Nancy D. Connell

Regulation of the cytoskeleton in mesothelial cells: reversible loss of keratin and increase in vimentin during rapid growth in culture

Human mesothelial cells grew rapidly in culture when provided with serum, EGF, and hydrocortisone, adopting a fibroblastoid shape and forming parallel, multilayered arrays at saturation density. In the absence of EGF, the cells grew slowly to a flat, epithelioid monolayer similar to their normal pattern in vivo. Mesothelial cells normally have a high keratin and a low vimentin content in vivo. In culture, rapidly growing cells greatly reduced synthesis and content of their four major keratins to levels undetectable by immunofluorescence in most cells, but keratin synthesis and content returned to high levels whenever growth slowed. Vimentin synthesis and content was high during serial culture, but decreased several-fold in nondividing cells. The unique ability of the mesothelial cell to reversibly alter its morphology and intermediate filament composition is of unknown function and mechanism, but accounts for the morphological heterogeneity and the presence of keratin-negative cells in mesotheliomas.

Characterization of a Fish Antimicrobial Peptide: Gene Expression, Subcellular Localization, and Spectrum of Activity

ABSTRACT Antimicrobial peptides are proposed to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against pathogenic microbes. Pleurocidin, a new 25-residue linear antimicrobial peptide, was recently isolated from the skin secretions of winter flounder (Pleuronectes americanus). The present study identifies the cDNA and gene encoding pleurocidin. The pleurocidin gene comprises four exons. Its upstream region demonstrates consensus binding sequences for transcription factors found in host defense genes in mammals, including sequences identical to the NF-IL6 and alpha and gamma interferon response elements. Pleurocidin is predicted to exist as a 68-residue prepropeptide that undergoes proteolytic cleavage of its amino-terminal signal and carboxy-terminal anionic propiece to form the active, mature peptide. Transmission electron microscopy localized pleurocidin to the mucin granules of skin and intestinal goblet cells. Significant synergy was shown to occur between pleurocidin and d-cycloserine targetingMycobacterium smegmatis. Pleurocidin was functionally active at physiologic concentrations of magnesium and calcium; however, high concentrations of these divalent cations ablated pleurocidins activity against a standard test strain, Escherichia coliD31. Pleurocidin was tested against bacterial and fungal clinical isolates and showed broad-spectrum antimicrobial activity. Together, these data support the hypothesis that pleurocidin participates in innate mucosal immunity, and it may prove to be a beneficial therapeutic agent.

An E. coli promoter induced by the cessation of growth

The production of the bacterial DNA replication inhibitor Microcin B17 is induced as cultures enter stationary phase. Using SI nuclease protection assays we have shown that this induction is the result of increased levels of transcription initiation from a promoter located upstream from mcbA, the structural gene for Microcin B17. Upstream from the start site of transcription there is a rather typical ‐35 region. However, there is no good homology to the consensus –10 region. While most of the cells transcription is shut off as a result of the cessation of growth, transcription from the mcbA promoter continues for several hours in stationary phase. A single‐copy gene fusion between mcbA and lacZ was used to monitor the response of the promoter to different nutritional conditions and in different host backgrounds altered in metabolic regulatory loci. Starvation for nitrogen, phosphate or carbon sources all induced transcription from the promoter. Levels of transcription were reduced in ompR backgrounds, in contrast, mutations in other global regulatory loci, fnr, relA and cya had little or no effect.

Human β-Defensin 2 Induces a Vigorous Cytokine Response in Peripheral Blood Mononuclear Cells

ABSTRACT β-Defensins are a family of small cationic peptides involved in the innate response to microbial infection. Although their role in microbial killing is well established, the mechanisms through which this occurs remain largely undefined. Here, using protein array technology, we describe a role for human β-defensins in the induction of an inflammatory cytokine response by human peripheral blood mononuclear cells (PBMCs). Human β-defensins 1, 2, and 3 were examined for induction of an array of cytokines and chemokines. Some cytokines, such as interleukin 8 (IL-8) and monocyte chemoattractant protein 1, were up-regulated by all three defensins, while others, such as IL-6 and IL-10, were induced more selectively. It was notable that each defensin induced a unique pattern of cytokines. This report documents, for the first time, an analysis of the composite cytokine response of human PBMCs to β-defensins. The induction or up-regulation of a number of cytokines involved in the adaptive immune response suggests a possible role for these defensins in linking innate and acquired immunity.

Mycobacterium: isolation, maintenance, transformation, and mutant selection.

Publisher Summary This chapter discusses methods for culturing Mycobacterium , isolating genetic mutants, and moving DNA in and out of the cells. Experimental methods for culturing and genetically manipulating M. avium , M. tuberculosis , M. smegmatis , and bacille Calmette Guerin (BCG) is discussed in the chapter. The isolation of mycobacteria from contaminated sources requires decontamination because specimens usually contain mixed bacterial flora and some degree of organic debris such as body fluids, blood cells, and tissue fragments. There are a number of isolation protocols but all require digestion, decontamination, and concentration steps. The mycobacteria must be recovered undamaged while organic debris is liquefied and contaminating micororganisms are killed. The chapter discusses protocol for treatment of sputum samples and is suitable for use in clinical biosafety level (BSL-2) and experimental BSL-3 laboratories for the treatment of clinical samples. The mycobacteriophages have been isolated and developed for the purpose of strain typing. Their use in transfer of genetic material for the construction of strains is discussed.

