Biomedical subjects

Ian L Ross

Publications and source records attributed to Ian L Ross.

6 recordsLinked to original sources

PubMed2006

Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector.

Gateway technology is a powerful system for converting a single entry vector into a wide variety of expression vectors. We expressed recombinant influenza matrix protein M1 (FMP), a potent antigen for cytotoxic T cells, using the Gateway vector pET-DEST42 containing the FMP cDNA, and purified the expressed FMP as a single 32 kDa recombinant protein. N-terminal and internal protein sequencing, however, showed that the recombinant FMP contained an extra 10 amino acids fused to the N-terminal of native FMP. Further investigation of the DNA sequence adjacent to the 5'-FMP cDNA indicated that the "TTG" in the attB1 site (30 bp upstream of the "ATG" in the 5'-FMP cDNA) behaved as a dominant translation start site, resulting in a 10 amino acid extension of the recombinant FMP. Thus, it is possible that recombinant proteins produced by this Gateway vector contain unexpected vector-derived peptides, which may affect experimental outcomes.

Base Sequence↗

PubMed2006

Expression of human DEC-205 (CD205) multilectin receptor on leukocytes.

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single approximately 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')2 also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to FcgammaR. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

Animals↗

PubMed2005

Discrimination within phenotypically closely related definitive types of Salmonella enterica serovar typhimurium by the multiple amplification of phage locus typing technique.

Multilocus sequence typing (MLST) is a relatively new high-resolution typing system employed for epidemiological studies of bacteria, including Salmonella. Discrimination based on MLST of housekeeping genes may be problematical, due to the high identity of gene sequences of closely related Salmonella species. The presence of genomic sequences derived from stable temperate phages in Salmonella offers an alternative for MLST of Salmonella. We have used MLST of prophage loci in Salmonella enterica serovar Typhimurium to discriminate closely related isolates of serovar Typhimurium. We have compared these results to MLST of five housekeeping genes, as well as pulsed-field gel electrophoresis (PFGE). The presence or absence of prophage loci in the 73 serovar Typhimurium isolates tested, as well as allelic variation as detected by sequencing, provided greater discrimination between isolates than either MLST of housekeeping genes or PFGE. Amplification of prophage loci alone separated serovar Typhimurium isolates into 27 groups comprising multiple isolates or individual strains. Sequencing of isolates found within the clusters separated isolates even further. By contrast, PFGE could only divide the 73 isolates into five distinct groups. MLST using housekeeping genes did not provide any significant separation of isolates in comparison to amplification or MLST of prophage loci. The results demonstrate that the amplification and sequencing of prophage loci provides a high-resolution, objective method for the discrimination of closely related isolates of serovar Typhimurium. It is proposed that multiple amplification of phage locus typing may provide sufficient discrimination for epidemiological purposes without recourse to MLST.

Bacterial Proteins↗

PubMed2003

A fluorescent amplified fragment length polymorphism study of Salmonella enterica serovar Sofia, the major Salmonella serovar isolated from chickens in Australia.

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed on 68 isolates of Salmonella enterica subsp. salamae serovar Sofia (S. Sofia). Fifty eight isolates were obtained over a period of approximately 15 years from a range of human, chicken industry and environmental sources throughout Australia. A further ten isolates were identified from human and poultry sources in Israel from 1972 to 1987. Analysis of FAFLP profiles for fragments between 50 to 500 base pairs in length indicated distinct clusters of isolates. All but seven isolates clustered into four groups of >90% similarity and all isolates displayed at least 70% similarity with each other. No cluster could be attributed to a particular geographical, temporal or source-of-isolation origin. It is concluded that S. Sofia is genetically variable with certain clones persisting over time but no group appears unique to Australia.

Animals↗

PubMed2003

Overt hypoadrenalism is uncommon in patients with stage 3 and 4 bronchogenic carcinoma.

INTRODUCTION: Lung cancer is the leading cause of cancer mortality in most countries. The adrenal glands are common sites of metastatic lung cancer as approximately 40% of subjects with stage 4 bronchogenic carcinoma have adrenal metastases. The prevalence of biochemical hypoadrenalism is, however, remarkably poorly documented. OBJECTIVES: Our study aimed to determine the prevalence of primary hypoadrenalism, as defined by a subnormal cortisol response to the 250 micrograms adrenocorticotrophic hormone (ACTH) stimulation test, in patients with stage 3 and 4 lung cancer. METHODS: Thirty patients with stage 3 and 4 bronchogenic carcinoma were prospectively recruited from the bronchus clinic. Demographic data and electrolytes were recorded and each patient had a 250 micrograms ACTH stimulation test to determine the prevalence of overt adrenal insufficiency, defined as a +30 minute cortisol of less than 550 nmol/l. RESULTS: The median age and quartile deviation was 62 (10) years and the median basal cortisol was 429.5 (321) nmol/l. The median peak cortisol was 828.5 (342) nmol/l (range 536-1,675 nmol/l). Twenty-eight patients (93.3%) had an appropriate rise of cortisol to greater than 550 nmol/l following 250 micrograms ACTH stimulation. Two patients (6.7%) had mild primary adrenal failure with a peak cortisol between 500 and 550 nmol/l associated with a raised plasma ACTH concentration (131.4 and 10.5 pmol/l, normal 2.2-10 pmol/l). Twenty-eight patients (92.9%) were normonatraemic, while the two hyponatraemic patients had biochemical evidence of the syndrome of inappropriate antidiuretic hormone secretion. CONCLUSION: In conclusion, despite evidence that the adrenal glands of patients with disseminated bronchogenic carcinoma are frequently affected by metastatic disease, biochemical evidence of clinically significant hypoadrenalism is relatively uncommon and is not accurately predicted by electrolyte abnormalities.

Adrenal Insufficiency↗

PubMed2002

The mononuclear phagocyte system revisited.

The mononuclear phagocyte system (MPS) was defined as a family of cells comprising bone marrow progenitors, blood monocytes, and tissue macrophages. In this review, we briefly consider markers for cells of this lineage in the mouse, especially the F4/80 surface antigen and the receptor for macrophage colony-stimulating factor. The concept of the MPS is challenged by evidence that there is a separate embryonic phagocyte lineage, the blurring of the boundaries between macrophages and other cells types arising from phenotypic plasticity and transdifferentiation, and evidence of local renewal of tissue macrophage populations as opposed to monocyte recruitment. Nevertheless, there is a unity to cells of the MPS suggested by their location, morphology, and shared markers. We discuss the origins of macrophage heterogeneity and argue that macrophages and antigen-representing dendritic cells are closely related and part of the MPS.

Animals↗