The relationship of multifocality and tumor burden with various tumor characteristics and survival in early breast cancer.

The presence of multifocality and the aggregate tumor size were retrospectively analysed in a database of 1071 operated breast cancers. Around a quarter of all these cancers involved multiple foci, while a tenth of the total demonstrated more than one invasive focus. Although the multifocal cancers were smaller and more often screen-detected than the unifocal cancers, their aggregate tumor size was larger, and they more frequently displayed casting-type calcifications in the mammogram and HER2 positivity. Lobular histology favoured larger tumor burden. The invasive multifocal cancers were more commonly lymph node-positive than the other tumors. In a subgroup of 584 patients with a median follow-up time of 5 years, the larger size of the invasive tumor, the presence of LVI or lymph node involvement, HER2 positivity and triple negativity were associated with a poorer RFS and OS, while the outcome of screen-detected tumors was superior to that of non-screen-detected or interval cancers. A large tumor size, lymph node positivity and HER2 positive or triple negative phenotypes were independent determinants of a poorer survival rate.

A connection between stress and development in the multicellular prokaryote Streptomyces coelicolor A3(2).

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sigmaH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sigmaH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2-egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sigmaH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2-egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sigmaH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.

Identification and characterization of the mre gene region of Streptomyces coelicolor A3(2).

During a search for new differentiation factors in Streptomyces coelicolor A3(2), a locus at 11 o'clock on the S. coelicolor map was identified which harbours several genes that show extensive similarity to cell division and differentiation genes from Escherichia coli and Bacillus subtilis. From the sequence data it was concluded that the region contains the genes mireB, mreC, mreD (murein formation gene cluster E), pbp83 (high-molecular-weight penicillin-binding protein) and sfr (member of the spoVE/ftsW/rodA family). Mre gene products are reported to be responsible for determining cell shape in E. coli and Bacillus. The S. coelicolor mreC gene was inactivated by gene disruption, resulting in mutants which showed significant growth retardation in comparison to the wild type. Inactivation of the mreB gene was incompatible with viability, and thus mreB represents a Streptomyces cell division gene that is essential for survival. Promoter-probe experiments led to the identification of an operon structure, with promoters located upstream of mreB, pbp83 and sfr. Detailed studies of mreB transcription revealed the existence of three promoters; two of them are constitutively transcribed, whereas the third is developmentally regulated.

A developmentally regulated gene encoding a repressor-like protein is essential for sporulation in Streptomyces coelicolor A3(2).

whiH is one of several known loci specifically needed for the orderly multiple sporulation septation of aerial hyphae of Streptomyces coelicolor A3(2) and for the expression of at least some late sporulation genes. DNA complementing whiH mutants was located immediately upstream on hrdB, which encodes the principal sigma factor of S. coelicolor. Sequencing revealed a gene whose disruption gave rise to a typical whiH mutant phenotype. Four whiH mutants contained base changes or a frameshift in this gene. The deduced product of whiH related to a large family of bacterial regulatory proteins, the most similar being several repressors (such as GntR of Bacillus subtilis) responsive to carboxylate-containing intermediates in carbon metabolism. Transcription of whiH was initiated at a single promoter, PwhiH. Levels of whiH mRNA were developmentally regulated, increasing sharply when aerial mycelium was present, and reaching a maximum approximately when spores were first detectable. Transcript levels were markedly increased in a whiH mutant, indicating the possible involvement of WhiH in negative regulation of its own production. PwhiH was directly dependent on the sigma factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.

Developmental regulation of transcription of whiE, a locus specifying the polyketide spore pigment in Streptomyces coelicolor A3 (2)

