A rhodanine agent active against non-replicating intracellular Mycobacterium avium subspecies paratuberculosis.

BACKGROUND: Antibiotic therapy targeting chronic mycobacterial disease is often ineffective due to problems with the emergence of drug resistance and non-replicating persistent intracellular antibiotic resistant phenotypes. Strategies which include agents able to enhance host cell killing mechanisms could represent an alternative to conventional methods with the potential for host clearance if active against dormant phenotypes. Investigations of agents with potential activity against non-replicating mycobacteria however are restricted due to a need for assays that can assess bacterial viability without having to culture. RESULTS: This study describes the development and use of a pre16S ribosomal gene RNA/DNA ratio viability assay which is independent of the need for culture, supported by a novel thin layer accelerated mycobacterial colony forming method for determining viability and culturability of MAP in intracellular environments. We describe the use of these tools to demonstrate intracellular killing activity of a novel rhodanine agent (D157070) against the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (MAP) and show that the culturability of MAP decreases relative to its viability on intracellular entry suggesting the induction of a non-culturable phenotype. We further demonstrate that D157070, although having no direct activity against the culturability of extracellular MAP, can bind to cultured MAP cells and has significant influence on the MAP transcriptome, particularly with respect of delta(L )associated genes. D157070 is shown to be taken up by bovine and human cells and able to enhance host cell killing, as measured by significant decreases in both culturability and viability of intracellular MAP. CONCLUSIONS: This work suggests that pre16srRNA gene ratios represent a viable method for studying MAP viability. In addition, the rhodanine agent D157070 tested is non-toxic and enhances cell killing activity against both growing and latent MAP phenotypes

Suppression of anchorage-independent growth after gene transfection.

A novel procedure for isolating anchorage-dependent cells has been developed. It involves negative selection of cells growing in suspension followed by clonal replica screening for anchorage-dependent growth. Cells which have regained anchorage-dependent growth have been isolated from a library of the Chinese hamster ovary cell line, CHO-K1, transfected with pSV2neo and human genomic DNA. One anchorage-dependent clone, 1042AC, has been studied in detail. Anchorage-dependent growth of 1042AC is stable when cultured as adherent monolayers, but revertants appear rapidly when cultured in suspension. Suppression is unlikely to be due to loss or mutation of hamster genes conferring anchorage-independent growth as hybrids between 1042AC and CHO-K1 have the suppressed phenotype of 1042AC. Furthermore, a population of cells obtained from the hybrid by selecting for revertants to anchorage-independent growth showed selective loss of the transgenome derived from 1042AC. The growth suppression was not due to transfection of the human Krev-1 gene, which has previously been shown to restore anchorage-dependent growth, nor was there any evidence of alteration in the endogenous hamster Krev-1 gene. However, evidence for a human gene being responsible for the suppressed phenotype has not been obtained yet

Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, also known as Johne’s disease, a chronic intestinal infection in cattle and other ruminants. Paratuberculosis is characterised by diarrhea and weight loss that occurs after a period of a few months up to several years without any clinical signs. The considerable economic losses to dairy and beef cattle producers are caused by reduced milk production and poor reproduction performance in subclinically infected animals. Early diagnosis of infected cattle is essential to prevent the spread of the disease. Efforts have been made to eradicate paratuberculosis by using a detection and cull strategy, but eradication is hampered by the lack of suitable and sensitive diagnostic methods. This thesis, based on five scientific investigations, describes the development of different DNA amplification strategies for detection and characterisation of M. paratuberculosis. Various ways to pre-treat bacterial cultures, tissue specimens and fecal samples prior to PCR analysis were investigated. Internal positive PCR control molecules were developed and used in PCR analyses to improve the reliability and to facilitate the interpretation of the results. The sensitivity of the ultimate methods was found to be approximate that of culture and allowed detection of low numbers of M. paratuberculosis expected to be found in subclinically infected animals. Genomic DNA of a Swedish mycobacterial isolate, incorrectly identified by PCR as M. paratuberculosis was characterised. The isolate was closely related to M. cookii and harboured one copy of a DNA segment with 94% similarity to IS900, the target sequence used in diagnostic PCR for detection of M. paratuberculosis. This finding highlighted the urgency of developing or evaluating PCR systems based on genes other than IS900. A PCR-based fingerprinting method using primers targeting the enterobacterial intergenic consensus sequence (ERIC) and the IS900 sequence was developed and successfully used to distinguish M. paratuberculosis from closely related mycobacteria, including the above mentioned mycobacterial isolate. In conclusion, the molecular biology techniques developed in these studies have proved useful for accelerating the diagnostic detection and characterisation of M. paratuberculosis

Establishment and characterization of primary human pancreatic carcinoma in continuous cell culture and in nude mice.

Primary human panceratic exocrine adenocarcinoma has been established in tissue culture and as xenografts in immune-deficient nu/nu mice. The cell line has a doubling time of 36 h and grows as a confluent monolayer together with a constant population of free-floating cells. Evidence of tumourigenicity was provided by growth on an early diploid fibroblast monolayer and in soft agar, and as solid tumours in immune-deficient nu/mu mice. Chromosome analysis of the cultured cells confirmed their tumour origin. Xenografts established from the cell line or directly from primary tumour tissue have retained a similar histology to the original tumour on serial transplantation. An electrophoretic study of exportable pancreatic digestive enzymes and a number of intracellular enzymes has shown that the cell line and xenografts maintain a human intracellular enzyme profile, but do not produce pancreatic digestive enzymes

Serial position and rated importance in the recall of text

Includes bibliographiesSupported in part by the National Institute of Education under contract no. HEW-NIE-C-400-76-011

Centennial Souvenir and History of Hermon, Penobscot County, Maine, 1814-1914

A history of Hermon, Maine, compiled by the Historical Committee: Frank A. Bishop, chairman; Roscoe W. Snow; Annie Mayhew