Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland.

Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools.

The modification of mammalian genomes is an important goal in gene therapy and animal transgenesis. To generate stable genetic and biochemical changes, the therapeutic genes or transgenes need to be incorporated into the host genome. Ideally, the integration of the foreign gene should occur at sites that ensure their continual expression in the absence of any unwanted side effects on cellular metabolism.

Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction.

A variety of technological advances in recent years have made permanent genetic manipulation of an organism a technical possibility. As the details of natural biological processes for genome modification are elucidated, the enzymes catalyzing these events (transposases, recombinases, integrases and DNA repair enzymes) are being harnessed or modified for the purpose of intentional gene modification. Targeted integration and gene repair can be mediated by the DNA-targeting specificity inherent to a particular enzyme, or rely on user-designed specificities.

Binding sites of VanRB and sigma70 RNA polymerase in the vanB vancomycin resistance operon of Enterococcus faecium BM4524.

The vanB operon of Enterococcus faecium BM4524 which confers inducible resistance to vancomycin is composed of the vanR(B)S(B) gene encoding a two-component regulatory system and the vanY(B)WH(B)BX(B) resistance genes that are transcribed from promoters P(RB) and P(YB) respectively. In this study, primer extension revealed transcription start sites at 13 and 48 bp upstream from the start codon of vanR(B) and vanY(B), respectively, that allowed identification of -10 and -35 promoter motifs. The VanR(B) protein was overproduced in Escherichia coli, purified and phosphorylated (VanR(B)-P) non-enzymically with acetylphosphate.

Osteopontin influences the invasiveness of pancreatic cancer cells and is increased in neoplastic and inflammatory conditions.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with an overall 5-year survival rate of less than 5%. Invasive tumor growth and early metastasis are two important reasons for this dismal prognosis. Osteopontin (OPN) is a secretory protein with a variety of functions, for example in cell adhesion and migration, inflammatory reaction and apoptosis.

Targeted modification of mammalian genomes.

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome.

[Fundus spectrometry in age-related maculopathy].

Background: Spectroscopic methods permit the non-invasive detection of fundus pigments by the wavelength-dependent absorption of fluorescence as well as by the fluorescence lifetime. From the relative concentrations of haemoglobin and oxyhaemoglobin, the oxygen saturation can be calculated. The onset of age-related maculopathy might be delayed by a high optical density of xanthophyll.

A virus-neutralising antibody is not cytotoxic in vitro.

Hybridoma cell lines are characterized by a preferential loss of the heavy chain gene. This observation has led to the theory that the immunoglobulin heavy chain possesses an intrinsic cytotoxic activity in some cell types. We have generated transgenic mice expressing the heavy and light chain genes of the virus-neutralising antibody A1 carrying constant domains of the human gamma1 and kappa isotype.

An unusual primary sigma factor in the Bacteroidetes phylum.

The presence of housekeeping gene promoters with a unique consensus sequence in Bacteroides fragilis, previously described by Bayley et al. (2000, FEMS Microbiol Lett 193: 149-154), suggested the existence of a particular primary sigma factor. The single rpoD-like gene observed in the B.

Substitutions in region 2.4 of sigma70 allow recognition of the sigmaS-dependent aidB promoter.

The strict dependence of transcription from the aidB promoter (PaidB) on the Esigma(S) form of RNA polymerase is because of the presence of a C nucleotide as the first residue of the -10 promoter sequence (-12C), which does not allow an open complex formation by Esigma(70). In this report, sigma(70) mutants carrying either the Q437H or the T440I single amino acid substitutions, which allow -12C recognition by sigma(70), were tested for their ability to carry out transcription from PaidB. The Gln-437 and Thr-440 residues are located in region 2.

Phage T4 early promoters are resistant to inhibition by the anti-sigma factor AsiA.

Phage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme Esigma(70). We studied in vitro the effects of the T4 anti-sigma(70) factor AsiA on the activity of several T4 early promoters. In single-round transcription, promoters motB, denV, mrh.

Comparative analysis of extracellular matrix proteins in chronic pancreatitis: differences between pancreatic head and tail.

Objectives: In patients with chronic pancreatitis (cP) with an inflammatory mass in the pancreatic head, the degree of fibrosis in the pancreatic head compared with the tail should be determined and differences in the expression pattern of collagen types I, III, and IV; laminin; vitronectin; and fibronectin should be evaluated.

Methods: From 12 patients with alcohol-induced and idiopathic cP who underwent surgery due to local complications, 24 corresponding cP tissue samples from the pancreatic head and the resection margin were obtained. The degree of fibrosis was calculated using a computer-assisted method (Adobe Photo Shop).

Mass spectrometry of Escherichia coli RNA polymerase: interactions of the core enzyme with sigma70 and Rsd protein.

The E. coli RNA polymerase core enzyme is a multisubunit complex of 388,981 Da. To initiate transcription at promoters, the core enzyme associates with a sigma subunit to form holo RNA polymerase.

Interactions between the 2.4 and 4.2 regions of sigmaS, the stress-specific sigma factor of Escherichia coli, and the -10 and -35 promoter elements.

