Biomedical subjects

Torsten Stein

Publications and source records attributed to Torsten Stein.

At least 19 recordsLinked to original sources

PubMed2006

Nature of Cx30-containing channels in the adult mouse mammary gland.

Oligonucleotide microarray analysis uniquely shows that several members of the connexin family of gap junction proteins are expressed by the epithelium during mouse mammary gland development. Connexin 26 (Cx26) is present throughout pregnancy and lactation, is then undetectable shortly after weaning, but reappears during involution. Additionally, Cx30 is abundant in late-pregnant and early lactating gland epithelium. From mid-pregnancy into early lactation, Cx26 and Cx30 co-localize in junctional plaques between epithelial cells, forming hemichannels of mixed connexin content. Microarray analysis also shows Cx32 is developmentally restricted to parturition, suggesting that specific modification of gap junction channel composition and/or intercellular communication pathways occurs at parturition. Specifically, heteromeric channels of all pairwise combinations are formed when these connexins are expressed within the same cells. Of these hemichannels, Cx26/Cx32 pores are increasingly sensitive to closure by taurine (an osmolyte implicated in milk protein synthesis) with increasing Cx26 content. In contrast, physiological taurine concentrations have no effect on Cx26/Cx30 and Cx30/Cx32 channel activity. Such changes in connexin expression and channel composition and their chemical modulation are discussed in relation to the various stages of mammary gland development in the adult mouse.

Animals↗

PubMed2006

Metabolic regulation in the lactating mammary gland: a lipid synthesizing machine.

The mammary gland of the lactating mouse synthesizes and secretes milk lipid equivalent to its entire body weight in a single 20-day lactation cycle, making it one of the most active lipid synthetic organs known. We test the hypothesis that multiple control points and potential regulatory mechanisms regulate milk lipid synthesis at the level of gene expression. The mammary transcriptome of 130 genes involved in glucose metabolism was examined at late pregnancy and early lactation, utilizing data obtained from microarray analysis of mammary glands from quadruplicate FVB mice at pregnancy day 17 and lactation day 2. To correlate changes with physiological parameters, the metabolome obtained from magnetic resonance spectroscopy of flash-frozen glands at day 17 of pregnancy was compared with that at day 2 of lactation. A significant increase in carbohydrates (glucose, lactose, sialic acid) and amino acids (alanine, aspartate, arginine, glutamate) with a moderate increase in important osmolytes (myo-inositol, betaine, choline derivatives) were observed in the lactating gland. In addition, diets containing 8% or 40% lipid were fed from lactation days 5-10 and mammary glands and livers of triplicate FVB mice prepared for microarray analysis. The results show that substantial regulation of lipid synthesis occurs at the level of mRNA expression and that some of the regulation points differ substantially from the liver. They also implicate the transcription factor SREBP-1c in regulation of part of the pathway.

Animals↗

PubMed2006

Involvement of axonal guidance proteins and their signaling partners in the developing mouse mammary gland.

Mammary morphogenesis in the mouse is driven by specialized structures at the ends of the developing ducts, the terminal end buds (TEB). The mechanisms controlling the precise branching and spacing of the ducts are, as yet, unknown. To identify genes that are associated with migration of TEB and differentiation of the subtending ducts, we developed a novel method of isolating TEB and ducts free of stroma, and compared the gene expression profiles of these two isolates using oligonucleotide microarrays. Ninety one genes were upregulated in TEB compared to ducts. Three of these genes, Sprr1A, Sema3B, and BASP1, are associated with axonal growth and guidance. Two additional members of the Sprr family, Sprr2A and 2B, not previously associated with axonal growth, were also highly expressed in TEB. Expression of these genes was confirmed by RT-PCR and Western blotting, and the cellular distribution of Sprr1A and BASP1 was demonstrated by immunohistochemistry. Other semaphorins, including Sema3C, 4A, 4F and the cancer invasion associated Sema 4D were also expressed in the mouse mammary gland along with the semaphorin receptors, Plexins A2, A3, B2, and D1, and Neuropilins 1 and 2. These results are discussed in the context of other proteins expressed in the developing gland that are known to be downstream effectors of these signaling molecules. We suggest that these genes may influence ductal growth and morphogenesis in the developing mammary gland.

Animals↗

PubMed2006

Proteomic analysis of the mouse mammary gland is a powerful tool to identify novel proteins that are differentially expressed during mammary development.

