Identification of a novel 7-cis-11-trans-lipoxin A4 generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of lipoxins A4 and B4

Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 microM) to human neutrophils led to the formation of both lipoxin A4 and lipoxin B4 as well as a novel 5,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A4 and lipoxin B4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA4 (7-cis-11-trans-LXA4). LXA4, 11-trans-LXA4, 7-cis-LXA4 and 7-cis-11-trans-LXA4 all evoked dose-dependent (0.1-10 microM) contractions of the guinea pig lung strip, whereas 6-cis-LXB4 and 6-cis-8-trans-LXB4 relaxed this preparation. LXA4 and 7-cis-LXA4 were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

A radioenzymatic assay for femtomole determination of catecholamines using α-methyldopamine as an internal standard

A radioenzymatic assay has been developed for the sensitive determination of plasma catecholamines in perchloric acid extracts using α-methyldopamine as an internal standard. With 25 μl of plasma extract in a total volume of 40 μl the assay gives blank values equivalent to approcximately 2 femtomoles (fmole) for epinephrine (E), norepinephrine (NE), 6 fmole for α-methyldopamine (MeDA) and approximately 15 femtomoles for dopamine (DA). Recoveries of 25 dpm/fmole NE, 40 dpm/fmole E, 56 dpm/fmole DA and 80 dpm/fmole MeDA have been obtained. The assay is linear to at least 1 picomole catecholamine (CA) and shows less than 0.5% crossover between E, NE and DA and a 4.7% crossover of αMeDA into DA. The interassay variability was ± 7% for DA, ± 4% for E and ±3% for NE.     

Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells

Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

Respiratory cytology in malignant lung disease – The thoracic oncologist’s perspective

Respiratory cytology continues to play a central role in the diagnosis and staging of thoracic malignancy, although over time indications have changed. Historically, sputum cytology and endobronchial brushings and washings figured prominently, but with the advent of endobronchial and endoscopic ultrasound much greater emphasis is placed on fine needle aspirates from lymph nodes. The advent of targeted sequencing panels for genomic profiling to identify driver mutations and PD-L1 directed immunotherapy means that there is a need to extract increasing amounts of diagnostic and predictive information from ever smaller amounts of diagnostic material. Recent work has demonstrated that cytology samples are well suited to delivering the information required, but in order to understand the limitations of clinical and laboratory techniques, a close working relationship between pathologist and thoracic oncologist is needed to optimise sample procurement and utilisation.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.