whiE is a complex locus that specifies the polyketide spore pigment in Streptomyces coelicolor A3(2). Two divergently oriented promoters, whiEP1 and whiEP2, were identified in the whiE gene cluster, and their activities were analyzed during colony development in wild-type and sporulation-deficient strains. Both promoters were developmentally regulated; whiEP1 and whiEP2 transcripts were detected transiently at approximately the time when sporulation septa were observed in the aerial hyphae, and transcription from both promoters depended on each of the six known "early" whi genes required for sporulation septum formation (whiA, -B, -G, -H, -I, and -J). Mutation of the late sporulation-specific sigma factor gene, sigF, had no effect on the activity of whiEP1 but blocked transcription from whiEP2. However, sigmaF-containing holoenzyme was not sufficient to direct transcription of whiEP2 in vitro. The whiEP2 promoter controls expression of whiE ORFVIII, encoding a putative flavin adenine dinucleotide-dependent hydroxylase that catalyzes a late tailoring step in the spore pigment biosynthetic pathway. Disruption of whiE ORFVIII causes a change in spore color, from grey to greenish (T.-W. Yu and D. A. Hopwood, Microbiology 141:2779-2791, 1995). Consistent with these observations, construction of a sigF null mutant of S. coelicolor M145 caused the same change in spore color, showing that disruption of sigF in S. coelicolor changes the nature of the spore pigment rather than preventing its synthesis altogether.

Initiation of aerial mycelium formation in Streptomyces.

In the past two years, the isolation of extracellular factors involved in the initiation of aerial mycelium formation, the identification of metabolic defects in certain developmental mutants, and the characterisation of three further bld genes and several gamma-butyrolactone receptor genes have led to new ideas about the mechanisms that initiate aerial mycelium formation in Streptomyces. The emerging picture suggests the integration of numerous signals from both inside and outside the cell.

Improved electrophoretic separation and immunoblotting of beta-amyloid (A beta) peptides 1-40, 1-42, and 1-43.

Beta-amyloid peptides (A beta peptides) form the main protein component of the amyloid deposits found in the brains of Alzheimer's disease (AD) patients. Soluble A beta peptides, which are proteolytic fragments of the amyloid-precursor protein (APP) are constitutively secreted by cells expressing APP during normal metabolism [1] and are also present in human plasma and cerebrospinal fluid [2]. Missense mutations in Codon 717 of the APP gene are responsible for a small percentage of inherited AD cases (FAD) and increase the amount of A beta peptides containing additional carboxy terminal amino acids (A beta 1-42, A beta 1-43) [3, 4]. Recent findings indicate that FAD mutations in the presenilin 1 and 2 genes also increase the amount of these longer A beta peptides [5]. A beta 1-42 polymerizes more rapidly in vitro [6] than A beta 1-40 and has been identified as the major component of the brain amyloid deposits [7-9]. We recently developed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system [10] for the separation of these two peptides. Here we describe a modified version of the original SDS-PAGE procedure, which allows the separation of A beta 1-40, A beta 1-42, and A beta 1-43 for the first time. Detection of the three A beta peptides in the lower ng and pg range is realized by optimized silver staining or immunoblot procedures. These nonradioactive methods may validate results obtained by ELISA procedures used to study the metabolic fate of APP. They may help to define the neurotoxic potential of the longer A beta peptides in relation to their aggregation state.

The positions of the sigma-factor genes, whiG and sigF, in the hierarchy controlling the development of spore chains in the aerial hyphae of Streptomyces coelicolor A3(2).

whiG and sigF encode RNA polymerase sigma factors required for sporulation in the aerial hyphae of Streptomyces coelicolor. Their expression was analysed during colony development in wild-type and sporulation-defective whi mutant strains. Each gene was transcribed from a single promoter. Unexpectedly, whiG mRNA was present at all time points, including those taken prior to aerial mycelium formation; this suggests that whiG may be regulated post-transcriptionally. Transcription of whiG did not depend upon any of the six known 'early' whi genes required for sporulation septum formation (whiA, B, G, H, I and J), placing it at the top of the hierarchy of whi loci. sigF expression appeared to be regulated at the level of transcription; sigF transcripts were detected transiently when sporulation septa were observed in the aerial hyphae. Transcription of sigF depended upon all six of the early whi genes, including whiG. The sigF promoter does not resemble the consensus sequence established for sigma WhiG-dependent promoters and E sigma WhiG did not transcribe from the sigF promoter in vitro. Consequently, the genetic dependence of sigF upon whiG is very likely to be indirect. These results show that there is a hierarchical relationship between sigma factors required for Streptomyces sporulation and also that at least five other genes are involved in this transcriptional network.

