Automated microscopic image analysis and the prognosis of preneoplasms and carcinomas of the mammary gland

Studies of preneoplasias and carcinomas of the mammary gland have been conducted by means of automated microscopic image analysis, on the basis of previous results as well as with references to the international literature, for 2 purposes: 1. Determination in the context of a clinical follow-up study of the individual carcinoma risk for patients with proliferative fibrocystic breast disease (mastopathy) and 2. Preparation of an objective automated grading of ductal breast carcinomas for better assessment of prognosis. Against the background of the assumption that the majority of carcinomas and precancerous lesions of the breast originate from the terminal ductal lobular unit, an effort is made to determine, independent severity of the mastopathy, the biological valence of solid, cribriform, and papillary ductal epithelial proliferations (no, possible or inevitable preneoplasia). Proliferation patterns without and with atypical features are checked for their similarity with intraductal and invasive carcinomas (similarity principle) and are additionally examined for differences, depending on localization and distance from tumour (topological principle). Automated histological tumour grading is to distinguish with greater subtlety within the large heterogeneous group of moderately differentiated carcinomas and is to objectify and thus facilitate the difficult task of delimitation of moderately and poorly differentiated carcinomas. Nuclear grading will be the major basis for classification along these lines. Objectified and reproducible tumour grading is believed to be extremely helpful in better prognostication of breast cancer.     

Influence of phloem transport, N-fertilization and ion accumulation on sucrose storage in the taproots of fodder beet and sugar beet

To investigate the factors governing the accumulation of sucroseand amino acids in the taproots of sugar beet, their contentswere measured in the leaves, phloem sap and the taproots ofsugar beet, fodder beet and a hybrid between both, grown oneither 3.0 or 0.5 mM nitrate. In the taproots the contents ofmalate, citrate and inorganic ions were also determined. Forthe high sucrose accumulation in sugar beet as compared to theother varieties three factors were found. (a) In sugar beet,less amino acids and more sucrose are taken up into the phloemthan in fodder beet. (b) In sugar beet, the sucrose and aminoacid syntheses are less sensitive to the nitrate concentrationsthat are required for optimal plant growth than in other varieties.In fodder beet, upon raising the nitrate concentration from0.5 mM to 3 mM, the synthesis and storage of sucrose is decreasedand that of amino acids increased. The corresponding valuesin sugar beet (0.5 mM) are similar to those in fodder beet andare not much affected by an increase of nitrate. (c) The sucroseaccumulation is limited by the accumulation of inorganic ionsin the taproots. The sucrose content in the taproots is negativelycorrelated to the total ion content. Whereas sucrose representstwo-third of all solutes in the taproots of sugar beet, it amountsto only one-third of the solutes in fodder beet taproots. Key words: Amino acids, Beta vulgans L, phloem sap, potassium, sucrose storage, sugar beet, taproots, transport     

Truncated hemoglobins in actinorhizal nodules of Datisca glomerata

Three types of hemoglobins exist in higher plants, symbiotic, non-symbiotic, and truncated hemoglobins. Symbiotic (class II) hemoglobins play a role in oxygen supply to intracellular nitrogen-fixing symbionts in legume root nodules, and in one case ( Parasponia Sp.), a non-symbiotic (class I) hemoglobin has been recruited for this function. Here we report the induction of a host gene, dgtrHB1, encoding a truncated hemoglobin in Frankia-induced nodules of the actinorhizal plant Datisca glomerata. Induction takes place specifically in cells infected by the microsymbiont, prior to the onset of bacterial nitrogen fixation. A bacterial gene (Frankia trHBO) encoding a truncated hemoglobin with O (2)-binding kinetics suitable for the facilitation of O (2) diffusion ( ) is also expressed in symbiosis. Nodule oximetry confirms the presence of a molecule that binds oxygen reversibly in D. glomerata nodules, but indicates a low overall hemoglobin concentration suggesting a local function. Frankia trHbO is likely to be responsible for this activity. The function of the D. glomerata truncated hemoglobin is unknown; a possible role in nitric oxide detoxification is suggested.     

Genome-Wide Transposon Mutagenesis Identifies a Role for Host Neuroendocrine Stress Hormones in Regulating the Expression of Virulence Genes in Salmonella

