Retroviral transduction of alveolar bone cells with a temperature-sensitive SV40 large T antigen

We have transduced adult human alveolar bone (AB) cells with a gene construct encoding a temperature-sensitive mutation of the SV40 large T antigen (tsT). Such cells divided rapidly, for more than 50 passages thus far, at a permissive low temperature (34.5 degrees C), comparable to the non-transduced parental cells at 37 degrees C. However, the tsT-transduced AB cells failed to grow at a non-permissive high temperature (39 degrees C) at which the T antigen is inactivated. Nevertheless, the cells formed mineralised nodules in vitro at both the low and high temperatures. Flow cytometry analysis showed that the transduced cells cultured at 34.5 degrees C, like the parental cells at 37 degrees C, were smaller and less granular than the transduced cells incubated at 39 degrees C. Moreover, the transduced cells grown at 34.5 degrees C were also found to express bone sialoprotein, osteopontin and type I collagen at levels similar to those of the parental cells at 37 degrees C, although osteonectin and fibronectin were down-regulated. When the transduced cells were incubated at 39 degrees C, the expression of all antigens was up-regulated, particularly osteonectin. Thus, we have obtained long-term cultures of tsT-transduced AB cells whose growth is temperature-dependent and which express certain features characteristic of bone-derived cells.     

Potential availability of cadaver organs for transplantation.

OBJECTIVE--To determine the potential number of cadaver kidney donors by applying defined donor criteria to people dying in hospital. DESIGN--Prospective study of all deaths occurring in 21 hospitals from 1 September 1988 to 31 August 1989. Questionnaires were administered to medical and nursing staff and families of potential donors aged 1-69. SETTING--Acute care hospitals in Gwent, South Glamorgan, Mid Glamorgan, West Glamorgan, Pembrokeshire, and East Dyfed health authorities, serving a population of 2.2 million. MAIN OUTCOME MEASURES--Cause of death, age, ventilation at time of death, diagnosis of brain death, and consideration of consent. RESULTS--Adequate data were available for 9840 of 10,095 hospital deaths (97.5% coverage). 188 patients aged 0-69 were identified as potential organ donors (widest definition), and of these 108 died without being ventilated at the time of death. Tests of brain stem death were formally completed in 57 cases, and organ donation was considered by the families of 47 of these potential donors. 26 patients became organ donors. Patients aged 50-69 with stroke were less likely to be ventilated than those aged less than or equal to 49 (21/96 v 24/34). Families of potential donors aged 20-39 were least likely to give permission. CONCLUSIONS--The supply of donor organs (specifically kidneys) could be increased by altering the management of patients aged 50-69 dying of severe cerebrovascular disease in general medical wards, in particular by increasing the proportion ventilated. The ethics of elective ventilation for the purposes of organ donation require discussion.     

Are we aware how contaminated our mobile phones with nosocomial pathogens?

Vibrio spp. is a pathogen rarely isolated in cancer patients, and in most cases it is associated with haematological diseases. Cutaneous manifestations of this organism are even rarer. We report a case of Non-O1 Vibrio cholerae inguinal skin and soft tissue infection presenting bullous skin lesions in a young type II diabetic patient with a penis squamous cell carcinoma having a seawater exposure history.     

GITR ligand provided by thrombopoietic cells inhibits NK cell antitumor activity

Thrombocytopenia inhibits tumor growth and especially metastasis in mice, whereas additional depletion of NK cells reverts this antimetastatic phenotype. It has therefore been speculated that platelets may protect hematogenously disseminating tumor cells from NK-dependent antitumor immunity. Tumor cells do not travel through the blood alone, but are rapidly coated by platelets, and this phenomenon has been proposed to shield disseminating tumor cells from NK-mediated lysis. However, the underlying mechanisms remain largely unclear. In this study, we show that megakaryocytes acquire expression of the TNF family member glucocorticoid-induced TNF-related ligand (GITRL) during differentiation, resulting in GITRL expression by platelets. Upon platelet activation, GITRL is upregulated on the platelet surface in parallel with the α-granular activation marker P-selectin. GITRL is also rapidly mobilized to the platelet surface following interaction with tumor cells, which results in platelet coating. Whereas GITRL, in the fashion of several other TNF family members, is capable of transducing reverse signals, no influence on platelet activation and function was observed upon GITRL triggering. However, platelet coating of tumor cells inhibited NK cell cytotoxicity and IFN-γ production that could partially be restored by blocking GITR on NK cells, thus indicating that platelet-derived GITRL mediates NK-inhibitory forward signaling via GITR. These data identify conferment of GITRL pseudoexpression to tumor cells by platelets as a mechanism by which platelets may alter tumor cell immunogenicity. Our data thus provide further evidence for the involvement of platelets in facilitating evasion of tumor cells from NK cell immune surveillance.     

