A feline large granular lymphoma and its derived cell line

Summary A lymphoma cell line (MCC) was derived from an abdominal mass from a 13-yr-old castrated male cat. The cells resemble natural killer precursor cells, have membrane-bound granules, and are positive for chloroacetate esterase, α-naphthyl butyrate esterase, and tartrate-resistant acid phosphatase activities. The MCC cells are negative for rearranged feline T-cell receptor genes, negative for feline T-cytotoxic antigen, Ia, and surface μ, τ, and lambda chains and do not form E-rosettes. The MCC cell line is negative for the feline leukemia virus (FeLV); e.g., negative for exogenous FeLV (exU3) sequences, negative for cytoplasmic and surface FeLV major core protein of 27 000 daltons (p27) by indirect, immunofluorescence assay, negative for helper FeLV by clone 81 assay, and negative for release of soluble FeLV p27 by enzyme-linked immunosorbent assay. Electron microscopy reveals budding type C retrovirus particles and MCC cells react with anti-RD-114 (anti-endogenous feline retrovirus) reference serum. After in vitro infection, MCC replicate FeLV readily, but replication is noncytopathic. This project has been funded, at least in part, with funds from the U.S. Department of Health and Human services under grants AI 25722, DK41939, and CA 35742 and contract AI-62525.     

Failure of an enriched nitrite-DPAO population to use nitrate as an electron acceptor

The metabolism of denitrifying polyphosphate accumulating organisms (DPAO) is not completely known. Recent reports suggest the existence of two types of DPAOs: those that can use nitrate and nitrite as electron acceptors (nitrate-DPAO) and those that can only use nitrite (nitrite-DPAO). Then, the survival of nitrite-DPAO in nitrate reducing environments is due to the existence of flanking denitrifying species, which reduce nitrate to nitrite. This works aims at a better understanding of the nitrite-DPAO population. For this aim, a nitrite-DPAO population was previously selected in a SBR using nitrite as electron acceptor. Then, nitrate utilisation by nitrite-DPAO was studied within a short-term period (4 days) and within a long-term period (50 days) with simultaneous nitrite and nitrate additions. The results obtained clearly indicate that nitrite-DPAO fail to use nitrate as electron acceptor even after 50 days of periodic dosing of nitrate and agree with the dual DPAO theory. Moreover, this failure casts doubts on the feasibility of nitrite based EBPR systems (i.e. partial nitrification + nitrite-DPAO) because these systems will not be able to denitrify an occasional nitrate inlet, which will remain in the effluent.     

Habitat use by radio-tagged Namib Desert golden moles (Eremitalpa granti namibensis)

The Namib Desert golden mole (Eremitalpa granti namibensis) is morphologically, physiologically and behaviourally specialized for living in the harsh loose‐sand desert. Ecological studies have relied on visually tracking animals on the surface of sand dunes. A radio tag would allow individuals to be more reliably located, even while under the sand. We developed a radio attachment and gathered preliminary data on winter habitat use by six individuals during 21 days. We compare data from previous studies and suggest that the greater diurnal activity, smaller home ranges, and more restricted movement patterns that we found are related to the unusual thermal and metabolic biology of Eremitalpa.     

Why do tubulin gene families lack diversity in flagellate/ciliate protists?

Summary Considerable amino acid sequence diversity is found among tubulin isotypes encoded by tubulin gene families in animal, higher plant, and fungal systems. In contrast, relatively little diversity is found among the isotypes produced by the gene families in a number of flagellate or ciliate protists. It is possible that proper assembly of the axoneme requires a homogeneous pool of tubulin subunits and that the axoneme thus provides a stringent selection against amino acid replacement substitutions among tubulin genes in these systems.     

H-ras-transformed NRK-52E renal epithelial cells have altered growth, morphology, and cytoskeletal structure that correlates with renal cell carcinoma in vivo