After lactation, the mouse mammary gland undergoes apoptosis and tissue remodelling as the gland reverts to its prepregnant state. This complex change was investigated using 2-DE. An integrated database was produced from lactation and involution proteomes. Forty-four molecular cluster indexes (MCIs) that showed altered expression from lactation to involution were selected for MS analysis. Of these, 32 gave protein annotations, 18 of which were unequivocal proteins. Selected proteins were then studied across all of development, including pregnancy, using data integrated from another proteome database. Two proteins, the RNA polymerase B transcription factor 3 (BTF3) and the minichromosome maintenance protein 3 (MCM3), although initially selected on the basis of the lactation/involution criteria, had expression profiles that indicated an additional role in mammary development and were further analysed. BTF3, a transcription factor previously not described in the mammary gland, was up-regulated strongly in pregnancy, indicating an involvement in alveolar growth. MCM3's expression was greatest in pregnancy and late involution, decreasing through lactation. Immunohistochemistry localised MCM3 to the mammary epithelium, where a greater proportion of cells stained than for the proliferation marker Ki67. MCM3 expression during lactation may identify cells that are licensed to repopulate the gland during cell loss in lactation and following involution.

Animals↗

PubMed2005

Annexin A8 is up-regulated during mouse mammary gland involution and predicts poor survival in breast cancer.

PURPOSE: Microarray studies have linked Annexin A8 RNA expression to a "basal cell-like" subset of breast cancers, including BRCA1-related cancers, that are characterized by cytokeratin 5 (CK5) and CK17 expression and show poor prognosis. We assessed Annexin A8's contribution to the overall prognosis and its expression in normal, benign, and cancerous tissue and addressed Annexin A8's physiologic role in the mammary gland. EXPERIMENTAL DESIGN: Using microarrays and reverse transcription-PCR, the Annexin A8 expression was studied during mouse mammary gland development and in isolated mammary structures. Reverse transcription-PCR on cultured human luminal and basal cells, along with immunocytochemistry on normal and benign breast tissues, was used for cellular localization. Annexin A8's prognostic relevance and its coexpression with CK5 were assessed on tissue arrays of 1,631 cases of invasive breast cancer. Coexpression was further evaluated on a small cohort of 14 BRCA1-related breast cancers. RESULTS: Annexin A8 was up-regulated during mouse mammary gland involution and in pubertal ductal epithelium. Annexin A8 showed preferred expression in cultured basal cells but predominant luminal expression in normal human breast tissue in vivo. Hyperplasias and in situ carcinomas showed a strong staining of basal cells. Annexin A8 expression was significantly associated with grade (P < 0.0001), CK5 (P < 0.0001), and estrogen receptor status (P < 0.0001); 85.7% BRCA1-related breast tumors coexpressed Annexin A8 and CK5. CONCLUSION: Annexin A8 is involved in mouse mammary gland involution. In humans, it is a luminally expressed protein with basal expression in cell culture and in hyperplasia/ductal carcinoma in situ. Expression in invasive breast carcinomas has a significant effect on survival (P = 0.03) but is not independent of grade or CK5.

Animals↗

PubMed2005

Engineering Bacillus subtilis ATCC 6633 for improved production of the lantibiotic subtilin.

To improve the production of the lantibiotic subtilin in Bacillus subtilis ATCC 6633, two genetic engineering strategies were followed. Firstly, additional copies of subtilin self-protection (immunity) genes spaIFEG have been integrated into the genome of the producer strain. Their expression significantly enhanced the subtilin tolerance level, and concomitantly, the subtilin yield 1.7-fold. Secondly, a repressor of subtilin gene expression, the B. subtilis general transition state regulator protein AbrB, was deleted. A sixfold enhancement of the subtilin yield could be achieved with the abrB deletion mutant; however, the produced subtilin fraction predominantly consists of succinylated subtilin species with less antimicrobial activity compared to unmodified subtilin.

ATP-Binding Cassette Transporters↗

PubMed2005

Carbon Source-dependent assembly of the Snf1p kinase complex in Candida albicans.