Genetic relationships among Pistacia species using AFLP markers

This study addresses the phylogenetic relationship between Pistacia species by amplified fragment length polymorphism (AFLP). The plant materials of this study consisted of a total of 44 accessions belonging to P. vera, P. eurycarpa, P. khinjuk, all subspecies of P. atlantica (atlantica, mutica, kurdica and cabulica), three unknown genotypes and three accessions, proposed to be hybrid from P. eurycarpa × P. atlantica. The accessions were from Iran, Turkey, USA and Syria. Six AFLP primer combinations produced a total of 475 fragments, with average of 79.16 fragments per primer pair, of which, 336 bands were polymorphic. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on jaccard’s similarity coefficient matrix and also average similarity of each species. According to the results, two main clusters were developed and P. vera, P. eurycarpa, P. atlantica (subsp. atlantica, kurdica, mutica, cabulica) and the hybrid genotypes located in the first main cluster. P. khinjuk accessions from Iran and USA localized in second main cluster. The hybrid accessions located between eurycarpa and atlantica species and their hybrid nature between these two species were confirmed. One of the unknown accessions clustered with the hybrid ones and the two other were grouped closely with P. Khinjuk. According to this study, the closest species to P. vera was Eurycarpa group, followed by P. atlantica. UPGMA analysis separated P. atlantica subsp. mutica and cabulica from P. atlantica and P. eurycarpa. Subspecies mutica and cabulica were two closest genotypes; hence, P. atlantica subsp. mutica could be classified as a distinct species as P. mutica and the cabulica as a subspecies of P. mutica. This study revealed that P. eurycarpa is synonym for P. atlantica subsp. kurdica and should be considered distinct from P. atlantica; however, P. atlantica showed a closer genetic similarity to P. eurycarpa than the other species.     

Evaluation of methicillin resistance by cefoxitin disk diffusion and PBP2a latex agglutination test in mecA-positive Staphylococcus aureus, and comparison of mecA with femA, femB, femX positivities

Methicillin resistance in staphylococci is primarily due to the presence of a mecA gene which encodes the novel penicillin binding protein2a. Some chromosomal factors such as femA and femB also participate in the expression of methicillin resistance. This study was designed to detect methicillin resistance by cefoxitin disk diffusion and penicillin binding protein2a latex agglutination methods, and to compare mecA, femA, femB and femX gene positivities. A total of 60 MRSA isolates were included in the evaluation. PCR analysis showed that all isolates were positive for mecA and femA genes. Seven of these isolates tested negative by the latex agglutination test. Fifteen isolates were positive for femB and 28 isolates for femX gene. This study implicated that for the determination of methicillin resistance, latex agglutination test is the least reliable method when compared to PCR and cefoxitin disk diffusion test. femA gene shows more correlation than femB and femX with methicillin resistance.     

Allelopathic effect of <Emphasis Type="Italic">Nepeta meyeri</Emphasis> Benth. extracts on seed germination and seedling growth of some crop plants

In order to evaluate the allelopathic potential of Nepeta meyeri Benth., the effects of aqueous extracts (0.125, 0.25, 0.5, 1, 2.5 and 5%) prepared from roots and leaves of N. meyeri were studied on the seed germination and seedling growth of several economically important crops (barley, wheat, canola, safflower, and sunflower). Both the root and leaf extracts of N. meyeri caused a general phytotoxic effect on the seed germination and seedling growth of barley and sunflower at all concentrations. However, both the root and leaf extracts significantly increased the seedling growth of wheat, especially at the lower concentrations 0.125, 0.25 and 0.5%, whereas the higher concentrations had a neutral effect. The seed germination and the seedling growth of canola and safflower were also generally improved by both extracts, especially at lower concentrations. However, these advantages were not observed at higher concentrations, at which the extracts mostly had a phytotoxic effect on canola and safflower. The Allelopathic activity of N. meyeri depended on whether the extract was derived from the leaf or root parts of the plant. The maximum allelopathic effect occurred with leaf extracts. The results demonstrate that the aqueous extracts from N. meyeri have allelopathic potential and should be evaluated as an allelopathic species, presenting a risk or advantage to seed germination and seedling growth of crop or weed plants.     

