Cytotoxicity of a GalNAc-specific C-type lectin CEL-I toward various cell lines

We found that CEL-I was a potent cytotoxic lectin. MDCK, HeLa, and XC cells were highly sensitive to CEL-I cytotoxicity and killed in a dose-dependent manner, whereas CHO, L929, and RAW264.7 cells were relatively resistant to CEL-I, and no significant toxicity was observed up to 10 microg/ml. Among these cell lines, MDCK cells showed the highest susceptibility to CEL-I cytotoxicity. A binding study using FITC-labeled CEL-I (F-CEL-I) revealed that the amounts of bound F-CEL-I on the sensitive cell lines were evidently greater than those on the resistant cell lines, suggesting that the different susceptibility of the cell lines to CEL-I cytotoxicity is partly explained by different efficiencies of binding of CEL-I to these cell lines. Interestingly, the cytotoxicity of CEL-I toward MDCK cells was more potent than those of other lectins such as WGA, PHA-L, and Con A, even though these lectins were capable of binding to MDCK cells at comparable levels to CEL-I. Since the cytotoxicity of CEL-I was strongly inhibited by GalNAc, the binding to cell surface specific carbohydrates is essential for the CEL-I cytotoxicity. The trypan blue dye exclusion test indicated that CEL-I caused a disorder of plasma membrane integrity as a relatively early event. CEL-I failed to induce the release of carboxyfluorescein (CF) from CF-loaded MDCK cells as seen for pore-forming hemolytic isolectin CEL-III, suggesting that the primary cellular target of CEL-I may be the plasma membrane, but its action mechanism differs from that of CEL-III. Although CEL-I induced dramatic cellular morphological changes in MDCK cells, neither typical apoptotic nuclear morphological changes nor DNA fragmentation was observed in CEL-I-treated MDCK cells even after such cellular changes. Our results demonstrated that CEL-I showed a potent cytotoxic effect, especially on MDCK cells, by causing plasma membrane disorder without induction of apoptosis.     

cda1+, encoding chitin deacetylase is required for proper spore formation in Schizosaccharomyces pombe

In Schizosaccharomyces pombe, a major role of chitin is to build up a complete spore. Here, we analyzed the cda1(+) gene (SPAC19G12.03), which encodes a protein homologous to chitin deacetylases, to know whether it is required for spore formation in S. pombe. The homothallic Deltacda1 strain constructed by homologous recombination was found to form a little amount of abnormal spores that contained one, two, or three asci, similar to (but not as strong as) the phenotype observed in a deletion mutant of chs1 encoding chitin synthase 1. This phenotype is reversed by expression of S. cerevisiae chitin deacetylase CDA1 or CDA2, suggesting that cda1 encodes a chitin deacetylase. To support the role of Cda1 in sporulation, the timing of expression of cda1(+) mRNA increased during sporulation process. We also found that the Cda1 protein self-associated when its binding was tested both by two-hybrid system and immunoprecipitation. Thus, these data indicated that cda1(+) is required for proper spore formation in S. pombe.     

Adrenomedullin induces matrix metalloproteinase-2 activity in rat aortic adventitial fibroblasts

Background

The delicate balance of the extracellular matrix (ECM) determines the stiffness of the vascular wall, and adventitial fibroblasts are involved in ECM formation by synthesizing and degrading matrix proteins. In the present study, we examined the effect of the bioactive peptide adrenomedullin (AM) on activity and expression of matrix metalloproteinases (MMPs) in cultured aortic adventitial fibroblasts.