The Snf1p/AMP-activated kinases are involved in transcriptional, metabolic, and developmental regulation in response to stress. In Saccharomyces cerevisiae, Snf1p (Cat1p) is one of the key regulators of carbohydrate metabolism, and cat1 (snf1) mutants fail to grow with non-fermentable carbon sources. In Candida albicans, Snf1p is an essential protein and cells depend on a functional Snf1 kinase even with glucose as carbon source. We investigated the CaSnf1p complex after tandem affinity purification and mass spectrometric analysis and show that the complex composition changes with the carbon source provided. Three subunits were identified, one of which was named CaSnf4p because of its homology to the ScSnf4 protein and the respective CaSNF4 gene could complement a S. cerevisiae snf4 mutant. The other two proteins revealed similarities to the S. cerevisiae kinase beta subunits ScGal83p, ScSip2p, and ScSip1p. Both genes complemented the scaffold function in a S. cerevisiae gal83,sip1,sip2 triple deletion mutant and were named according to their scaffold function as CaKIS1p and CaKIS2p. Matrix-assisted laser desorption ionization peptide mass fingerprint analysis indicated that CaKis2p is N-terminal myristoylated and the incorporation of CaKis2p in the Snf1p complex was reduced when compared with cells grown with glucose as a carbon source. To verify the different complex assemblies, a stable isotope labeling technique (iTraqtrade mark) was employed, confirming a 3-fold decrease of CaKis2p with ethanol. Yeast two-hybrid analysis confirmed the interaction partners, and these results showed an activator domain for the CaKis2 protein that has not been reported for S. cerevisiae scaffold subunits.

AMP-Activated Protein Kinases↗

PubMed2005

Basal cytokeratins and their relationship to the cellular origin and functional classification of breast cancer.

Recent publications have classified breast cancers on the basis of expression of cytokeratin-5 and -17 at the RNA and protein levels, and demonstrated the importance of these markers in defining sporadic tumours with bad prognosis and an association with BRCA1-related breast cancers. These important observations using different technology platforms produce a new functional classification of breast carcinoma. However, it is important in developing hypotheses about the pathogenesis of this tumour type to review the nomenclature that is being used to emphasize potential confusion between terminology that defines clinical subgroups and markers of cell lineage. This article reviews the lineages in the normal breast in relation to what have become known as the 'basal-like' carcinomas.

Breast↗

PubMed2005

Regulation of genes encoding proteolytic enzymes during mammary gland development.

The mammary gland undergoes extensive tissue remodelling during each lactation cycle. During pregnancy, the epithelial compartment of the gland is vastly expanded (Benaud et al. 1998). At the end of lactation the epithelial cells undergo apoptosis and adipocyte differentiation is induced (Lilla et al. 2002). Ductal and alveolar growth during puberty and pregnancy, and the involution process require the action of proteolytic enzymes (including matrix metalloproteinases, plasminogen and membrane-peptidases) and the corresponding genes are activated during these periods (Benaud et al. 1998; Alexander et al. 2001). Matrix metalloproteinases (MMP) are expressed in several cell types of the mammary gland including stromal fibroblasts (e.g., MMP3, MMP2), epithelial cells (e.g., MMP7 or MMP9), adipocytes (e.g., MMP2) and lymphoid cells (e.g., MMP9) (Crawford et al. 1996; Lund et al. 1996; Wiseman et al. 2003). A number of knock-out mice, which are deficient for individual MMP genes (e.g., MMP2, MMP3) or plasminogen, display alterations to mammary gland structure and impairment of lactation (Lund et al. 1999; Wiseman et al. 2003).

Animals↗

PubMed2005

Bacillus subtilis antibiotics: structures, syntheses and specific functions.

The endospore-forming rhizobacterium Bacillus subtilis- the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the "pure" anti-microbial action: Non-ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum-sensing, and a "killing factor" effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.

Amino Acid Sequence↗

PubMed2005

Expression and functional analysis of the subtilin immunity genes spaIFEG in the subtilin-sensitive host Bacillus subtilis MO1099.

Bacillus subtilis ATCC 6633 produces the cationic pore-forming lantibiotic subtilin, which preferentially acts on gram-positive microorganisms; self protection of the producer cells is mediated by the four genes spaIFEG. To elucidate the mechanism of subtilin autoimmunity, we transferred different combinations of subtilin immunity genes under the control of an inducible promoter into the genome of subtilin-sensitive host strain B. subtilis MO1099. Recipient cells acquired subtilin tolerance through expression of either spaI or spaFEG, which shows that subtilin immunity is based on two independently acting systems. Cells coordinately expressing all four immunity genes acquired the strongest subtilin protection level. Quantitative in vivo peptide release assays demonstrated that SpaFEG diminished the quantity of cell-associated subtilin, suggesting that SpaFEG transports subtilin molecules from the membrane into the extracellular space. Homology and secondary structure analyses define SpaFEG as a prototype of lantibiotic immunity transporters that fall into the ABC-2 subfamily of multidrug resistance proteins. Membrane localization of the lipoprotein SpaI and specific interaction of SpaI with the cognate lantibiotic subtilin suggest a function of SpaI as a subtilin-intercepting protein. This interpretation was supported by hexahistidine-mediated 0-A cross-linking between hexahistidine-tagged SpaI and subtilin.