Cloning and Expression of the Vitreoscilla Hemoglobin Gene in Enterobacter Aerogenes: Effect on Cell Growth and Oxygen Uptake

The hemoglobins found in unicellular organisms show a great deal of chemical reactivity, protecting cells against oxidative stress, and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than their VHb counterparts. Here, the expression of VHb, its effect on the growth and antioxidant enzyme status of cells under different culture conditions was studied by cloning the complete regulatory and coding sequences (vgb) for VHb in Enterobacter aerogenes. Contrary to what has been reported for Escherichia coli, the expression of vgb in E.aerogenes decreased several fold under 10% of atmospheric oxygen (2% oxygen) and its growth was not greatly improved by the presence of VHb. Measured either as viable cells or total cell mass, untransformed E. aerogenes grew better than the recombinant strains. At the late exponential phase, however, the vgb-bearing strain was determined to have a higher cell number and total cell mass than the strain bearing only the plasmid vector with no vgb insert. The VHb expressing strain also had an oxygen uptake rate several fold higher than its counterparts. Given that oxidative stress may occur upon elevated oxygen exposure and be balanced by the action of antioxi-dative compounds, the level of antioxidative response of E. aerogenes expressing VHb was also studied. The VHb expressing strain had substantially (1.5–2.6-fold) higher catalase activity than strains not expressing VHb. Both VHb+ and VHb- strains, however, showed similar levels of superoxide dismutase activity. The activity of both enzymes was also growth phase dependent. Stationary phase cells of all strains showed 2–5-fold higher activity for these enzymes than cells at the exponential phase.     

In situ non-invasive spectral discrimination between bone cell phenotypes used in tissue engineering

Raman micro-spectroscopy was used to discriminate between different types of bone cells commonly used in tissue engineering of bone, with the aim of developing a method of phenotypic identification and classification. Three types of bone cells were analysed: human primary osteoblasts (HOB), retroviral transfected human alveolar bone cells with SV40 large T antigen (SV40 AB), and osteoblast-like human osteosarcoma derived MG63 cell line. Unsupervised principal component analysis (PCA) and linear discriminant analysis (LDA) of the Raman spectra succeeded in discriminating the osteosarcoma derived MG63 cells from the non-tumour cells (HOB and SV40 AB). No significant differences were observed between the Raman spectra of the HOB and SV40 AB cells, confirming the biochemical similarities between the two cell types. Difference spectra between tumour and non-tumour cells suggested that the spectral discrimination is based on the fact that MG63 osteosarcoma derived cells are characterised by lower concentrations of nucleic acids and higher relative concentrations of proteins compared to the non-tumour bone cells. A supervised classification model (LDA) was built and showed high cross-validation sensitivity (100%) and specificity (95%) for discriminating the MG63 cells and the non-tumour cells, with 96% of the cells being correctly classified either as tumour or non-tumour derived cells. This study proves the feasibility of using Raman spectroscopy to identify in situ phenotypic differences in living cells.     

Site-specific mutations in the D1 polypeptide affect the susceptibility of Synechocystis 6803 cells to photoinhibition

Photoinhibition of photosystem II in the cyanobacterium Synechocystis 6803 was followed after site-specific mutagenesis of the D1 polypeptide. Mutations were created in the stromal/cytosolic loop connecting helices D and E. Two mutations E243K and CA1, a deletion of the three glutamates 242–244 and a substitution Q241H, were made in the putative cleavage area of the D1 polypeptide. A third mutation E229D was made in the PEST-like sequence. Mutants and control cells were illuminated and F_V/F_M was recorded. Compared to the control, the mutants were less photoinhibited. Fluorescence relaxation after a single flash was delayed in CA1. Restoration of F_V/F_M after photoinhibition in the mutants was totally dependent on protein synthesis while control cells were able to recover partially also when protein synthesis was inhibited. In addition, the protein synthesis-dependent recovery of CA1 was slowed down. Our results indicate a correlation between the mutated amino acids and photoinhibition of photosystem II.