High resolution magic angle spinning (MAS) 1 H nuclear magnetic resonance (NMR) spectra of whole pieces of human prostatic tissue ( < 40 mg per sample) from patients with benign prostatic hyperplasia (BPH) and malignant tumours have been obtained and compared with high resolution NMR solution state spectra obtained via a standard tissue extraction method. Well resolved MAS-NMR spectra of the tissue samples were obtained in which both cytosolic and membrane-related compounds could be identified. Two-dimensional 1 H MAS-NMR measurements, including J-resolved and shift correlation spectra, were also obtained to allow metabolite signal assignment. In all cases high quality spectra were obtained that were rich in bioanalytical information on a wide range of intracellular metabolites and lipids. Marked differences were observed between MAS spectra from tumours versus BPH tissue, particularly in the lipid signals, which were much more intense in tumours. This was in contrast to the protein-free extracts of the same samples which showed much more similar NMR profiles for the two disease classes. These data indicate that MAS-NMR spectra of intact tissue biopsy samples give a wider range of bioanalytical information on small and large molecules than conventional, more labour intensive cell extraction procedures, and this strengthens the case for the use of MAS-NMR in clinical diagnostics.
BackgroundMalaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite’s complex biology.MethodsThe expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears.ResultsThe Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes.ConclusionsThe results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation.
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