Amino Acid Sequence↗

PubMed2004

Altered permeability and modulatory character of connexin channels during mammary gland development.

Abrupt developmental changes occur in structural form and function of connexin (Cx) channels in the mouse mammary gland. Microarray study shows that the principal connexin isoform in epithelial cells during pregnancy is Cx26, up-regulated and persisting from the virgin. After parturition, there is rapid induction of Cx32. In epithelial plasma membranes, size exclusion chromatography reveals that Cx32 organizes initially with Cx26 as heteromeric (Cx26-Cx32) hemichannels and later in heteromeric and homomeric Cx32 channels. Dramatic alterations of connexin channel function following these developmental changes in channel composition are characterized using native channels reconstituted into liposomes. Changes to channel stoichiometry increase the allowable physical size limits of permeant after parturition; the new Cx32 channels are wider than channels containing Cx26. Most remarkably, heteromeric Cx26-Cx32 channels are selectively permeability to adenosine 3',5' cyclic phosphate (cAMP), guanosine 3',5' cyclic phosphate (cGMP), and inositol 1,4,5-triphosphate (IP(3)), whereas homomeric channels are not. Homomeric Cx26 and heteromeric channels with high Cx26/Cx32 stoichiometry are also inhibited by taurine, an osmolyte playing a key role in milk protein synthesis. Taurine effect is reduced where heteromeric channels contain Cx32 > Cx26 and eliminated when channels contain only Cx32. Connexin channel stoichiometry, permeability, and chemical gating character change in precisely the desired fashion after parturition to maximize molecular and electrical coupling to support coordinated milk secretion.

Adult↗

PubMed2004

Subtilosin production by two Bacillus subtilis subspecies and variance of the sbo-alb cluster.

Eight different Bacillus subtilis strains and Bacillus atrophaeus were found to produce the bacteriocin subtilosin A. On the basis of the subtilosin gene (sbo) sequences two distinct classes of B. subtilis strains were distinguished, and they fell into the two B. subtilis subspecies (B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii). The entire sequence of the subtilosin gene cluster of a B. subtilis subsp. spizizenii strain, B. subtilis ATCC 6633, was determined. This sequence exhibited a high level of homology to the sequence of the sbo-alb gene locus of B. subtilis 168. By using primer extension analysis the transcriptional start sites of sbo in B. subtilis strains ATCC 6633 and 168 were found to be 47 and 45 bp upstream of the sbo start codon, respectively. Our results provide insight into the incipient evolutionary divergence of the two B. subtilis subspecies.

Alleles↗

PubMed2003

Involution of the mouse mammary gland is associated with an immune cascade and an acute-phase response, involving LBP, CD14 and STAT3.

INTRODUCTION: Involution of the mammary gland is a complex process of controlled apoptosis and tissue remodelling. The aim of the project was to identify genes that are specifically involved in this process. METHODS: We used Affymetrix oligonucleotide microarrays to perform a detailed transcript analysis on the mechanism of controlled involution after withdrawal of the pups at day seven of lactation. Some of the results were confirmed by semi-quantitative reverse transcriptase polymerase chain reaction, Western blotting or immunohistochemistry. RESULTS: We identified 145 genes that were specifically upregulated during the first 4 days of involution; of these, 49 encoded immunoglobulin genes. A further 12 genes, including those encoding the signal transducer and activator of transcription 3 (STAT3), the lipopolysaccharide receptor (CD14) and lipopolysaccharide-binding protein (LBP), were involved in the acute-phase response, demonstrating that the expression of acute-phase response genes can occur in the mammary gland itself and not only in the liver. Expression of LBP and CD14 was upregulated, at both the RNA and protein level, immediately after pup withdrawal; CD14 was strongly expressed in the luminal epithelial cells. Other genes identified suggested neutrophil activation early in involution, followed by macrophage activation late in the process. Immunohistochemistry and histological staining confirmed the infiltration of the involuting mammary tissue with neutrophils, plasma cells, macrophages and eosinophils. CONCLUSION: Oligonucleotide microarrays are a useful tool for identifying genes that are involved in the complex developmental process of mammary gland involution. The genes identified are consistent with an immune cascade, with an early acute-phase response that occurs in the mammary gland itself and resembles a wound healing process.