Methods and results

In cultured adventitial fibroblasts isolated from aorta of adult Wistar rats, 10⁻⁶ mol/L angiotensin II (Ang II) significantly (p < 0.05) down-regulated MMP-2 activity as determined by in vitro gelatin zymography. In contrast, 10⁻⁷ mol/L synthetic rat AM significantly (p < 0.05) stimulated zymographic MMP-2 activity by 23%, increasing intracellular cAMP, and AM abolished the action of Ang II, augmenting the MMP-2 activity. Similarly, Ang II down-regulated MMP-2 protein expression assessed by Western blotting, whereas AM increased it. Furthermore, 8-bromo-cAMP, an analogue of cAMP, mimicked the effect of AM, and H-89, an inhibitor for protein kinase A (PKA), significantly decreased the basal and AM-induced MMP-2 activity.

Conclusion

This study provides a new insight into the biological action of AM and its intracellular signaling system of cAMP/PKA stimulating the matrix degrading enzyme MMP-2, suggesting an important role for this molecule in modulating ECM deposition in the adventitial layer.     

Novel metabolism of 1 alpha,25-dihydroxyvitamin D3 with C24-C25 bond cleavage catalyzed by human CYP24A1

Our previous study revealed that human CYP24A1 catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway [Sakaki, T., Sawada, N., Komai, K., Shiozawa, S., Yamada, S., Yamamoto, K., Ohyama, Y. and Inouye, K. (2000) Eur. J. Biochem. 267, 6158-6165]. In this study, by using the Escherichia coli expression system for human CYP24A1, we identified 25,26,27-trinor-23-ene-D(3) and 25,26,27-trinor-23-ene-1alpha(OH)D(3) as novel metabolites of 25(OH)D(3) and 1alpha,25(OH)(2)D(3), respectively. These metabolites appear to be closely related to the C-23 hydroxylation pathway, because human CYP24A1 produces much more of these metabolites than does rat CYP24A1. We propose that the C(24)-C(25) bond cleavage occurs by a unique reaction mechanism including radical rearrangement. Namely, after hydrogen abstraction of the C-23 position of 1alpha,25(OH)(2)D(3), part of the substrate-radical intermediate is converted into 25,26,27-trinor-23-ene-1alpha(OH)D(3), while a major part of them is converted into 1alpha,23,25(OH)(3)D(3). Because the C(24)-C(25) bond cleavage abolishes the binding affinity of 1alpha,25(OH)D(3) for the vitamin D receptor, this reaction is quite effective for inactivation of 1alpha,25(OH)D(3).     

Metabolism of A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 by CYP24A1

The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1alpha,3alpha) and (1beta,3alpha) was 1:1, although the ratio for (1alpha,3beta) and (1beta,3beta) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1alpha,3beta)>(1alpha,3alpha)>(1beta,3beta)>(1beta,3alpha), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs.     

Inhibition of poly (ADP-ribose) polymerase and caspase-3, but not caspase-1, prevents apoptosis and improves spatial memory of rats with twice-repeated cerebral ischemia

The effect of inhibition of PARP [(poly (ADP-ribose) polymerase], caspase-3 and caspase-1 on twice-repeated ischemia-induced apoptosis and memory impairment were examined. The twice repeated ischemia was induced by four-vessel occlusion method in which a 10 min ischemic episode was repeated once after 60 min. The spatial memory was assessed using 8-arm radial maze. The results of this study showed that the repeated ischemia impaired memory and induced apoptosis in hippocampus CA1 field after 7 days. Moreover, 3-aminobezamide (10 mg/kg i.v.), a PARP inhibitor, and Ac-DEVD-CHO (8.4 microg/5 microL i.c.v., bilaterally), a caspase-3 inhibitor, decreased apoptosis by 45% and 58% respectively. Both drugs reduced the error choices, but 3-aminobezamide additionally increased the correct choices and improved the memory when either drug was injected immediately after the ischemic insult. The results also showed that inhibition of interleukin-1beta-converting enzyme, ICE (caspase-1) by Z-ASP-DCB-CH2 (100 microg/kg i.c.v., bilaterally) neither decreased apoptosis (13% reduction) nor improved memory of the ischemic rats. These results suggest that direct inhibition of PARP and caspase-3, but not of caspase-1, prevents apoptosis and improves spatial memory impaired by repeated ischemia.     

