Effect of magnesium on the growth and cell cycle of transformed and non-transformed epithelial rat liver cells in vitro

The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epithelial-like rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 × the concentration in the regular RPMI 1640 media (100mg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G₁ and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G₁ with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G₁ nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics.     

Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

Reciprocal homologous junctions generated in mouse cells

Summary We analysed pairs of reciprocal homologous junctions resulting from intermolecular conservative homologous recombination in mouse cells. The assay used did not rely on the reconstitution of a selectable gene. This permitted the introduction of multiple markers in the parental homologous sequences which in turn enabled us to compare the contribution of each parent to the reciprocal products of a given recombination event. In all recombinants analysed we found, when comparing the reciprocal junctions, a middle segment originating from only one parent. This segment of uniparental origin occurred randomly throughout the region of homology and could extend over a thousand base pairs. These results are consistent with a gap repair process like the one proposed for homologous recombination in yeast. However, introducing a double-strand break in the region of homology did not enhance but rather decreased the proportion of recombinants with reciprocal homologous junctions relative to other types of recombinants.     

The titres of lectins in the haemolymph of Lacanobia oleracea and the effects of parasitism by the ectoparasitoid wasp Eulophus pennicornis

Serum from larvae of Lacanobia oleracea L. (Lepidoptera; Noctuidae) parasitized by Eulophus pennicornis (Hymenoptera; Eulophidae) and from normal non‐parasitized larvae is capable of agglutinating rabbit, sheep, calf, goat, chicken, horse and human erythrocytes, but not yeast. Studies with a range of inhibitory carbohydrates showed that serum lectins(s) had specificity for sugars containing galactose and for rhamnose, and for the glycosubstances fetuin and asialofetuin. Lectin activity is heat‐labile and is not dependent on calcium. Parasitism by E. pennicornis caused an increase in the agglutination titre of the serum from larvae of L. oleracea but not an increase in specific activity (titre per mg protein per ml). However, when venom from the venom gland of female wasps was injected into L. oleracea larvae, both the agglutinating activity and the specific activity of the larval serum increased. The possible causes of this increase are discussed. It is suggested that venom contains antigenic components which, when injected into the haemocoel of the L. oleracea larva, may be increasing lectin synthesis and/or release into the serum.     

Breeding biology, resource partitioning and reproductive effort of a dioecious shrub, Clutia pulchella L. (Euphorbiaceae)

 The flowering of Clutia pulchella was studied in a coastal scarp forest community in southern KwaZulu-Natal, South Africa. Sex dependent differences in the frequency and magnitude of floral output were recorded, with male individuals producing 4.32 times more flowers per leaf axis than females. Increased node production, as a manifestation of significantly increased branching, allowed for prolific flower production in males. Energy investment per flower was marginally greater in females (0.045 J/mg), despite copious pollen production and glandular secretion in males (0.035 J/mg). Differential flower production, reproductive organ dry mass and an unbalanced branching ratio revealed that “per plant” expenditure towards flowering was more pronounced in males (♀ flowering × 5.04). The elevated cost of fruit production (♀ flowering × 5.5), inclusive of imperfect fruit set, resulted in overall reproductive expense being slightly higher for females. Such sex-dependent reproductive investment was shown to have minimal influence on the population sex ratio, with sex frequencies and nearest neighbor distances yielding a non-significant male bias of 1.16. No evidence of spatial asymmetry was found. Received December 1, 1999 Accepted September 27, 2000     

Nanoparticle-Delivered Multimeric Soluble CD40L DNA Combined with Toll-Like Receptor Agonists as a Treatment for Melanoma