Acute-Phase Proteins↗

PubMed2003

Activation of subtilin precursors by Bacillus subtilis extracellular serine proteases subtilisin (AprE), WprA, and Vpr.

The maturation of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 includes posttranslational modifications of the propeptide and proteolytic cleavage of the leader peptide. To identify subtilin processing activities, we used antimicrobial inactive subtilin precursors consisting of the leader peptide which was still attached to the fully matured propeptide. Two extracellular B. subtilis proteases were able to activate subtilin precursors, the commercially available serine protease prototype subtilisin (AprE) and WprA. The latter was isolated from B. subtilis WB600, a strain deficient in six extracellular proteases. Surprisingly, the aprE wprA double mutant of the ATCC 6633 strain was still able to produce active subtilin, however, with a reduced production rate. No subtilin processing was found within the culture supernatant of the WB800 strain, which is deficient in eight extracellular proteases. Vpr was identified as the third protease capable to process subtilin.

Amino Acid Sequence↗

PubMed2003

The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis.

The subtilin gene cluster (spa) of Bacillus subtilis ATCC 6633 is organized in transcriptional units spaBTC, spaS, spaIFEG and spaRK. Specific binding of the response regulator protein SpaR to spaB, spaS and spaI DNA promoter fragments was shown by means of electromobility shift assays. A repeated pentanucleotide sequence spaced by six nucleotides was identified as SpaR binding motif (spa-box). Saturating mutational analysis of the spa-box by single- and multiple-base-pair substitutions revealed the consensus motif (A/T)TGAT for optimal SpaR binding with the second, third and fifth position being absolutely conservative. Variations in the spacer size between the two pentanucleotide repeats revealed a strong conservation of their relative location. Only DNA with a proximal arrangement of two pentanucleotide repeats showed affinity to SpaR. A 2:1 stoichiometry between SpaR and DNA was obtained by optical biosensor analyses, which corresponds to the binding of two SpaR proteins per spa-box.

Amino Acid Sequence↗

PubMed2002

Function of Lactococcus lactis nisin immunity genes nisI and nisFEG after coordinated expression in the surrogate host Bacillus subtilis.

Nisin-producing Lactococcus lactis strains show a high degree of resistance to the action of nisin, which is based upon expression of the self-protection (immunity) genes nisI, nisF, nisE, and nisG. Different combinations of nisin immunity genes were integrated into the chromosome of a nisin-sensitive Bacillus subtilis host strain under the control of an inducible promoter. For the recipient strain, the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes. But either the lipoprotein NisI or the ABC transporter-homologous system NisFEG, respectively, were also able to protect the Bacillus host cells. The acquired immunity was specific to nisin and provided no tolerance to subtilin, a closely related lantibiotic. Quantitative in vivo peptide release assays demonstrated that NisFEG diminished the quantity of cell-associated nisin, providing evidence that one role of NisFEG is to transport nisin from the membrane into the extracellular space. NisI solubilized from B. subtilis membrane vesicles and recombinant hexahistidine-tagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.

ATP-Binding Cassette Transporters↗

PubMed2002

Several regions of p53 are involved in repression of RNA polymerase III transcription.

The tumour suppressor p53 has been shown to regulate RNA polymerase (pol) III transcription both in vitro and in vivo. We have characterized the regions of p53 that contribute to this effect. Repression of pol III transcription in vivo does not require residues 13-19 near the N-terminus of p53 that are highly conserved through evolution. However, amino acids 22 and 23 in the adjacent transactivation domain do contribute to the inhibition of pol III activity. Deletions within the central DNA-binding core domain (residues 102-292) of p53 can entirely abolish the repression function in these assays, despite the fact that pol III templates contain no recognized p53 binding site. Deletion or substitution within the C-terminal domain of p53 can also compromise its ability to repress pol III activity in vitro and in transfected cells. These observations reveal that repression of pol III transcription is a complex function involving multiple regions of p53 extending throughout much of the protein.

Chloramphenicol O-Acetyltransferase↗