Expression of mouse Coiled-coil-DIX1 (Ccd1), a positive regulator of Wnt signaling, during embryonic development

Wnt signaling plays an important role in cell growth, differentiation, polarity formation, and neural development. We have recently identified the Coiled-coil-DIX1 (Ccd1) gene encoding a third type of a DIX domain-containing protein. Ccd1 forms homomeric and heteromeric complexes with Dishevelled and Axin, and positively regulates the Wnt/beta-catenin pathway. Here, we examined the spatiotemporal expression pattern of Ccd1 mRNA in mouse embryos from embryonic day 6.5 (E6.5) to E17.5 by in situ hybridization. Ccd1 expression was detected in the node region in gastrula embryos, in the cephalic mesenchyme and tail bud at E8.5, and in the branchial arch and forelimb bud at E9.5. In the central nervous system, Ccd1 expression began and persisted in the regions where the neurons differentiated, so that it was observed throughout the brain and spinal cord at E17.5. Ccd1 expression was also strong in the peripheral nervous system, including sensory cranial ganglia (trigeminal, facial, and vestibulocochlear ganglia), dorsal root ganglia, and autonomic ganglia (sympathetic ganglia, celiac ganglion, and hypogastric plexus). Ccd1 was detected in the sensory organs, such as the inner nuclear layer of the neural retina, saccule and cochlea of the inner ear, and nasal epithelium. Outside the nervous system, Ccd1 mRNA was observed in the cartilage, tongue, lung bud, stomach, and gonad at E12.5-E14.5, and in the tooth bud, bronchial epithelium, and kidney at E17.5. Taken together, these findings demonstrate that Ccd1 expression is observed in all the neurons in the nervous system, closely associated with neural crest-derived tissues, and largely overlapping with the regions where several Wnt genes are reported to play a role.     

Life history of the nonnative convict cichlid <Emphasis Type="Italic">Amatitlania nigrofasciata</Emphasis> in the Haebaru Reservoir on Okinawa-jima Island,Japan

Life history parameters associated with reproductive biology, age, and growth of the convict cichlid (also known as the zebra cichlid) Amatitlania nigrofasciata, which was introduced into the Haebaru Reservoir on Okinawa-jima Island, were estimated using 437 specimens that ranged from 13.7 to 82.9 mm standard length (SL). Lengths of females at first maturity (SL) and 50% maturity (L ₅₀) were estimated to be 32.2 and 37.3 mm SL, respectively. The spawning period continued throughout the year, with a peak spawning cycle from March to May 2006–2007. Observations of postovulatory follicles and tertiary yolk stage oocytes indicate that convict cichlids spawn multiple times within a year. Female cichlids that hatched during the peak spawning seasons matured after October of the same year. Batch fecundity of females (32.2–61.2 mm SL) ranged from 65 to 345 (mean ± SD = 155 ± 63). Opaque zones along the outer margins of otoliths formed annually. The maximum age of male and female cichlids was 3 years. The von Bertalanffy growth formulae (VBGF) were expressed as L_t = 57.4( 1 - e ^{- 0.78( t + 0.91 )} ) {{\hbox{L}}_{\rm{t}}}{ = 57}{.4}\left( {1 - {e^{ - 0.78\left( {t + 0.91} \right)}}} \right) for females and L_t = 69.5( 1 - e ^{- 1.07( t + 0.24 )} ) {{\hbox{L}}_{\rm{t}}}{ = 69}{.5}\left( {1 - {e^{ - 1.07\left( {t + 0.24} \right)}}} \right) for males. Males grew larger than females beginning from the first year. Certain life history characteristics, such as year-round spawning and early maturation, probably contributed to the successful establishment of the convict cichlid, and this species in particular is thought to adapt and become established quickly upon introduction to freshwater systems on Okinawa-jima Island.