Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, we characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. We found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts.     

Pharmacokinetics of bestatin and oral activity for treatment of experimental metastases

Summary Bestatin is a low molecular weight aminopeptidase inhibitor originally isolated from culture filtrates of Streptomyces olivoreticuli. The serum pharmacokinetics in mice are dependent on route of administration, with a short t1/2 (1.69 min t1/2 and 12.8 min t1/2), but a high initial serum level following i.v. administration. When administered via the i.p., s.c., i.m., or p.o. routes of administration, bestatin had serum t1/2s of 8.56, 16.91, 19.25, or 15.4 min, respectively. The maximum area under the curve (concentration×time) occurred following i.v. and i.m. administration, with a lower level following p.o. or i.p. administration. Bestatin had therapeutic activity for experimental metastases, not only following i.v., i.p., and i.m. routes of administration but also following oral administration. Because of its brief serum t1/2, bestatin's therapeutic activity depends on aggressive (either daily or twice daily injection, especially following p.o. administration) and high-dose administration. Thus, the rate-limiting aspect of bestatin's therapeutic activity appears to be associated with its pharmacokinetics.This research was supported by the National Cancer Institute, DHHS, under contract No. N01-23910 with Program Resources, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government     

Suramin sensitizing cells to ionizing radiation by inactivating DNA-dependent protein kinase

Here we report that suramin sensitizes LM217, MDA-MB-468, T98G and A431 cells to ionizing radiation. Suramin sensitized cells to X radiation in a dose-dependent fashion, and longer exposure to suramin before X irradiation resulted in more efficient sensitization. The dose-modifying factors calculated from the survival curves were 1.18 in LM217 cells and 1.37 in MDA-MB-468 cells. Suramin did not sensitize Scid cells that had no DNA-dependent protein kinase activity. Suramin inhibited DNA-dependent protein kinase activity in vitro and in vivo. The concentration of suramin resulting in 50% inhibition in vitro was 1.7 microM in LM217 cells and 2.4 microM in MDA-MB-468 cells. Exposure of LM217 and MDA-MB-468 cells to suramin did not affect the level of Ku70 (G22P1) or Ku80 (XRCC5), but it increased the level of DNA-PKcs(PRKDC). Suramin did not sensitize LM217 or MDA-MB-468 cells to UV radiation. Suramin's effects were not caused by accumulation of cells in a specific phase of the cell cycle. These results suggest that suramin sensitizes cells to ionizing radiation by inhibiting DNA-dependent protein kinase activity.     

Isolation of Orientia tsutsugamushi from Leptotrombidium fuji and its characterization

In our attempts to isolate Orientia tsutsugamushi from trombiculid mites, a strain was successfully isolated from Leptotrombidium fuji collected in Aichi Prefecture, Japan. This is the first case of isolation of O. tsutsugamushi from L. fuji. A phylogenetic analysis based on the base-sequence homology of the 56-kDa type-specific antigen-gene indicated that the strain is a new type which is not closely related to any strains analyzed previously. Three strains isolated from Leptotrombidium pallidum harvested at the same area were identified as being closely related to the JP-2 type (subtype-2 of Karp type in Japan) by phylogenetic analysis.     

Development of a genotyping method for potato scab pathogens based on multiplex PCR

Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S-23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.     

Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.     

Isolation of two forms of activated C1s, a subcomponent of the first component of rabbit complement.

Two forms of activated C1s, a subcomponent of the first component of complement, were present in preparations of C1 specifically purified from rabbit serum by affinity chromatography on IgG-Sepharose 6B and were separated by DEAE-cellulose chromatography in the presence of EDTA. These two activated C1s, designated C1s(I) and C1s(II), were indistinguishable with regard to hemolytic activity as well as C1s esterase activity, though they had different molecular weights. C1s(I) had a molecular weight of 106,000, consisting of H and L chains connected by disulfide bonds; the molecular weights of the chains were 70,000 and 36,000, respectively. On the other hand, C1s(II), with a molecular weight of 72,000, consisted of two chains each with a molecular weight of about 37,000, which were also connected by disulfide bonds. These results suggest that, in the case of rabbit C1s, the primary product of activation with C1r, C1s(I), may be susceptible to further cleavage of its H chain without any loss of C1s activity, resulting in the formation of C1s(II), though the active principle responsible for this conversion remains to be elucidated.     

