Dissection of fission yeast microtubule associating protein p93Dis1: regions implicated in regulated localization and microtubule interaction

Background : Fission yeast microtubule associating protein (MAP) p93^Dis1 functions for sister chromatid separation: dis1 mutants fail to separate chromosomes, while the spindle elongates but without cyclin destruction. p93^Dis1 localizes along microtubules in interphase cytoplasm, but shifts to the spindle pole body (SPB) and spindle microtubules upon the entry into mitosis. In this study, regions of p93^Dis1 were dissected to examine their role.
Results : Nitrocellulose filter blotting shows that recombinant Dis1 binds to bovine brain microtubules in vitro . A basic central region rich in S, T and P is essential for this association. However, the whole p93^Dis1 with N- and C-termini containing a conserved repeat motif and heptad repeats, respectively, is necessary for normal microtubule association in vivo . The N-truncated region also binds to microtubules but only to the portions near the SPBs. Overproduction phenotypes indicate that p93^Dis1 greatly affects spindle formation and cell morphogenesis. The central region is essential but, by itself, not sufficient for generating such effects.
Conclusions : We propose that p93^Dis1 consists of three regions which carry distinct properties for localization: the N-region for cell cycle dependent localization, the central region for direct microtubule association, and the C-region for SPB and nuclear localization. The essential role of p93^Dis1 is carried out in the C-region, while the N-region acts as a regulator.     

Enhanced production of transforming growth factor-beta (TGF-beta) during autologous mixed lymphocyte reaction of systemic sclerosis patients.

Systemic sclerosis (SSc) is characterized by systemic fibrosis and microvascular lesions. As TGF-beta is suggested to be related to skin fibrosis, we examined the production of TGF-beta from peripheral mononuclear cells (MNC) of SSc patients. Since anti-TGF-beta neutralizing antibody improved the defective proliferative response in autologous mixed lymphocyte reaction (AMLR) of SSc patients, TGF-beta was thought to participate in the decreased AMLR of SSc patients. Greater amounts of TGF-beta in the active as well as in the latent forms were produced during AMLR of SSc patients than that of normal subjects. It was suggested that TGF-beta excessively produced from the MNC of SSc patients might play a major role in the fibrosis of the patients during AMLR-like in vivo responses.     

Induction of fibroblast-like cells from CD34(+) progenitor cells of the bone marrow in rheumatoid arthritis

To assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA), we examined the capacity of CD34(+) cells from bone marrow to generate fibroblast-like type B synoviocytes. CD34(+) cells from the bone marrow of 22 RA patients differentiated into cells with fibroblast-like morphology, which expressed prolyl 4-hydroxylase, in the presence of stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha), much more effectively than CD34(+) cells from bone marrow of 15 control subjects (10 patients with osteoarthritis and 5 healthy individuals). The generation of fibroblast-like cells was not at all observed in cultures with SCF, GM-CSF, and interleukin 4 (IL-4) with or without TNF-alpha. Generation of fibroblast-like cells was correlated with matrix metalloproteinase (MMP)-1 levels in culture supernatants. Thus, MMP-1 levels were significantly higher in TNF-alpha-stimulated cultures of bone marrow CD34(+) cells from patients with RA than in those from the control group. These results indicate that bone marrow CD34(+) cells from patients with RA have abnormal capacities to respond to TNF-alpha and to differentiate into fibroblast-like cells producing MMP-1, suggesting that bone marrow CD34(+) progenitor cells might generate type B synoviocytes and thus could play an important role in the pathogenesis of RA.     

Influence of sperm immobilization on onset of Ca(2+) oscillations after ICSI

Sperm immobilization prior to intracytoplasmic sperm injection (ICSI) is thought to be necessary for efficient fertilization. A variety of methods of sperm immobilization (pipetting, squeezing and piezo application) are currently employed in ICSI. The effect of differences in immobilization method on the timing of initial Ca(2+) oscillations of oocytes in ICSI was investigated. Motile spermatozoa were immobilized in eosin Y solution using pipetting, squeezing and piezo application. Complete staining of the sperm head was achieved after 220.7, 42.2 and 5.0 s respectively. Oscillations after ICSI were measured fluorometrically for each method. The onset of Ca(2+) oscillations was observed at 4.8 to 80.4 min after ICSI. Ca(2+) oscillations developed earlier with the piezo method (14.4 +/- 6.4 min) than other methods (pipetting, 43.1 +/- 20.2 min, P < 0.01; squeezing, 18.4 +/- 3.8 min, P = NS). The piezo method produced the earliest staining of the sperm head and may have caused the most damage to the sperm membrane. A more rapid onset of Ca(2+) oscillations was also observed with the piezo method. The method of sperm immobilization may be important for the rapid release of sperm factors that initiate oocyte activation. This study also showed that Ca(2+) oscillations develop earlier in human oocytes treated by ICSI than indicated in previous reports.     

IgE antibody to fish gelatin (type I collagen) in patients with fish allergy

BACKGROUND: Most children with anaphylaxis to measles, mumps, and rubella vaccines had shown sensitivity to bovine gelatin that was included in the vaccines. Recently, it was found that bovine type I collagen, which is the main content in the gelatin, is a major allergen in bovine gelatin allergy. Fish meat and skin also contain type I collagen. OBJECTIVE: The present study was designed to investigate IgE antibody to fish gelatin in children with fish allergy. METHODS: Serum samples were taken from patients in 3 groups: (1) 10 patients with fish allergy and specific IgE to fish meat; (2) two patients with allergies to both fish meat and bovine gelatin and specific IgE to fish meat and bovine gelatin; and (3) 15 patients with atopic dermatitis and specific IgE to fish meat. Various fish gelatins (type I collagen) were prepared from fish skin. IgE antibody to fish gelatin was analyzed by using ELISA and immunoblotting. RESULTS: Of 10 patients with fish allergy, 3 had specific IgE to fish gelatin. Of two patients with fish allergy and bovine gelatin allergy, all had specific IgE to fish gelatin. Of 15 patients with atopic dermatitis and specific IgE to fish meat, 5 had specific IgE to fish gelatin. Furthermore, IgE from pooled serum of the patients reacted with both the alpha1 and alpha2 chains of fish type I collagen in immunoblots. There is cross-reactivity among gelatins from various fishes, but there is little cross-reactivity between fish and bovine gelatins. CONCLUSION: Some fish-sensitive patients possessed IgE antibody to fish gelatin. Fish gelatin (type I collagen) might be an allergen in subjects with fish allergy.     

Dibutyrylcytidine 3',5'-cyclic monophosphate stimulates neurite outgrowth in rat pheochromocytoma PC12

Dibutyrylcytidine 3',5'-cyclic monophosphate (Bt2cCMP) stimulated neurite outgrowth in rat pheochromocytoma PC12 cells in a dose-dependent manner at the dose range from 0.5 mM to 6 mM. About 25% of the cells had neurites in response to 6 mM Bt2cCMP. In contrast with the effect of nerve growth factor (NGF) which increased the percentage of cells with neurites gradually during a 6-day culture period, the stimulating effect of Bt2cCMP reached a plateau 2 days after plating. Staurosporine inhibited the neurite outgrowth induced by NGF, but not that by Bt2cCMP. These results suggest that Bt2cCMP stimulates neuronal differentiation by a mechanism different from that by NGF.