The sigmas subunit of Escherichia coli RNA polymerase holoenzyme (EsigmaS) is a key factor of gene expression upon entry into stationary phase and in stressful conditions. The selectivity of promoter recognition by EsigmaS and the housekeeping Esigma70 is as yet not clearly understood. We used a genetic approach to investigate the interaction of sigmaS with its target promoters.

Studies of the Escherichia coli Rsd-sigma70 complex.

Escherichia coli Rsd protein was previously identified on the basis of its binding to the RNA polymerase sigma(70) subunit. The Rsd-sigma(70) complex has been studied using different methods. Our data show that Rsd associates with sigma(70) to form a complex with a stoichiometry of 1:1.

Enhanced isolation of low frequency herpes simplex virus recombinants using green-fluorescent protein and FACS.

The generation of recombinant herpes simplex virus to study the effect of engineered mutations on viral biology relies on the isolation of recombinants from a mixed population of viruses following a marker transfer procedure. Currently, the E. coli lacZ or green-fluorescent protein (GFP) genes are most frequently used as markers for isolation and isolation of recombinants relies on visual screening of plaques.

Biochemical characterization of a new melanoma model--the minipig MeLiM strain.

Hereditary minipig melanomas, which have many histopathological and other features in common with their human counterparts, have recently become preferred melanoma models. The MeLiM (Melanoma-bearing Libĕchov Minipig) strain was selected by partial inbreeding. A high tumour incidence and malignant behaviour on the one hand together with the occurrence of spontaneous regression and high susceptibility to the devitalization technique as a new strategy in melanoma therapy, make the MeLiM strain a superior melanoma model to other hereditary swine melanomas.

Molecular determinants of the hpa regulatory system of Escherichia coli: the HpaR repressor.

The HpaR-mediated regulation of the hpa-meta operon (Pg promoter) of the 4-hydroxyphenylacetic acid catabolic pathway of Escherichia coli has been studied. The HpaR regulator was purified to homogeneity showing that it is able to bind selectively to 4-hydroxyphenylacetic, 3-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids, which act as inducers of the system. The role of HpaR as a repressor and the requirement for cAMP receptor protein for maximal activity have been confirmed by in vitro transcription analyses.

Tyrosine 116 of the herpes simplex virus type 1 IEalpha22 protein is an ocular virulence determinant and potential phosphorylation site.

Purpose: To determine whether tyrosine 116 of the HSV-1 alpha22 protein is involved in virulence and is a potential phosphorylation site.

Methods: Site-directed mutagenesis was used to revert the Y116C mutation in the alpha22 gene of the strain OD4 to wild type (C116Y), and the effect of virulence was tested by using a marker transfer-infection protocol in mice. Immunoblot analysis, tryptic phosphopeptide mapping, and phosphotyrosine pulldown-immunoblot protocols were used to assess the OD4 alpha22 isoforms.

The prospects of modifying the antimicrobial properties of milk.

Milk contains a variety of substances, which inhibit the infection of pathogens. This is of benefit to the mother, safeguarding the integrity of the lactating mammary gland, but also of huge importance for protection of the suckling offspring. The antimicrobial substances in milk can be classified into two categories.

Nucleotides from -16 to -12 determine specific promoter recognition by bacterial sigmaS-RNA polymerase.

The alternative sigma factor sigmaS, mainly active in stationary phase of growth, recognizes in vitro a -10 promoter sequence almost identical to the one for the main sigma factor, sigma70, thus raising the problem of how specific promoter recognition by sigmaS-RNA polymerase (EsigmaS) is achieved in vivo. We investigated the promoter features involved in selective recognition by EsigmaS at the strictly sigmaS-dependent aidB promoter. We show that the presence of a C nucleotide as first residue of the aidB -10 sequence (-12C), instead of the T nucleotide canonical for sigma70-dependent promoters, is the major determinant for selective recognition by EsigmaS.

The first intron of the murine beta-casein gene contains a functional promoter.

Caseins are the major milk proteins in most mammals. Together with calcium and phosphate they form the casein micelle. The corresponding casein genes are clustered in mammalian genomes and their expression is coordinately regulated with regard to developmental and tissue specificity.

Multiple determinants contribute to the virulence of HSV ocular and CNS infection and identification of serine 34 of the US1 gene as an ocular disease determinant.

Purpose: The virulence of any given strain of herpes simplex virus (HSV) is probably due to the effects of the constellation of genes in that strain and how they act in concert to promote disease. The goal of this work was to develop a system to identify and study the role of multiple genes in HSV disease.

Methods: Mixed ocular infection with HSV-1 strains CJ394 and OD4 yield recombinants with increased ocular and central nervous system (CNS) virulence.

Transcription regulation coupling of the divergent argG and metY promoters in Escherichia coli K-12.

The cAMP-catabolite activator protein (CAP) complex is a pleiotropic regulator that regulates a vast number of Escherichia coli genes, including those involved in carbon metabolism. We identified two new targets of this complex: argG, which encodes the arginosuccinate synthase involved in the arginine biosynthetic pathway, and metY, which encodes one of the two methionine tRNA initiators, tRNAf2Met. The cAMP-CAP complex activates argG transcription and inhibits metY transcription from the same DNA position.