Naftopidil reduced the proliferation of lung fibroblasts and bleomycin‐induced lung fibrosis in mice

Naftopidil, an α‐1 adrenoceptor antagonist with few adverse effects, is prescribed for prostate hyperplasia. Naftopidil inhibits prostate fibroblast proliferation; however, its effects on lung fibroblasts and fibrosis remain largely unknown. Two normal and one idiopathic pulmonary fibrosis human lung fibroblast lines were cultured with various naftopidil concentrations with or without phenoxybenzamine, an irreversible α‐1 adrenoceptor inhibitor. We examined the incorporation of 5‐bromo‐2?‐deoxyuridine into DNA and lactic acid dehydrogenase release by enzyme‐linked immunosorbent assay, cell cycle analysis by flow cytometry, scratch wound‐healing assay, and mRNA expressions of type IV collagen and α‐smooth muscle actin by polymerase chain reaction. Effects of naftopidil on bleomycin‐induced lung fibrosis in mice were evaluated using histology, micro‐computed tomography, and surfactant protein‐D levels in serum. Naftopidil, dose‐dependently but independently of phenoxybenzamine, inhibited 5‐bromo‐2?‐deoxyuridine incorporation in lung fibroblasts. Naftopidil induced G1 cell cycle arrest, but lactic acid dehydrogenase release and migration ability of lung fibroblasts were unaffected. Naftopidil decreased mRNA expressions of type IV collagen and α‐smooth muscle actin in one normal lung fibroblast line. Histological and micro‐computed tomography examination revealed that naftopidil attenuated lung fibrosis and decreased serum surfactant protein‐D levels in bleomycin‐induced lung fibrosis in mice. In conclusion, naftopidil may have therapeutic effects on lung fibrosis.     

Analysis of proteases involved in phagocytic activity of macrophages through the use of various amino acid esters

Inhibitory effects of various amino acid esters on the phagocytic activity of guinea pig peritoneal macrophages were studied with sensitized 51Cr-sheep erythrocytes (51Cr-EAb) as well as 125I-alpha-amylase complexed with homologous IgG2 antibody (Ag-Ab complex). The intracellular uptake of 51Cr-EAb was markedly inhibited by N-acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt), N-acetyl-L-tryptophan ethyl ester (Ac-Trp-OEt) and N-benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt), but not by N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt), N-alpha-acetyl-L-arginine methyl ester (Ac-Arg-OMe), N-alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) or N-alpha-acetyl-L-lysine methyl ester (Ac-Lys-OMe). When phagocytosis of the Ag-Ab complex was assayed by measuring the amount of digested products released from macrophage cells, Ac-Tyr-OEt also inhibited it as markedly as Ac-Phe-OEt, Ac-Trp-OEt, and Bz-Tyr-OEt did, whereas Bz-Arg-OEt again did not show any effect. The results of analysis of the intracellular fate of the Ag-Ab complex taken up by macrophages through the use of analytical density gradient fractionation of the homogenized cells suggest that Ac-Phe-OEt inhibits the ingestive process since the distribution of Ag-Ab complex showed a single peak, closely accompanying the plasma membrane. Ac-Tyr-OEt, on the other hand, caused a marked accumulation of Ag-Ab complex in the lysosome fraction, reflecting the inhibition of intralysosomal digestion of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Raman spectra and conformations of the cis-unsaturated fatty-acid chains

Raman spectra of low (13°C) and high (16°C) m.p. crystals of oleic acid were recorded and the spectral differences were ascribed to different conformations around a pair of sp², CC axes, i.e. (skew, skew′) and (skew, skew). Crystalline modifications (m.p. 29°C and 29.5°C) of petroselinic acid were found for the first time; after spectral comparison with oleic acid conformations in those crystals were predicted to be (skew, skew′) and (skew, skew). Raman spectra of dioleoyl- and dipetroselinoyl-l-α-phosphatidylcholines were measured for different crystalline phases and the conformation was examined.The skeletal vibration bands of the polymethylene chains of cis- and trans-unsaturated fatty-acids were analysed by using the frequency-phase difference relationships of saturated fatty-acids. The ν₄ (stretching) vibrations were localised within each polymethylene chain and the bands of an acid CH₃(CH₂)_m?2CHCH(CH₂)_n?2COOH were explained in terms of the set of phase differences δ = kπ/m and kπ/n (k = 1, 2,..). A unique ν₄ vibration with δ = π/2m was also found. The ν₅ (bending) vibrations sometimes couple strongly with each other to form overall vibrations characterised by δ = kπ/(m + n).Implications of the cis-olefin group for the physical properties of phospholipid bilayers and the applicability of Raman spectroscopy in probing chain conformations were discussed.