Bacterial sensing of environmental signals plays a key role in regulating virulence and mediating bacterium-host interactions. The sensing of the neuroendocrine stress hormones epinephrine (adrenaline) and norepinephrine (noradrenaline) plays an important role in modulating bacterial virulence. We used MudJ transposon mutagenesis to globally screen for genes regulated by neuroendocrine stress hormones in Salmonella enterica serovar Typhimurium. We identified eight hormone-regulated genes, including yhaK, iroC, nrdF, accC, yedP, STM3081, and the virulence-related genes virK and mig14. The mammalian α-adrenergic receptor antagonist phentolamine reversed the hormone-mediated effects on yhaK, virK, and mig14 but did not affect the other genes. The β-adrenergic receptor antagonist propranolol had no activity in these assays. The virK and mig14 genes are involved in antimicrobial peptide resistance, and phenotypic screens revealed that exposure to neuroendocrine hormones increased the sensitivity of S. Typhimurium to the antimicrobial peptide LL-37. A virK mutant and a virK mig14 double mutant also displayed increased sensitivity to LL-37. In contrast to enterohemorrhagic Escherichia coli (EHEC), we have found no role for the two-component systems QseBC and QseEF in the adrenergic regulation of any of the identified genes. Furthermore, hormone-regulated gene expression could not be blocked by the QseC inhibitor LED209, suggesting that sensing of hormones is mediated through alternative signaling pathways in S. Typhimurium. This study has identified a role for host-derived neuroendocrine stress hormones in downregulating S. Typhimurium virulence gene expression to the benefit of the host, thus providing further insights into the field of host-pathogen communication.Bacterial sensing of environmental signals plays a key role in regulating virulence gene expression and bacterium-host interactions. It is increasingly recognized that detection of host-derived molecules, such as the neuroendocrine stress hormones (catecholamines) epinephrine (adrenaline) and norepinephrine (noradrenaline), plays an important role in modulating bacterial virulence (29, 42).Physical and psychological stress has been linked to increased severity and susceptibility to infection in humans and other animals (23, 42), and epinephrine/norepinephrine levels are an important factor in this. Stress triggers an increase in plasma epinephrine levels (31), and plasma levels of epinephrine and norepinephrine have been reported to increase with patients suffering from postoperative sepsis compared to patients with no complications (32). Administration of norepinephrine and epinephrine to otherwise healthy subjects increases the severity of bacterial infections, including Clostridium perfringens in humans and enterohemorrhagic Escherichia coli (EHEC) in calves (42, 63, 65). Treatment with norepinephrine also increases the virulence of Salmonella enterica serovar Enteritidis in chicks and Salmonella enterica serovar Typhimurium in mice, with a substantial increase in bacterial numbers recovered from the cecum and liver in both cases (47, 65).Norepinephrine is found in large concentrations in the gut due to release by gastrointestinal neurones; indeed up to half the norepinephrine in the body may be produced in the enteric nervous system (ENS) (3). Epinephrine, while not normally found in the gut, is present in the bloodstream and is also produced by macrophages in response to bacteria-derived lipopolysaccharide (LPS) (12, 26). S. Typhimurium is an enteropathogen, can also cross the epithelial barrier to cause systemic infection, and will therefore encounter both these molecules in the normal infection cycle.Phenotypes induced by stress hormones in bacteria include increased adherence of EHEC to bovine intestinal mucosa (63), upregulation of type III secretion and Shiga toxin production in EHEC (22, 60), upregulation of type III secretion in Vibrio parahaemolyticus (51), increase in invasion of epithelial cells and breakdown of epithelial tight junctions by Campylobacter jejuni (15), affected motility and expression of iron uptake genes in S. Typhimurium (8, 9, 36), and modulated virulence in Borrelia burgdorferi (59). Epinephrine and norepinephrine can overcome the growth inhibition of many bacteria, including Salmonella, in serum-containing media (13, 43), due to the ability to act as a siderophore to facilitate iron uptake (13, 28, 47).Norepinephrine and epinephrine also interact with bacterial quorum-sensing (QS) systems. QS is a process of bacterial cell-cell communication in which each cell produces small signal molecules termed “autoinducers” (AIs), which regulate gene expression when a critical threshold concentration and therefore population density have been reached. QS affects diverse processes, including motility, virulence, biofilm formation, type III secretion, and luminescence (6, 64).The EHEC AI-3 QS system is important for motility and expression of the type III secretion system encoded by the locus of enterocyte effacement (LEE) (60). AI-3 sensing and signal transduction are mediated via the QseBC and QseEF two-component systems, respectively. Epinephrine and norepinephrine can substitute for AI-3, causing cross talk between the two signaling systems and induction of type III secretion and motility (57, 60). The sensor kinase QseC is autophosphorylated upon binding either epinephrine or norepinephrine (14), demonstrating the presence of adrenergic receptors in bacteria. These adrenergic phenotypes can also be blocked by the mammalian α- and β-adrenergic antagonists phentolamine and propranolol, although it should be noted that QseC is blocked only by the former (14, 60). This suggests the occurrence of cross talk between bacterial and mammalian cell signaling systems and the existence of multiple bacterial adrenergic sensors.To elucidate the role of host-derived stress hormones in the physiology and pathogenicity of S. Typhimurium, we used MudJ transposon mutagenesis to screen globally for epinephrine- and norepinephrine-regulated genes in S. Typhimurium.     

The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa

LecA (PA-IL) is a cytotoxic lectin and adhesin produced by Pseudomonas aeruginosa which binds hydrophobic galactosides with high specificity and affinity. By using a lecA-egfp translation fusion and immunoblot analysis of the biofilm extracellular matrix, we show that lecA is expressed in biofilm-grown cells. In static biofilm assays on both polystyrene and stainless steel, biofilm depth and surface coverage was reduced by mutation of lecA and enhanced in the LecA-overproducing strain PAO-P47. Biofilm surface coverage by the parent strain, PAO-P47 but not the lecA mutant on steel coupons was also inhibited by growth in the presence of either isopropyl-beta-D-thiogalactoside (IPTG) or p-nitrophenyl-alpha-D-galactoside (NPG). Furthermore, mature wild-type biofilms formed in the absence of these hydrophobic galactosides could be dispersed by the addition of IPTG. In contrast, addition of p-nitrophenyl-alpha-L-fucose (NPF) which has a high affinity for the P. aeruginosa LecB (PA-IIL) lectin had no effect on biofilm formation or dispersal. Planktonic growth of P. aeruginosa PAO1 was unaffected by the presence of IPTG, NPG or NPF, nor was the strain able to utilize these sugars as carbon sources, suggesting that the observed effects on biofilm formation were due to the competitive inhibition of LecA-ligand binding. Similar results were also obtained for biofilms grown under dynamic flow conditions on steel coupons, suggesting that LecA contributes to P. aeruginosa biofilm architecture under different environmental conditions.