17beta-Estradiol responsiveness of MCF-7 laboratory strains is dependent on an autocrine signal activating the IGF type I receptor

BACKGROUND: Human MCF-7 cells have been studied extensively as a model for breast cancer cell growth. Many reports have established that serum-starved MCF-7 cells can be induced to proliferate upon the sole addition of 17beta-estradiol (E2). However, the extent of the mitogenic response to E2 varies in different MCF-7 strains and may even be absent. In this study we compared the E2-sensitivity of three MCF-7 laboratory strains. RESULTS: The MCF-7S line is non-responsive to E2, the MCF-7 ATCC has an intermediate response to E2, while the MCF-7 NKI is highly E2-sensitive, although the levels and activities of the estrogen receptor (ER) are not significantly different. Both suramin and IGF type I receptor blocking antibodies are able to inhibit the mitogenic response to E2-treatment in MCF-7 ATCC and MCF-7 NKI cells. From this we conclude that E2-induced proliferation is dependent on IGF type I receptor activation in all three MCF-7 strains. CONCLUSIONS: The results presented in this article suggest that E2-responsiveness of MCF-7 cells is dependent on the secretion of an autocrine factor activating the IGF-IR. All three strains of MCF-7 breast cancer cells investigated do not respond to E2 if the IGF-RI-pathway is blocked. Generally, breast cancer therapy is targeted at inhibiting estrogen action. This study suggests that inhibition of IGF-action in combination with anti-estrogen-treatment may provide a more effective way in treatment or even prevention of breast cancer.     

Cloning,Sequencing, and Characterization of the Genetic Region Relevant to Biosynthesis of the Lipopeptides Iturin A and Surfactin in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.Received: 29 October 2002 / Accepted: 6 December 2002     

拐芹根化学成分研究Ⅱ

从伞型科当归属植物拐芹(Angelica polymorpha Maxim)的根及根茎中又分得4个结晶性化合物。经物理常数测定、光谱分析,分别鉴定为欧前胡素Ⅰ,异氧化前胡内酯Ⅱ,Pabulenol Ⅲ,Phellopterin Ⅳ。     

Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step.

Transglutaminase (TGase) from Streptomyces mobaraensis is secreted as a precursor protein which is completely activated by the endoprotease TAMEP, a member of the M4 protease family [Zotzel, J., Keller, P. & Fuchsbauer, H.-L. (2003) Eur. J. Biochem. 270, 3214-3222]. In contrast with the mature enzyme, TAMEP-activated TGase exhibits an additional N-terminal tetrapeptide (Phe-Arg-Ala-Pro) suggesting truncation, at least, by a second protease. We have now isolated from the culture broth of submerged colonies a tripeptidyl aminopeptidase (SM-TAP) that is able to remove the remaining tetrapeptide. The 53-kDa peptidase was purified by ion-exchange and phenyl-Sepharose chromatography and subsequently characterized. Its proteolytic activity was highest against chromophoric tripeptides at pH 7 in the presence of 2 mm CaCl2. EDTA and EGTA (10 mm) both diminished the proteolytic activity by half. Complete inhibition was only achieved with 1 mm phenylmethanesulfonyl fluoride, suggesting that SM-TAP is a serine protease. Alignment of the N-terminal sequence confirmed its close relation to the Streptomyces TAPs. That removal of Phe-Arg-Ala-Pro from TAMEP-activated TGase by SM-TAP occurs in a single step was confirmed by experiments using various TGase fragments and synthetic peptides. SM-TAP was also capable of generating the mature N-terminus by cleavage of RAP-TGase. However, AP-TGase remained unchanged. As SM-TAP activity against chromophoric amino acids such as Pro-pNA or Phe-pNA could not be detected, the tetrapeptide of TAMEP-activated TGase must be removed without formation of an intermediate.     

A quenched fluorescent dipeptide for assaying dispase- and thermolysin-like proteases

Metalloproteases such as dispase and thermolysin play a crucial role in the life cycle of bacteria. Commonly, they prefer hydrophobic amino acids at P1' of substrate proteins, thereby cleaving the peptide bond at the alpha amino group. Activity of such proteases has been measured by the use of tailor-made oligopeptides provided with fluorescence resonance energy transfer dyes. We can now show that the short dipeptide Dabcyl-Ser-Phe-EDANS is an appropriate substrate of dispase and thermolysin. It was cleaved by both enzymes at the single peptide bond accompanied by a steep increase in fluorescence. Substantial quenching effects of the formed products were observed only when more than 80microM substrate was hydrolyzed. High affinity of the proteases for the dipeptide resulted in low K(m) values of 91+/-9 and 104+/-18microM, which are comparable to those measured for longer peptides. Dabcyl-Ser-Phe-EDANS was also used to determine the pH and optimal temperature of dispase, which were found at pH 7.0 and 50 degrees C. Buffer substances such as acetate, citrate, and tris(hydroxymethyl)aminomethane had no significant effect on enzyme activity. Measurements up to 100 degrees C revealed that hydrolysis of the quenched fluorescent dipeptide took place only in the presence of active dispase.     

