A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus

We have characterized a family of GHF45 cellulases from the pine wood nematode Bursaphelenchus xylophilus. The absence of such genes from other nematodes and their similarity to fungal genes suggests that they may have been acquired by horizontal gene transfer (HGT) from fungi. The cell wall degrading enzymes of other plant parasitic nematodes may have been acquired by HGT from bacteria. B. xylophilus is not directly related to other plant parasites and our data therefore suggest that horizontal transfer of cell wall degrading enzymes has played a key role in evolution of plant parasitism by nematodes on more than one occasion.     

Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo

Silencing gene expression by siRNAs is rapidly becoming a powerful tool for the genetic analysis of mammalian cells. However, the rapid degradation of siRNA and the limited duration of its action call for an efficient delivery technology. Accordingly, we describe here that Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing. Site-specific in vivo administration of an anti-luciferase siRNA/Atelocollagen complex reduced luciferase expression in a xenografted tumor. Furthermore, Atelocollagen-mediated transfer of siRNA in vivo showed efficient inhibition of tumor growth in an orthotopic xenograft model of a human non-seminomatous germ cell tumor. Thus, for clinical applications of siRNA, an Atelocollagen-based non-viral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.     

JNK inhibitory kinase is up-regulated in retinal ganglion cells after axotomy and enhances BimEL expression level in neuronal cells

Optic nerve transection results in retinal ganglion cell (RGC) death in adult mammals, after the alteration of gene expression of RGCs. To elucidate the molecular mechanism by which axotomy induces RGC death, we isolated the molecules up-regulated after optic nerve transection. One of these, axotomy-related [corrected] gene (ARG)357, an 898-amino-acid [corrected] protein containing a complete serine-threonine kinase domain, was isolated from a subtraction library of the rat retina. The sequence showed that this gene was a rat homolog of human c-Jun N-terminal kinase (JNK) inhibitory kinase and so belonged to the germinal center kinase-VIII subfamily of Sterile20s protein kinase. We designated ARG357 as rat JNK inhibitory kinase (JIK). Rat JIK was expressed ubiquitously in various tissues and was highly expressed in the retina, with selective expression in RGCs. After axotomy, BimEL and Hrk, which are BH3-only proteins, and rat JIK were up-regulated in RGCs. Overexpression of rat JIK in neuronal cells up-regulated the expression of BimEL, but not that of Hrk. These results indicate that JIK may contribute to axotomy-induced RGC death by up-regulating the expression of BH3-only protein.     

Production,Binding Characteristics and Protective Immunity of Monoclonal Antibody to Pneumococcal Type-9V Conjugate

A monoclonal antibody (MAb) to pneumococcal type-9V polysaccharide (PS) was produced using PS conjugated to inactivated pneumolysin as the immunogen. The MAb to 9V PS was of the IgG₁ subclass. The antigen-antibody reaction increased rapidly at low concentrations and reached a plateau at 10 μg PS/ml as measured by nephelometry of the group 9 PS against 9V MAb binding. In contrast, the binding of group 9 PS against rabbit 9V antiserum (AS) increased proportionally and continued to increase up to the highest concentration of PS tested (20 μg PS/ml). The 9V MAb reacted with all group 9 PSs (9A, 9L, 9N and 9V) by immunodiffusion. In the homologous 9V Ag-MAb reaction, there were marked differences in the inhibition of binding by the cross-reactive 9L PS (19.2% inhibition) and the 9N PS (0.2%). In contrast, inhibition of the homologous 9V Ag-rabbit AS binding by cross-reactive 9L and 9N PSs ranged from 57.8 to 62.7%. Removal of the O-acetyl group from 9V PS by alkali hydrolysis resulted in decreased binding with rabbit 9V AS. However, the binding reaction with 9V MAb was less affected by the loss of O-acetyl content. The 9V MAb was both opsonic and passively protected young mice against challenge with type-9V pneumococci.     

Presence of a Tetrahymena growth promoting activity in fetal bovine serum

Treatment of cultures of the ciliate Tetrahymena with fetal bovine serum (FBS) enhanced the rate of cell proliferation. The growth promoting activity was partially purified from FBS as a high Mr complex including four components with apparent Mr values of 180 kDa, 68 kDa, 60 kDa and 30 kDa by a 4-step procedure. The 180 kDa component was identified by amino acid sequencing as α2-macroglobulin. The addition of purified α2-macroglobulin from bovine plasma to cultures of Tetrahymena was also found to enhance the rate of cell proliferation. This report is the first dealing with the direct identification of a mammalian factor which promotes the growth of free-living protozoa.     

