Involvement of umuDCST genes in nitropyrene-induced -CG frameshift mutagenesis at the repetitive CG sequence in the hisD3052 allele of Salmonella typhimurium

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons, umuDC _ST on the chromosome and samAB on a 60-MDa cryptic plasmid. The roles of theumuDC-like operons in chemically induced frameshift mutagenesis of the hisD3052 allele of S. typhimurium were investigated. Introduction of a pBR322-derived plasmid carrying umuDCST increased the rate of reversion of hisD3052, following treatment with 1-nitropyrene (1-NP) or 1,8-dinitropyrene (1,-8DNP) tenfold and fivefold, respectively, whereas it did not substantially increase the rate of reversion induced by other frameshift mutagens, i.e. 2-nitrofluorene (2NF) and 2-amino- 3-methyldipyrido[1,2-a:3 ,2-d]imi-dazole (Glu-P-1). Introduction of a pBR322-derived plasmid carrying samAB did not increase the incidence of reversion of hisD3052 observed with any of the mutagens examined. Deletion of umuDC _STSubstantially lowered the reversion rate induced by l-NP or 1,8-DNP, but it did not affect reversion induced by 2-NF, Glu-P-1 or N-hydroxyacetylaminofluorene (N-OH-AAF). Deletion of samAB had little impact on reversion incidence induced by any of the five frameshift mutagens. DNA amplification using the polymerase chain reaction technique followed by restriction enzyme analysis using BssHII, suggested that the mutations induced by the five frameshift mutagens were all CG deletions at the CGCGCGCG sequence in hisD3052. These results suggest that umuDCST, but not samAB, is involved in the -2 frameshift mutagenesis induced by l-NP and 1,8-DNP at the repetitive CG sequence, whereas neither operon participates in induction of the same type of mutations by 2-NF, Glu-P-1 or N-OH-AAF.     

Purification and properties of thiamine-binding proteins from sesame seed

Three thiamine-binding proteins of 17-19 kDa (STBP-I, II, and III) were purified from sesame seed (Sesamum indicum L.). Each of the proteins was composed of two subunits of equal molecular mass and each subunit consisted of a large polypeptide and a small polypeptide linked by a disulfide bond(s). They were rich in glutamic acid (or glutamine) and arginine. Their binding activities were optimal at neutral pH. They bound specifically free thiamine but not thiamine phosphates. STBP-I had higher affinity for thiamine than STBP-II or STBP-III. STBP-II and STBP-III bound one molecule of thiamine per molecule, and STBP-I bound 0.5 molecule. The amino acid composition and structure of the STPBs were similar to those of 2S storage proteins.     

Isolation and Characterization of Hardening-Induced Proteins in Chlorella vulgaris C-27: Identification of Late Embryogenesis Abundant Proteins

Hardening-induced soluble proteins of Chlorella vulgaris BeijerinkIAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27) wereisolated and purified by two-dimensional high-performance liquidchromatography (2D-HPLC) on an anion-exchange column, with subsequentreversed-phase chromatography. Some of the proteins were resolvedby SDS-PAGE, characterized by amino-terminal sequencing andidentified by searching for homologies in databases. Separationof the soluble proteins during the hardening of Chlorella bya combination of 2D-HPLC and SDS-PAGE revealed that at least31 proteins were induced or increased in abundance. Of particularinterest was the induction after 12 h of a 10-kDa protein withthe amino-terminal amino acid sequence AGNKPITEQISDAVGAAGQKVGand the induction after 6 h of a 14-kDa protein with the amino-terminalsequence ALGEESLGDKAKNAFEDAKDAVKDAAGNVKEAV. The amino-terminalsequences of these proteins indicated that they were homologousto late embryogenesis abundant (LEA) proteins. Furthermore,the level of a 22-kDa protein also increased after 12 h. Theamino-terminal sequence of this protein, AAPLVGGPAPDFTAAAVFD,indicated that it was homologous to thioredoxin peroxidase. (Received June 9, 1995; Accepted September 12, 1995)     

Relationship between actions of transforming growth factor (TGF)-β and cell surface expression of its receptors in clonal osteoblastic cells

Various osteoblastic cell lines were examined for the relationship between the presence of cell-surface transforming growth factor (TGF)-β receptors and the synthesis of matrix proteins with their responsiveness to TGF-β. Treatment with TGF-β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3-E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF-β1 markedly stimulated the synthesis of decorin in MC3T3-E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF-β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF-β1 enhanced fibronectin synthesis. TGF-β1 did not show any of these effects in UMR 106 cells. Receptor cross-linking studies revealed that only MC3T3-E1 and MG 63 cells had both types I and II signal-transducing receptors for TGF-β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3-E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF-β1 under these conditions. UMR 106 cells with no response to TGF-β1 had neither of the signal-transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF-β1, that the responsiveness of osteoblastic cells to TGF-β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF-β1 on fibronectin synthesis can develop with little binding of TGF-β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell-surface receptors for TGF-β determines the responsiveness of osteoblastic cells to TGF-β and that changes in cell-surface TGF-β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley-Liss, Inc.     

Petunia guarapuavensis (Solanaceae): A new species from planalto of Paraná and Santa Catarina,Brazil

Petunia guarapuavensis, a new species fromplanalto (high plateau) of Paraná and Santa Catarina in Brazil, is described, and its morphological distinction from related species, features of the habitats, and geographical distribution are discussed.     

Immunohistochemical localization of Na+-dependent glucose transporter in the rat digestive tract

Summary Glucose is actively absorbed in the intestine by the action of the Na⁺-dependent glucose transporter. Using an antibody against the rabbit intestinal Na⁺-dependent glucose transporter (SGLT1), we examined the localization of SGLT1 immunohistochemically along the rat digestive tract (oesophagus, stomach, duodenum, jejunum, ileum, colon and rectum). SGLT1 was detected in the small intestine (duodenum, jejunum and ileum), but not in the oesophagus, stomach, colon or rectum. SGLT1 was localized at the brush border of the absorptive epithelium cells in the small intestine. Electron microscopical examination showed that SGLT1 was localized at the apical plasma membrane of the absorptive epithelial cells. SGLT1 was not detected at the basolateral plasma membrane. Along the crypt-villus axis, all the absorptive epithelial cells in the villus were positive for SGLT1, whose amount increased from the bottom of the villus to its tip. On the other hand, cells in the crypts exhibited little or no staining for SGLT1. Goblet cells scattered throughout the intestinal epithelium were negative for SGLT1. These observations show that SGLT1 is specific to the apical plasma membrane of differentiated absorptive epithelial cells in the small intestine, and suggest that active uptake of glucose occurs mainly in the absorptive epithelial cells in the small intestine.     

Basic steroid saponins from aerial parts of Fritillaria Thunbergii

From the aerial parts of Fritillaria thunbergii, three glycosidal Solanum alkaloids (basic steroid saponins) were isolated together with minor     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.