Stimulation of CD40 or Toll-Like Receptors (TLR) has potential for tumor immunotherapy. Combinations of CD40 and TLR stimulation can be synergistic, resulting in even stronger dendritic cell (DC) and CD8+ T cell responses. To evaluate such combinations, established B16F10 melanoma tumors were injected every other day X 5 with plasmid DNA encoding a multimeric, soluble form of CD40L (pSP-D-CD40L) either alone or combined with an agonist for TLR1/2 (Pam₃CSK₄ ), TLR2/6 (FSL-1 and MALP2), TLR3 (polyinosinic-polycytidylic acid, poly(I:C)), TLR4 ( monophosphoryl lipid A, MPL), TLR7 (imiquimod), or TLR9 (Class B CpG phosphorothioate oligodeoxynucleotide, CpG). When used by itself, pSP-D-CD40L slowed tumor growth and prolonged survival, but did not lead to cure. Of the TLR agonists, CpG and poly(I:C) also slowed tumor growth, and the combination of these two TLR agonists was more effective than either agent alone. The triple combination of intratumoral pSP-D-CD40L + CpG + poly(I:C) markedly slowed tumor growth and prolonged survival. This treatment was associated with a reduction in intratumoral CD11c+ dendritic cells and an influx of CD8+ T cells. Since intratumoral injection of plasmid DNA does not lead to efficient transgene expression, pSP-D-CD40L was also tested with cationic polymers that form DNA-containing nanoparticles which lead to enhanced intratumoral gene expression. Intratumoral injections of pSP-D-CD40L-containing nanoparticles formed from polyethylenimine (PEI) or C32 (a novel biodegradable poly(B-amino esters) polymer) in combination with CpG + poly(I:C) had dramatic antitumor effects and frequently cured mice of B16F10 tumors. These data confirm and extend previous reports that CD40 and TLR agonists are synergistic and demonstrate that this combination of immunostimulants can significantly suppress tumor growth in mice. In addition, the enhanced effectiveness of nanoparticle formulations of DNA encoding immunostimulatory molecules such as multimeric, soluble CD40L supports the further study of this technology for tumor immunotherapy.     

Promoting Smoke-Free Homes: A Novel Behavioral Intervention Using Real-Time Audio-Visual Feedback on Airborne Particle Levels

Interventions are needed to protect the health of children who live with smokers. We pilot-tested a real-time intervention for promoting behavior change in homes that reduces second hand tobacco smoke (SHS) levels. The intervention uses a monitor and feedback system to provide immediate auditory and visual signals triggered at defined thresholds of fine particle concentration. Dynamic graphs of real-time particle levels are also shown on a computer screen. We experimentally evaluated the system, field-tested it in homes with smokers, and conducted focus groups to obtain general opinions. Laboratory tests of the monitor demonstrated SHS sensitivity, stability, precision equivalent to at least 1 µg/m³, and low noise. A linear relationship (R² = 0.98) was observed between the monitor and average SHS mass concentrations up to 150 µg/m³. Focus groups and interviews with intervention participants showed in-home use to be acceptable and feasible. The intervention was evaluated in 3 homes with combined baseline and intervention periods lasting 9 to 15 full days. Two families modified their behavior by opening windows or doors, smoking outdoors, or smoking less. We observed evidence of lower SHS levels in these homes. The remaining household voiced reluctance to changing their smoking activity and did not exhibit lower SHS levels in main smoking areas or clear behavior change; however, family members expressed receptivity to smoking outdoors. This study established the feasibility of the real-time intervention, laying the groundwork for controlled trials with larger sample sizes. Visual and auditory cues may prompt family members to take immediate action to reduce SHS levels. Dynamic graphs of SHS levels may help families make decisions about specific mitigation approaches.     

Detection of coccidioidal antibodies in serum of a small rodent community in Baja California,Mexico

Coccidioidomycosis (Valley Fever) represents a serious threat to inhabitants of endemic areas of North America. Despite successful clinical isolations of the fungal etiological agent, Coccidioides spp., the screening of environmental samples has had low effectiveness, mainly because of the poor characterization of Coccidioides ecological niche. We explored Valle de las Palmas, Baja California, Mexico, a highly endemic area near the U.S.–Mexico border, where we previously detected Coccidioides via culture-independent molecular methods. By testing the serum from 40-trapped rodents with ELISA, we detected antibodies against Coccidioides in two species: Peromyscus maniculatus and Neotoma lepida. This study comprises the first report of wild rodent serum tested for coccidioidal antibodies, and sets the basis to analyze this pathogen in its natural environment and explore its potential ecological niche.     

Chemokine Receptor Utilization by Human Immunodeficiency Virus Type 1 Isolates That Replicate in Microglia

The role of human immunodeficiency virus (HIV) strain variability remains a key unanswered question in HIV dementia, a condition affecting around 20% of infected individuals. Several groups have shown that viruses within the central nervous system (CNS) of infected patients constitute an independently evolving subset of HIV strains. A potential explanation for the replication and sequestration of viruses within the CNS is the preferential use of certain chemokine receptors present in microglia. To determine the role of specific chemokine coreceptors in infection of adult microglial cells, we obtained a small panel of HIV type 1 brain isolates, as well as other HIV strains that replicate well in cultured microglial cells. These viruses and molecular clones of their envelopes were used in infections, in cell-to-cell fusion assays, and in the construction of pseudotypes. The results demonstrate the predominant use of CCR5, at least among the major coreceptors, with minor use of CCR3 and CXCR4 by some of the isolates or their envelope clones.