Human thermoregulatory responses during prolonged walking in water at 25, 30 and 35°C

Eight healthy and physically well-trained male students exercised on a treadmill for 60 min while being immersed in water to the middle of the chest in a laboratory flowmill. The water velocity was adjusted so that the intensity of exercise correspond to 50% maximal oxygen uptake of each subject, and experiments were performed once at each of three water temperatures: 25, 30, 35°C, following a 30-min control period in air at 25°C, and on a treadmill in air at an ambient temperature of 25°C. Thermal states during rest and exercise were determined by measuring rectal and skin temperatures at various points, and mean skin temperatures were calculated. The intensity of exercise was monitored by measuring oxygen consumption, and heart rate was monitored as an indicator for cardiovascular function. At each water temperature, identical oxygen consumption levels were attained during exercise, indicating that no extra heat was produced by shivering at the lowest water temperature. The slight rise in rectal temperature during exercise was not influenced by the water temperature. The temperatures of skin exposed to air rose slightly during exercise at 25°C and 30°C water temperature and markedly at 35°C. The loss of body mass increased with water temperature indicating that both skin blood flow and sweating during exercise increased with the rise in water temperature. The rise in body temperature provided the thermoregulatory drive for the loss of the heat generated during exercise. Heart rate increased most during exercise in water at 35°C, most likely due to enhanced requirements for skin blood flow. Although such requirements were certainly smallest at 25°C water temperature, heart rate at this temperature was slightly higher than at 30°C suggesting reflex activation of sympathetic control by cold signals from the skin. There was a significantly greater increase in mean skin and rectal temperatures in subjects exercising on the treadmill in air, compared to those exercising in water at 25°C. Accepted: 22 May 1998     

Biologically active oligodeoxyribonucleotides. Part 11: The least phosphate-modification of quadruplex-forming hexadeoxyribonucleotide TGGGAG, bearing 3′- and 5′-end-modification, with anti-HIV-1 activity

We have found that a hexadeoxyribonucleotide (5′TGGGAG3′, R-95288), Koizumi, M. et al. Bioorganic & Medicinal Chemistry, 1997, 5, 2235, bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5′-end and a 2-hydroxyethylphosphate at the 3′-end, has high anti-HIV-1 activity and the least cytotoxicity in vitro and in vivo. In order to synthesize more potent hexadeoxyribonucleotides, we substituted phosphodiester (P---O) bonds in the 6-mer with the least phosphorothioate (P---S), phosphoramidate (P---N), or methylphosphonate (P---Me) bonds. When more than two P---N or P---Me bonds were introduced into a 6-mer, the phosphate-modified 6-mers had weak or no anti-HIV-1 activity, in spite of quadruplex structure formation. However, when P---S bonds were substituted for P---O bonds, anti-HIV-1 activity of their 6-mers did not dramatically decrease, compared with compounds substituted with P---N or P---Me bonds. The results suggest that the formation of a quadruplex structure is not always sufficient for anti-HIV-1 activity of the 6-mer, and that net negative charges derived from P---O or P---S bonds in the quadruplex are important for anti-HIV-1 activity. Moreover, among various phosphate-modified ODNs, we found that the anti-HIV-1 activity of ODN PS7 with only one P---S bond was the same as that of R-95288, both having a high stability in human plasma.     

F9 Cells Can be Differentiated toward Two Distinct, Mutually Exclusive Pathways by Retinoic Acid and Sodium Butyrate

Both retinoic acid (RA) and sodium butyrate (NaB) induce differentiation in embryonal carcinoma F9 cells. Phenotypic changes caused by RA are irreversible, whereas those of NaB are rapid and reversible. In this study, we investigated the effects of combinations of these two agents on F9 cell differentiation and showed that RA had no effect on the cells induced to differentiate with NaB and vice versa. Thus, F9 cells are induced to differentiate along two distinct pathways which are mutually exclusive.     

Potential involvement of drought-induced Ran GTPase CLRan1 in root growth enhancement in a xerophyte wild watermelon

Enhanced root growth is known as the survival strategy of plants under drought. Previous proteome analysis in drought-resistant wild watermelon has shown that Ran GTPase, an essential regulator of cell division and proliferation, was induced in the roots under drought. In this study, two cDNAs were isolated from wild watermelon, CLRan1 and CLRan2, which showed a high degree of structural similarity with those of other plant Ran GTPases. Quantitative RT-PCR and promoter-GUS assays suggested that CLRan1 was expressed mainly in the root apex and lateral root primordia, whereas CLRan2 was more broadly expressed in other part of the roots. Immunoblotting analysis confirmed that the abundance of CLRan proteins was elevated in the root apex region under drought stress. Transgenic Arabidopsis overexpressing CLRan1 showed enhanced primary root growth, and the growth was maintained under osmotic stress, indicating that CLRan1 functions as a positive factor for maintaining root growth under stress conditions.