Deletion of DNA lying close to the glkA locus induces ectopic sporulation in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) J1668 sporulated ectopically in the substrate hyphae (the Esp phenotype) with the same time course as sporulation in the aerial hyphae. Examination of related strains implied that the Esp phenotype was caused by the deletion of DNA that lies close to, but is distinct from, the glucose kinase gene (glkA). Co-transduction of the Esp phenotype with the deletion present in J1668 confirmed this hypothesis. The size of the deletion was found to be 7.4 kb. Construction of a strain carrying both the J1668 deletion and a whiG mutation demonstrated that the Esp phenotype depends on at least one of the genes required for the differentiation of aerial hyphae into spores.

A new RNA polymerase sigma factor, sigma F, is required for the late stages of morphological differentiation in Streptomyces spp.

A gene (sigF) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well-conserved region 2.2 of the eubacterial sigma 70 family. Homologues were present in other Streptomyces spp., and that of the genetically well studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 (S. coelicolor) and 295 (S. aureofaciens) amino acid residues, both showing greatest similarity to sigma B of Bacillus subtilis. However, while sigma B is involved in stationary-phase gene expression and in the general stress response in B. subtilis, sigma F affects morphological differentiation in Streptomyces. Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. sigma F is proposed to control the late stages of spore development in Streptomyces.

Transcriptional attenuation control of the tylosin-resistance gene tlrA in Streptomyces fradiae.

The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase-coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome-mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD.

Cloning and characterization of gentamicin-resistance genes from Micromonospora purpurea and Micromonospora rosea.

Aminoglycoside-resistance genes (grm) were cloned from a gentamicin producer Micromonospora purpurea and a sisomicin producer Micromonospora rosea. The nucleotide (nt) sequences of both genes were determined and the similarity between them was very high (90.4% identity). In either case, the transcription start point was localised to about 11 nt upstream from the likely translation start codons of grm, which is expressed as a polycistronic transcript. In studies to be reported elsewhere, it has been established that the M. purpurea grm gene encodes a ribosomal RNA methyltransferase. Here, we confirmed that the similarity of the two genes exists not only at the structural but also at the functional level.

Agents inducing high Mg2+-ATPase activity of isolated coupling factor 1 from spinach chloroplasts.

Conditions are reported under which purified coupling factor 1 (CF1) from spinach chloroplasts exhibits Mg2+-dependent ATPase activity of about 120 mumoles/min/mg protein. It is shown that CF1, partially activated by treatment with heat and dithiothreitol (DTT), can be further activated by octyl glucoside. The Mg2+-dependent ATPase activity increases linearly as a function of the concentration of octyl glucoside from about 20 mumoles/min/mg protein in the absence of detergent to 120 mumoles/min/mg protein in the presence 15 mM octyl glucoside. This concentration is below the critical micellar concentration (CMC) of the detergent, indicating that the monomeric form is responsible for the activation. Without treatment with heat and DTT, the Mg2+-dependent ATPase activity of CF1 is virtually zero, but can be stimulated by octyl glucoside. In this case, however, only concentrations around CMC give a substantial increase in activity (about 50 mumoles/min/mg at 28 mM octyl glucoside). Concentrations higher than CMC inhibit both latent and heat-activated CF1.

High-performance liquid chromatography of isopeptides.

High-performance liquid chromatographic (HPLC) methods are described for the structural analysis of clavicepamines, their analogues and branched-chain polypeptides, the analysis in synthetic stages of isopeptides (analysis of half-protected derivatives and purity control of active esters) and the differentiation between alpha- and iso-peptides (such as alpha- and gamma-glutamyl peptides). Pre-column derivatization was used to label the free amino groups for the structural investigation. Dansylated and hydrolysed isopeptides were analysed by HPLC methods based on isocratic separation of alpha-, epsilon- and bis-Dns-lysines using Hypersil, ODS-Hypersil and Partisil PAC columns. For the analysis of peptide active esters, an RP-HPLC method was developed, with methanol-acetonitrile-water mobile phases containing an acidic buffer. Peptides containing alpha-glutamyl residues were analysed for gamma-isomer content in the same system.