Improving the long‐term storage of a mammalian biosensor cell line via genetic engineering

The unique properties of mammalian cells make them valuable for a variety of applications in medicine, industry, and diagnostics. However, the utility of such cells is restricted due to the difficulty in storing them non‐frozen for an extended time and still maintaining their stability and responsiveness. In order to extend the active life span of a mammalian biosensor cell line at room and refrigerated temperatures, we have over expressed genes that are reported to provide protection from apoptosis, stress, or oxidation. We demonstrated that over expression of genes from the extremophile, Artemia franciscana, as well as GADD45β, extends room‐temperature storage of fully active cells 3.5‐fold, while over production of several anti‐apoptotic proteins extended 4°C storage 2‐ to 3‐fold. Methodologies like these that improve the stability of mammalian‐cell‐based technologies in the absence of freezers may enable widespread use of these tools in applications that have been considered impractical based solely on limited storage characteristics. Biotechnol. Bioeng. 2010; 106: 474–481. © 2010 Wiley Periodicals, Inc.     

Cytotaxonomy of Eurypyga helias (Gruiformes,Eurypygidae): First Karyotypic Description and Phylogenetic Proximity with Rynochetidae

The sunbittern (Eurypyga helias) is a South American Gruiformes, the only member of Family Eurypigidae. In most phylogenetic proposals, it is placed in a more distant position than other families of the so-called “core Gruiformes”. Different studies based on molecular, morphological and biogeographical data suggest that the Eurypigidae is closely related to the kagu (Rhynochetos jubatus), the only species in Rynochetidae, another family not included in the core Gruiformes. Here, the karyotype of the sunbittern is described for the first time, by classical and molecular cytogenetics, using whole chromosome probes derived from Gallus gallus and Leucopternis albicollis. We found a diploid number of 80, with only one pair of biarmed autosomal macrochromosomes, similar to that observed in the kagu. Chromosome painting revealed that most syntenies found in the avian putative ancestral karyotype (PAK) were conserved in the sunbittern. However, PAK1, PAK2, and PAK5 corresponded to two chromosome pairs each. Probes derived from L. albicollis confirm that fissions in PAK1 and PAK2 were centric, whereas in PAK5 the fission is interstitial. In addition, there is fusion of segments homologous to PAK2q and PAK5. From a phylogenetic point of view, comparisons of our results with two other Gruiformes belonging to family Rallidae suggest that the PAK5q fission might be a synapomorphy for Gruiformes. Fissions in PAK1 and PAK2 are found only in Eurypigidae, and might also occur in Rynochetidae, in view of the similar chromosomal morphology between the sunbittern and the kagu. This suggests a close phylogenetic relationship between Eurypigidae and Rynochetidae, whose common ancestor was separated by the Gondwana vicariancy in South America and New Caledonia, respectively.     

Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1

We describe the first lipase structure from a thermophilic organism. It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold. The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability. Zinc binding is mediated by two histidine and two aspartic acid residues. These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana. The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.     

Phase II study of vaccinia melanoma cell lysates (VMCL) as adjuvant to surgical treatment of stage II melanoma

Summary Patients with stage II melanoma were vaccinated with vaccinia virus-induced melanoma cell lysates (VMCL). The vaccine contained viable vaccinia virus, membranous fragments and no intact nuclei. A number of antigens defined by monoclonal antibodies were detected in the vaccine including the ganglioside GD3 and DR antigens. Administration of the vaccine was associated with depression of natural killer cell activity against melanoma and K562 target cells in the first 3–6 months of treatment. Leucocyte dependent antibody (LDA) activity against melanoma cells was induced or increased in titre in approximately half of the patients studied. Continued vaccination was associated in a number of patients with a decrease in LDA titres. Studies on a small sample of patients revealed that this was associated with the development of serum factors which inhibited LDA activity. LDA activity appeared directed to non-MHC antigens on melanoma cells which were of at least two specificities. One specificity which was shared with antigens on a number of non-melanoma carcinoma cells was removed by absorption on fetal brain and may be similar to oncofetal antigens described by other workers. Reactivity against melanocytes was induced in some patients and may underlie the development of vitiligo in several patients. These results suggest that vaccines prepared from VMCL may be a favourable method for increasing immune responses against melanoma.     

Host response to malaria during pregnancy: placental monocyte recruitment is associated with elevated beta chemokine expression

Malaria during pregnancy is associated with poor birth outcomes, particularly low birth weight. Recently, monocyte infiltration into the placental intervillous space has been identified as a key risk factor for low birth weight. However, the malaria-induced chemokines involved in recruiting and activating placental monocytes have not been identified. In this study, we determined which chemokines are elevated during placental malaria infection and the association between chemokine expression and placental monocyte infiltration. Placental malaria infection was associated with elevations in mRNA expression of three beta chemokines, macrophage-inflammatory protein 1 (MIP-1) alpha (CCL3), monocyte chemoattractant protein 1 (MCP-1; CCL2), and I-309 (CCL1), and one alpha chemokine, IL-8 (CXCL8); all correlated with monocyte density in the placental intervillous space. Placental plasma concentrations of MIP-1 alpha and IL-8 were increased in women with placental malaria and were associated with placental monocyte infiltration. By immunohistochemistry, we localized placental chemokine production in malaria-infected placentas: some but not all hemozoin-laden maternal macrophages produced MIP-1 beta and MCP-1, and fetal stromal cells produced MCP-1. In sum, local placental production of chemokines is increased in malaria, and may be an important trigger for monocyte accumulation in the placenta.     

A disposable corer for sampling organic soils

A disposable and inexpensive corer is described that offers a versatile method for sampling soft soils with a large organic content. The cans are suitable for the immediate collection of soil and for field incubations. Fine roots are sliced easily during insertion of corers by the sharp bottom edge, and the thin walls and the high thermal conductivity of steel aid transfer of heat energy between the bulk soil and the sample. If desired the cylinders may be perforated, have the upper cap removed and also can be used as an indicator of anaerobic soil conditions. This revised version was published online in June 2006 with corrections to the Cover Date.