Anticoagulant activity of sulfoalkyl derivatives of curdlan.

Curdlan is a natural beta-1,3-glucan produced by Agrobacterium biovar 1. In this study, the anticoagulant activity of sulfoalkyl derivatives of curdlan was investigated by carrying out activated partial thromboplastin time (APTT) assay and compared with that of o-sulfonated curdlan. Approximately 100-fold higher concentration of o-sulfonated curdlan than heparin was required to obtain the same level of the clotting time. Anticoagulant activity of curdlan derivatives was dependent on the degree of sulfation in prolonging the clotting time. However, the chain length of the substituent did not play a role in prolonging the clotting time. The curdlan derivatives enhanced thrombin inhibition by mediating through antithrombin III. The inhibition of thrombin by o-sulfonated curdlan was found to be approximately 10-fold weaker than that by heparin.

Isolation and characterization of proteoglycan derived from human placenta and its biological activities.

Chondroitin sulfates proteoglycans were isolated from human placenta. For the identification of enzymatic digestion products of isolated proteoglycan, strong anion, exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitin ABC and chondroitin B lyase, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (deltaDi-OS), 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (deltaDi-6S) and 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (deltaDi-4S) were produced from the human placenta proteoglycan. The anticoagulant activity of chondroitin sulfate proteoglycan was evaluated by activated partial thromboplastin time (aPTT) assay and thrombin time (TT) assay. The clotting times of aPTT and TT were increased from 72 to 144 sec and 19 to 27 sec, respectively. The immuno-modulating activity of chondroitin sulfate proteoglycan was examined by cell proliferation assay and these results suggest that it may play a role in suppression of the function of immune-related cells.

Isolation and identification of chondroitin sulfates from the mud snail.

Chondroitin sulfates were isolated from the mud snail. For the quantitative analysis of enzymatic digestion products of isolated chondroitin sulfates, strong anion exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitinase ABC, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS), 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S) and 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S) were produced from the mud snail chondroitin sulfates. The analysis showed that relative proportion of delta Di-OS/delta Di-6S/delta Di-4S was 58.7/3.1/38.2. The immunomodulating activity of chondroitin sulfate was examined by cell proliferation assay and these results suggest that it might be a immunosuppressant.

Glucocorticoid regulation of androgen binding protein expression in primary Sertoli cell cultures from rats.

Glucocorticoids are known to inhibit testicular function, and its receptor is also localized in the Sertoli cells. To evaluate possible role of glucocorticoid in Sertoli cells, the effects of dexamethasone on the expression of androgen binding protein (ABP) have been investigated in primary Sertoli cell cultures. Dexamethasone increased ABP mRNA levels, with maximal stimulation reached at 36 hr. The induction of ABP mRNA was dependent on the low concentration (10(-8) and 10(-7) M) of dexamethasone but gradually reduced in the cells treated with high concentration (10(-6) and 10(-5) M). Dexamethasone-induced ABP mRNA level was no change in the cells after addition of cycloheximide but almost reduced by actinomycin-D pretreatment. Steady-state levels of ABP mRNA gradually increased in the Sertoli cells prepared from 14- and 21-days of age corresponding to rat puberty, and ABP mRNA was induced by dexamethasone. These results suggest that ABP gene is transcriptionally regulated by dexamethasone in primary Sertoli cell cultures.

Reduced level of octamer binding transcription factor (Oct-1) is correlated with H2B histone gene repression during differentiation of HL-60 cells by all-trans retinoic acid.

To gain insight on the role of transacting factors in the regulatory mechanism of H2B histone gene expression during the differentiation of HL-60 cells by all-trans retinoic acid (retinoic acid), the binding pattern of the nuclear proteins to various elements in the human H2B histone upstream region has been investigated with DNase I footprinting and DNA mobility shift assay. The level of H2B histone mRNA was markedly reduced at 48 hr in retinoic acid-treated HL-60 cells. The H2B histone mRNA was repressed in proportion to the concentration of retinoic acid. In DNase I footprinting analysis, a nuclear factor (octamer binding transcription factor, Oct-1) bound at -42 bp (ATTTGCAT) both before and after retinoic-acid-induced differentiation of HL-60 cells. One DNA-protein complex was formed by DNA mobility shift assay, and the level of Oct-1 decreased during retinoic-acid-induced differentiation. In the cycloheximide-treated HL-60 cells, the level of Oct-1 also reduced. These results suggest that the transcriptional repression of H2B histone gene during retinoic-acid-induced differentiation in HL-60 cells may be mediated by reduced level of Oct-1.

Promotion of microtubule assembly in vitro by a novel 35-kDa protein purified from human term placenta.

Microtubule assembly promoting-protein (taxol-like protein, TALP) was purified by combination of high salt extraction, phosphocellulose chromatography and hydroxyapatite chromatography from human term placenta. Molecular weight of purified protein was identified as 35kDa on SDS-polyacrylamide gel electrophoresis. In vitro, TALP promoted microtubule assembly in dose-dependent manner in spite of the absence of GTP. Both TALP (0.5 microM) and taxol (10 microM) gave hyperbolic kinetics and shortened the lag time for microtubule polymerization. TALP-stabilized microtubules were resistant to depolymerization by cold (4 degrees C) and CaCl2 (4 mM) like taxol-stabilized microtubules. TALP showed its direct binding on the microtubule in cosedimentation assay. These results suggest that the action mechanism of TALP on the microtubule assembly is similar to taxol in vitro and the binding site of TALP is available on the intact microtubule.

Testosterone regulation of proto-oncogene c-myc expression in primary Sertoli cell cultures from prepubertal rats.

The expression of c-myc has been associated with cell proliferation through changes of nuclear function. To evaluate the possibility that the proto-oncogene c-myc plays a role in testosterone-dependent gene regulation, the effects of testosterone on the expression of c-myc have been investigated in primary Sertoli cell cultures. Testosterone increased c-myc mRNA levels, with maximal stimulation reached in 16 hours. The induction of c-myc mRNA was dependent on the concentration of testosterone. Testosterone-induced c-myc mRNA levels were also increased in cells after addition of cycloheximide but reduced by actinomycin-D pretreatment. Even in the absence of hormone in culture medium, c-myc mRNA was clearly detectable in Sertoli cells from 8-day-old rats but hardly detectable in cells from 14 and 28 days of age. Testosterone stimulated c-myc mRNA expression in the Sertoli cells from only 8-/and 14-day-old rats. These results suggest that testosterone induces c-myc mRNA levels in the primary Sertoli cells from prepubertal rats, and then transient expression of c-myc may be responsible for some of the regulatory roles of testosterone-dependent genes in the Sertoli cells. The biological significance of testosterone-dependent c-myc induction is not known.

Regenerating liver-specific transacting factors of H2B histone gene are newly synthesized during liver regeneration.

We have investigated DNA synthesis and levels of H2B histone mRNA, and the binding pattern of nuclear proteins to various elements in the rat H2B histone gene upstream region with DNase I footprinting assay. Both DNA synthesis and H2B histone mRNA level were increased with maximal stimulation reached at 24 hrs and 36 hrs after partial hepatectomy, respectively. In DNase I footprinting analysis, the nuclear factors interacting with the three elements, TATA at -19 bp (site AR), site B at -29 bp, and CAAT at -69 bp (site C) were required during maximal increase of H2B histone mRNA level after partial hepatectomy. The DNase I protection pattern by nuclear extract of the cycloheximide-treated regenerating liver showed the same results with normal liver. These results suggest that transcriptional regulation of H2B histone gene during liver regeneration may be mediated by nuclear factors that are newly induced by partial hepatectomy.