Cloning, sequencing, and characterization of the genetic region relevant to biosynthesis of the lipopeptides iturin A and surfactin in Bacillus subtilis

Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.     

Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease.

Streptomyces mobaraensis secretes a Ca2+-independent transglutaminase (TGase) that is activated by removing an N-terminal peptide from a precursor protein during submerged culture in a complex medium [Pasternack, R., Dorsch, S., Otterbach, J. T., Robenek, I. R., Wolf, S. & Fuchsbauer, H.-L. (1998) Eur. J. Biochem. 257, 570-576]. However, an activating protease could not be identified, probably because of the presence of a 14-kDa protein (P14) belonging to the Streptomyces subtilisin inhibitor family. In contrast, if the microorganism was allowed to grow on a minimal medium, several soluble proteases were extracted, among them the TGase-activating protease (TAMEP). TAMEP was purified by sequential chromatography on DEAE- and Arg-Sepharose and used to determine the cleavage site of TGase. It was clearly shown that the peptide bond between Phe(-4) and Ser(-5) was hydrolyzed, indicating that at least one additional peptidase is necessary to complete TGase processing, even if TAMEP cleavage was sufficient to obtain total activity. Sequence analysis from the N-terminus of TAMEP revealed the close relationship to a zinc endo-protease from S. griseus. The S. griseus protease differs from other members of the M4 protease family, such as thermolysin, in that it may be inhibited by the Streptomyces subtilisin inhibitor. P14 likewise inhibits TAMEP in approximately equimolar concentrations, suggesting its important role in regulating TGase activity.     

Inhibition of bacterial transglutaminase by its heat-treated pro-enzyme

Transglutaminases form a unique family of cross-linking enzymes which may be interesting for pharmaceutical and technical purposes. Bacterial transglutaminase, differing from the eucaryotic counterparts in being independent from Ca2+ ions, is excreted by several Streptomyces species. Until now an endogenous factor regulating activated transglutaminase could not be detected. Here, we investigated whether an inhibitor of transglutaminase is excreted into the culture fluid of Streptomyces mobaraensis. We could demonstrate that heat-resistant inhibitory activity is produced after 24h of growth reaching a maximum after 72h. A two-step ion exchange chromatography purification procedure revealed co-elution of the heat-treated inhibitor with pro-transglutaminase. Experiments with wild-type and recombinant pro-transglutaminase confirmed that the precursor protein indeed inhibits the activity of the mature enzyme.     

Redescription and palaeobiology of Palaeoscorpius devonicus Lehmann, 1944 from the Lower Devonian Hunsrück Slate of Germany

Abstract: Palaeoscorpius devonicus Lehmann, 1944 is known from only a single specimen, found in the Eschenbach Pit near Bundenbach in the Lower Devonian Hunsrück Slate of Germany. It is a key fossil, having been interpreted both as the most basal member of the Scorpiones and as one of the order’s most likely candidates for an aquatic mode of life. Prepared both ventrally and dorsally, some aspects of its morphology remain problematic. Here, with the aid of new techniques, including computed tomography, we present a re‐investigation of this scorpion’s anatomy and a new reconstruction, with a particular focus on the species’ original habitat. On the basis of the environmental interpretation of the Hunsrück Slate and the completeness of the specimen, previous authors concluded that P. devonicus was marine, but none offered convincing morphological evidence. Recent studies of the deposit’s environment suggest that the Hunsrück Sea was part of an intrashelf basin, relatively close to the coastline, and fossils of land plants show that terrestrial wash‐in occasionally occurred. Our revised interpretation of the fossil’s morphology demonstrates that the scorpion was most probably terrestrial. Internal mesosomal organs are interpreted as book lungs, but other terrestrial adaptations are lacking. The absence of both coxapophyses and gnathobases makes determining the scorpion’s feeding mechanism difficult. Interpreting the scorpion’s character states within a phylogenetic framework, especially the possible presence of book lungs, implies either that the plesiomorphic position of P. devonicus is no longer supported or that the development of book lungs had already taken place early in the scorpion lineage.     

Variants of Myxococcus virescens Exhibiting Dispersed Growth. Growth and Production of Extracellular Enzymes and Slime

The growth of two strains of Myxococcus virescens exhibiting dispersed growth was followed in casamino acids (N III-C) media and casitone media. The changes in optical density, pH, pigmentation as well as the secretion of bacteriolytic and proteolytic enzymes, DNA and polysaccharides during growth were recorded. In both media the bacteria grew exponentially with a generation time of 4 (casitone) and 20 hours (N III-C) respectively. The maximal cell mass was about 4 times higher in casitone than in casamino acids media. The amounts of bacteriolytic enzymes produced by the two strains in N III-C medium were different but in casitone medium they were about equal and considerably higher. The maximal values of proteolytic enzymes were about the same in both media and always occurred later than the bacteriolytic maxima. Both activity peaks appeared before the phase of decline. The polysaccharide production reached a maximum during the stationary growth phase in both media. A higher value was reached during growth in casitone medium than in N III-C medium. During the phase of decline a second increase of polysaccharide in the medium appeared. No DNA could be detected in the cell-free solutions until the beginning of the phase of decline.