Down-regulation of osteoprotegerin production in bone marrow macrophages by macrophage colony-stimulating factor

Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) induce the differentiation of bone marrow macrophages (BMMs) into osteoclasts. To delineate mechanisms involved, the effect of M-CSF on the production of osteoprotegerin (OPG), decoy receptor of RANKL, in BMMs was investigated. Mouse bone marrow cells were cultured with M-CSF for 4 days and adherent cells formed were used as BMMs. BMMs were cultured with or without M-CSF, and analyzed for expression of OPG and receptor activator of NF-kappaB (RANK; receptor for RANKL) mRNAs by real-time polymerase chain reaction and secretion of OPG by enzyme-linked immunosorbent assay. BMMs expressed macrophage markers, CD115 (c-fms), Mac-1 and F4/80, and showed phagocytotic activity. In addition, BMMs expressed OPG mRNA and secreted OPG into medium. M-CSF inhibited both the OPG mRNA expression and the OPG secretion dose-dependently and reversibly. The expression of RANK mRNA was not significantly affected by M-CSF. The results showed that M-CSF suppresses the OPG production in BMMs, which may increase the sensitivity of BMMs to RANKL.     

Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.     

Temporal progression of hypothalamic patterning by a dual action of BMP

In the developing chick hypothalamus, Shh and BMPs are expressed in a spatially overlapping, but temporally consecutive, manner. Here, we demonstrate how the temporal integration of Shh and BMP signalling leads to the late acquisition of Pax7 expression in hypothalamic progenitor cells. Our studies reveal a requirement for a dual action of BMPs: first, the inhibition of GliA function through Gli3 upregulation; and second, activation of a Smad5-dependent BMP pathway. Previous studies have shown a requirement for spatial antagonism of Shh and BMPs in early CNS patterning; here, we propose that neural pattern elaboration can be achieved through a versatile temporal antagonism between Shh and BMPs.     

Unexpected A-form formation of 4'-thioDNA in solution, revealed by NMR, and the implications as to the mechanism of nuclease resistance

Fully modified 4′-thioDNA, an oligonucleotide only comprising 2′-deoxy-4′-thionucleosides, exhibited resistance to an endonuclease, in addition to preferable hybridization with RNA. Therefore, 4′-thioDNA is promising for application as a functional oligonucleotide. Fully modified 4′-thioDNA was found to behave like an RNA molecule, but no details of its structure beyond the results of circular dichroism analysis are available. Here, we have determined the structure of fully modified 4′-thioDNA with the sequence of d(CGCGAATTCGCG) by NMR. Most sugars take on the C3′-endo conformation. The major groove is narrow and deep, while the minor groove is wide and shallow. Thus, fully modified 4′-thioDNA takes on the A-form characteristic of RNA, both locally and globally. The only structure reported for 4′-thioDNA showed that partially modified 4′-thioDNA that contained some 2′-deoxy-4′-thionucleosides took on the B-form in the crystalline form. We have determined the structure of 4′-thioDNA in solution for the first time, and demonstrated unexpected differences between the two structures. The origin of the formation of the A-form is discussed. The remarkable biochemical properties reported for fully modified 4′-thioDNA, including nuclease-resistance, are rationalized in the light of the elucidated structure.     

Enzymatic repair of 5-formyluracil. II. Mismatch formation between 5-formyluracil and guanine during dna replication and its recognition by two proteins involved in base excision repair (AlkA) and mismatch repair (MutS).

5-Formyluracil (fU), a major methyl oxidation product of thymine, forms correct (fU:A) and incorrect (fU:G) base pairs during DNA replication. In the accompanying paper (Masaoka, A., Terato, H., Kobayashi, M., Honsho, A., Ohyama, Y., and Ide, H. (1999) J. Biol. Chem. 274, 25136-25143), it has been shown that fU correctly paired with A is recognized by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II). In the present work, mispairing frequency of fU with G and cellular repair protein that specifically recognized fU:G mispairs were studied using defined oligonucleotide substrates. Mispairing frequency of fU was determined by incorporation of 2'-deoxyribonucleoside 5'-triphosphate of fU opposite template G using DNA polymerase I Klenow fragment deficient in 3'-5' exonuclease. Mispairing frequency of fU was dependent on the nearest neighbor base pair in the primer terminus and 2-12 times higher than that of thymine at pH 7.8 and 2.6-6.7 times higher at pH 9.0 with an exception of the nearest neighbor T(template):A(primer). AlkA catalyzed the excision of fU placed opposite G, as well as A, and the excision efficiencies of fU for fU:G and fU:A pairs were comparable. In addition, MutS protein involved in methyl-directed mismatch repair also recognized fU:G mispairs and bound them with an efficiency comparable to T:G mispairs, but it did not recognize fU:A pairs. Prior complex formation between MutS and a heteroduplex containing an fU:G mispair inhibited the activity of AlkA to fU. These results suggest that fU present in DNA can be restored by two independent repair pathways, i.e. the base excision repair pathway initiated by AlkA and the methyl-directed mismatch repair pathway initiated by MutS. Biological relevance of the present results is discussed in light of DNA replication and repair in cells.     

Properties of the erythrocyte receptors for influenza C virus.

Properties of the receptor for influenza C virus were studied. Although the receptor for influenza C virus on chicken erythrocytes was destroyed by the homologous virion, neuraminidase activity could not be detected in any of the influenza C virus strains tested. The receptor activity of chicken erythrocytes for influenza C virus was diminished by formaldehyde treatment but not by periodate oxidation. There was a considerable variation in the pattern and the titer of hemagglutination of influenza C virus when human erythrocytes of different blood types were used; the virus agglutinated most type B erythrocytes but not type A erythrocytes. By using human type B erythrocytes, differences among strains of influenza C virus in the hemagglutinating activity were also demonstrated. These results showed that both the receptor for and the receptor-destroying activity of influenza C virus were completely different from those of influenza A or B virus and also that carbohydrates were not involved in the receptor for influenza C virus.