Airborne infection with Bacillus anthracis--from mills to mail.

The lack of identified exposures in 2 of the 11 cases of bioterrorism-related inhalation anthrax in 2001 raised uncertainty about the infectious dose and transmission of Bacillus anthracis. We used the Wells-Riley mathematical model of airborne infection to estimate 1) the exposure concentrations in postal facilities where cases of inhalation anthrax occurred and 2) the risk for infection in various hypothetical scenarios of exposure to B. anthracis aerosolized from contaminated mail in residential settings. These models suggest that a small number of cases of inhalation anthrax can be expected when large numbers of persons are exposed to low concentrations of B. anthracis. The risk for inhalation anthrax is determined not only by bacillary virulence factors but also by infectious aerosol production and removal rates and by host factors.

Glutathione and Nitrosoglutathione in Macrophage Defense against Mycobacterium tuberculosis

ABSTRACT We demonstrate that Mycobacterium tuberculosis grown in vitro is sensitive to glutathione and its derivative S-nitrosoglutathione. Furthermore, our infection studies with J774.1 macrophages indicate that glutathione is essential for the control of the intracellular growth of M. tuberculosis. This study indicates the important role of glutathione in the control of macrophages by M. tuberculosis.

Role of Glutathione in Macrophage Control of Mycobacteria

ABSTRACT Reactive oxygen and nitrogen intermediates are important antimicrobial defense mechanisms of macrophages and other phagocytic cells. While reactive nitrogen intermediates have been shown to play an important role in tuberculosis control in the murine system, their role in human disease is not clearly established. Glutathione, a tripeptide and antioxidant, is synthesized at high levels by cells during reactive oxygen intermediate and nitrogen intermediate production. Glutathione has been recently shown to play an important role in apoptosis and to regulate antigen-presenting-cell functions. Glutathione also serves as a carrier molecule for nitric oxide, in the form of S-nitrosoglutathione. Previous work from this laboratory has shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. A mutant strain of Mycobacterium bovis BCG, defective in the transport of small peptides such as glutathione, is resistant to the toxic effect of glutathione and S-nitrosoglutathione. Using the peptide transport mutant as a tool, we investigated the role of glutathione and S-nitrosoglutathione in animal and human macrophages in controlling intracellular mycobacterial growth.

Characterization of a Glutathione Metabolic Mutant of Mycobacterium tuberculosis and Its Resistance to Glutathione and Nitrosoglutathione

Glutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form of S-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione and S-nitrosoglutathione is brought about by the action of the enzyme gamma-glutamyl transpeptidase. The function of gamma-glutamyl transpeptidase is to cleave glutathione and S-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain of Mycobacterium tuberculosis lacking this metabolic enzyme. We investigated the sensitivity of this strain to glutathione and S-nitrosoglutathione compared to that of the wild-type bacteria. In addition, we examined the role of glutathione and/or S-nitrosoglutathione in controlling the growth of intracellular M. tuberculosis inside mouse macrophages.

A Peptide Permease Mutant of Mycobacterium bovis BCG Resistant to the Toxic Peptides Glutathione and S-Nitrosoglutathione

ABSTRACT Oligopeptides play important roles in bacterial nutrition and signaling. Using sequences from the available genome database forMycobacterium tuberculosis H37Rv, the oligopeptide permease operon (oppBCDA) of Mycobacterium bovis BCG was cloned from a cosmid library. An opp mutant strain was constructed by homologous recombination with an allele ofoppD interrupted by kanamycin and streptomycin resistance markers. The deletion was complemented with a wild-type copy of theopp operon. Two approaches were taken to characterize the peptide transporter defect in this mutant strain. First, growth of wild-type and mutant strains was monitored in media containing a wide variety of peptides as sole source of carbon and/or nitrogen. Among 25 peptides ranging from two to six amino acids in length, none was capable of supporting measurable growth as the sole carbon source in either wild-type or mutant strains. The second approach exploited the resistance of permease mutants to toxic substrates. The tripeptide glutathione (γ-glutamyl-l-cyteinylglycine [GSH]) is toxic to wild-type BCG and was used successfully to characterize peptide uptake in the opp mutant. In 2 mM GSH, growth of the wild-type strain is inhibited, whereas the opp mutant is resistant to concentrations as high as 10 mM. Similar results were found with the tripeptide S-nitrosoglutathione (GSNO), thought to be a donor of NO in mammalian cells. Using incorporation of [3H]uracil to monitor the effects of GSH and GSNO on macromolecular synthesis in growing cells, it was demonstrated that theopp mutant is resistant, whereas the wild type and the mutant complemented with a wild-type copy of the operon are sensitive to both tripeptides. In uptake measurements, incorporation of [3H]GSH is reduced in the mutant compared with wild type and the complemented mutant. Finally, growth of the three strains in the tripeptides suggests that GSH is bacteriostatic, whereas GSNO is bacteriocidal.