thymidine-modified antisense oligodeoxynucleotide targeting midkine. Its design and application for cancer therapy

Oligodeoxynucleotides modified at both 5'- and 3'-ends with inverted thymidine (5'-,3'-inverted T) were introduced as new reagents for antisense strategies. These modifications were performed to make the oligodeoxynucleotides resistant to nucleases. The effectiveness of these oligodeoxynucleotides was evaluated in terms of inhibition of synthesis of midkine (MK), a heparin-binding growth factor, and consequent inhibition of growth of CMT-93 mouse rectal carcinoma cells. 5'-,3'-Inverted T antisense MK suppressed synthesis of MK by CMT-93 cells and their growth in culture. Furthermore, 5'-,3'-inverted T oligodeoxynucleotides exhibited less cytotoxicity and better stability than phosphorothioate oligodeoxynucleotides. When 5'-,3'-inverted T antisense MK was mixed with atelocollagen, and injected into CMT-93 tumors pregrown in nude mice, tumor growth was markedly suppressed as compared with tumors injected with sense controls. The suppressive effect of 5'-,3'-inverted T antisense MK on tumor growth was stronger than that of phosphorothioate antisense MK. These findings indicated the usefulness of inverted thymidine-modified antisense oligodeoxynucleotides as a new reagent instead of phosphorothioate-modified oligodeoxynucleotides.     

Binding of mibolerone to androgen receptor of benign hypertrophic human prostate. Comparison with R1881

The binding nature of mibolerone in cytosols and nuclear extracts from hypertrophic human prostate was examined in comparison with that of R 1881. The binding of mibolerone in the cytosol and nuclear extract was single and of high affinity when evaluated by the method of Scatchard (1949). Binding of mibolerone with testosterone-binding globulin was not detected. The sedimentation coefficients of the binder for mibolerone in the cytosol and nuclear extract were 10.6 S and 3.6 S, respectively. When triamcinolone acetonide was induced in the binding medium, inhibition of mibolerone binding in the cytosol by testosterone and dihydrotestosterone was potentiated and this may imply that the binding observed in the presence of triamcinolone acetonide was responsible for the binding of the androgen receptor. In the nuclear extract, the binding was attributable mainly to the androgen receptor irrespective of the presence or absence of triamcinolone acetonide. These properties of the binding observed in the hypertrophic human prostate were almost same as those of the binding with R 1881. Although maximum binding sites measured using mibolerone were correlated with those using R 1881 in the cytosols as well as in the nuclear extracts, the values obtained with mibolerone were slightly greater than those with R 1881. Thus, mibolerone seems to be a suitable ligand for measuring the androgen receptor, but when compared with R 1881 no special merits in using mibolerone were detected.     

Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution

The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs–Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.     

Effect of Bordetella pertussis leukocytosis (lymphocytosis)-promoting factor (LPF) on the physical lymphoepithelial-cell association studied with the use of an in vitro model of mouse thymus

The effect of highly purified leukocytosis (lymphocytosis)-promoting factor (LPF) of Bordetella pertussis on physical lymphocyte and reticuloepithelial (RE) cell association was studied in an in vitro thymus model. First, a simplified in vitro system to assess the lympho-RE-cell association was developed. A completely confluent layer of thymic RE cells was formed by cultivating trypsinized thymus cell suspensions from 2- to 7-day-old mice. When thymic lymphoid cells were seeded on this cell layer and cultivated overnight, a significant proportion of them were found underneath the RE cell layer. This physical lympho-RE-cell association was quantitated by counting the lymphoid cells underneath the RE cell layers. Second, the effect of LPF on this physical lympho-RE-cell association phenomenon was investigated. Addition of LPF to the culture markedly inhibited the formation of the lympho-RE-cell complex; that is, it inhibited the infiltration of lymphoid cells under the RE cell layer. LPF rendered a nearly maximal level of inhibitory effect at a dose of 0.1 ng/ml. Furthermore, LPF enhanced the liberation of lymphoid cells from preformed lympho-RE-cell complexes. On the other hand, LPF had no direct cytotoxic effect on lymphoid cells at doses below 1 microgram/ml. In order to investigate whether LPF produced the effect by acting on lymphoid cells, RE cells, or both, the following experiments were performed. When lymphoid cells were pretreated with LPF and added to normal RE cell layers, the lympho-RE-cell association was maximally inhibited above the dose of 1 ng/ml. Treatment of these LPF-treated lymphoid cells with anti-LPF antibodies failed to abrogate the effect of LPF. When RE cell layers were similarly pretreated with LPF and were cultivated with normal lymphoid cells, however, much higher doses of LPF, above 100 ng/ml, were required for maximal inhibition. Furthermore, treatment of these LPF-treated RE cells with anti-LPF antibodies abrogated the effect of LPF. Therefore, the apparent effect of LPF on RE cells was considered to be due to the carry-over by RE cells of LPF, which should directly act on lymphoid cells at extremely low doses. On the basis of these results, it was concluded that LPF acted directly on lymphoid cells without mediation of RE cells. These in vitro results appear to parallel the effects of LPF in vivo, where it induces a depletion of cells in the thymus. The model may be useful to study this phenomenon and the concomitant accumulation of blood lymphocytes.     

Effect of dietary oils enriched with n-3 fatty acids on survival of mice

Female mice were fed a conventional diet, shifted at 119 days of age to a diet supplemented with 10 wt % lard (Lar), high-linoleic (n-6) safflower oil (Saf), rapeseed oil (low-erucic, Rap), high-alpha-linolenic (n-3) perilla oil (Per) or a mixture (1:9) of ethyl docosahexaenoate (n-3) and soybean oil (DHA/Soy). Weight gain was less in the Per group than in the other groups at 497 days of age. In the Rap group, proteinuria was more severe than in the Saf, Per and DHA/Soy group, and hepatic triacylglycerol accumulation was greater than in the other groups. The mean survival time of the DHA/Soy group (753 days) was significantly longer than in the Lar group (672 days) and Saf group (689 days); the differences among other groups (e.g., 701 days in the Per group and 712 days in the Rap group) were not statistically significant. Although DHA is more susceptible to auto-oxidation than other major fatty acids in the air, an oil containing DHA was found to increase the survival of mice. Rapeseed oil that decreases the survival time of SHRSP rats was found to be safe in the mouse strain used in this study when survival was an end point.     

Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.