Summary We studied the effect of the ras oncogene on the growth kinetics, morphology, cytoskeletal structure, and tumorigenicity of the widely used NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transformed cell lines, H/1.2-NRK-52E (H/1.2) and H/6.1-NRK-52E (H/6.1). Population doubling times of NRK-52E, H/1.2, and H/6.1 cells were 28, 26, and 24 h, respectively, with the transformed cells reaching higher saturation densities than the parent cells. NRK-52E cells had typical epithelial morphology with growth in colonies. H/1.2 and H/6.1 cell colonies were more closely packed, highly condensed, and had increased plasma membrane ruffling compared to parent cell colonies. NRK-52E cells showed microfilament, microtubule, and intermediate filament networks typical of epithelial cells, while H/1.2 and H/6.1 cells showed altered cytoskeleton architecture, with decreased stress fibers and increased microtubule and intermediate filament staining at the microtubule organizing center. H/1.2 and H/6.1 cells proliferated in an in vitro soft agar transformation assay, indicating anchorage-independence, and rapidly formed tumors in vivo with characteristics of renal cell carcinoma, including mixed populations of sarcomatoid, granular, and clear cells, H/6.1 cells consistently showed more extensive alterations of growth kinetics, morphology, and cytoskeleton than H/1.2 cells, and formed tumors of a more aggressive phenotype. These data suggest that analysis of renal cell characteristics in vitro may have potential in predicting tumor behavior in vivo, and significantly contribute to the utility of these cell lines as in vitro models for examining renal epithelial cell biology and the role of the ras proto-oncogene in signal transduction involving the cytoskeleton.     

Anti-Trichomonas vaginalis activity of Hypericum polyanthemum extract obtained by supercritical fluid extraction and isolated compounds

The anti-Trichomonas vaginalis activity of Hypericum polyanthemum extract obtained by supercritical fluid extraction (50 °C, 150 bar) and the chemical compounds isolated and purified from this extract (benzopyrans HP1, HP2, HP3, and phloroglucinol derivative uliginosin B) were assessed. All samples had anti-T. vaginalis activity; however, HP1 demonstrated the best selectivity against this protozoan (metronidazole-resistant and susceptible isolates), with no cytotoxicity on mammalian cells (selectivity index of 73.97). Moreover, HP1 had activity against a metronidazole-resistant isolate (52% of viable trophozoites), and this effect was higher when tested with a low concentration of metronidazole (23% of viable trophozoites). Experiments demonstrated that all isolated compounds caused damage to the parasites' membrane (> 90% of LDH release) and do not present a notable hemolytic effect, although HP2 and uliginosin B exhibited cytotoxicity against mammalian cells. Therefore, the analyzed molecules are promising prototypes for new antiprotozoal drugs, especially HP1, which seems to improve metronidazole's effect on a resistant T. vaginalis isolate.     

The DRY Box and C-Terminal Domain of the Human Cytomegalovirus US27 Gene Product Play a Role in Promoting Cell Growth and Survival

Human cytomegalovirus (HCMV) is a widespread pathogen that can lay dormant in healthy individuals and establish lifelong latent infection. This successful co-existence is facilitated by a number of viral gene products that manipulate host cellular functions and immune responses. Among these immunomodulatory genes are four G-protein coupled receptors (GPCRs) encoded by HCMV, designated US27, US28, UL33, and UL78. Studies have shown the US28 gene product to be a functional chemokine receptor that signals both constitutively and in a ligand-dependent manner, resulting in a wide range of cellular effects. In previous work, we have found that US27 expression results in at least two biological effects: enhanced CXCR4 signaling and increased in cellular proliferation in HEK293 cells. Here, we examined the involvement of two protein domains, the DRY box and the C-terminal intracellular domain (CTD) of US27, in mediating both cell proliferation and survival. While both domains were required for a proliferative effect, loss of either domain only moderately impacted cell survival, suggesting that US27 may interact with cell survival pathways through protein regions other than the DRY box and CTD. Quantitative RT-PCR arrays were used to profile changes in cellular gene expression in the HEK293-US27 cell line, and down-regulation of cell cycle regulators CDKN1A/p21/CIP1 (cyclin dependent kinase inhibitor 1A) and SESN (Sestrin2 or Hi95) was observed. These results indicate that increased cell proliferation due to US27 may be linked to suppression of negative growth regulators, and that these interactions require the DRY box and CTD.     

Optimization of NMR analysis of biological fluids for quantitative accuracy

With the rising interest in the use of nuclear magnetic resonance (NMR) for the study of biological fluids such as urine and serum for metabonomic or diagnostic purposes, new challenges have arisen concerning the efficacy of NMR data acquisition and analysis. In particular the quantification of sample constituents such as metabolites is of great importance. This study compares five one-dimensional proton NMR pulse sequences using synthetic urine samples to determine appropriate acquisition parameters for reasonable sample throughput and accuracy. Each pulse sequence has its own advantages and limitations with respect to solvent suppression, stable baseline, exchangeable protons, and quantization of resonances near the residual water peak. Hardware issues such as low-pass filters, unique to each spectrometer, also impact quantitation accuracy. Metabolite concentrations were determined using integration referenced to an added internal standard, and using the Chenomx NMR Suite software package. Since nuclei in different metabolites and the internal standard all have different longitudinal relaxation rates (T ₁) we included a mathematical correction factor for quantitation.