Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.     

Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.     

In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.     

The radioenhancement of two human head and neck squamous cell carcinomas by 2'-2' difluorodeoxycytidine (gemcitabine; dFdC) is mediated by an increase in radiation-induced residual chromosome aberrations but not residual DNA DSBs

PURPOSE: The present study aimed at investigating if 2'-2' difluorodeoxycytidine (dFdC) radioenhancement was mediated by an effect on induction and/or repair of radiation-induced DNA DSBs and chromosome aberrations in cells with different intrinsic radiosensitivity. METHODS: Confluent human head and neck squamous cell carcinoma cell lines designated SCC61 and SQD9 were treated with 5 microM dFdC for 3 or 24 h prior to irradiation. DNA DSBs induction and repair were analyzed by PFGE. Radiation-induced chromosome aberrations were examined with a FISH technique. RESULTS: In both cell lines, dFdC did not modify radiation-induced DNA DSBs in a dose range between 0 and 40 Gy. After a single dose of 40 Gy, dFdC affected neither the kinetic of repair nor the residual amount of DNA DSBs up to 4 h after irradiation. Whereas dFdC did not increase the induction of chromosome aberrations, after a single dose of 5 Gy, the percentage of aberrant cells and the number of aberrations per aberrant cells were significantly higher in combination with dFdC. CONCLUSION: Our data suggest that under experimental conditions yielding substantial radioenhancement, dFdC decreases the repair of genomic lesions inducing secondary chromosome breaks but has no effect on DNA DSBs repair as measured by PFGE.     

Extended-term cultures of human T-lymphocytes and the comet assay: a useful combination when testing for genotoxicity in vitro?

Extended-term cultures of human lymphocytes provide a source of uniform human cells that can be used for several experiments performed over a long time, avoiding the variability arising from taking blood samples for individual experiments. The use of extended-term cultures of human T-lymphocytes in the alkaline single-cell gel electrophoresis assay (comet assay) was evaluated as a test for the potential genotoxicity of chemicals. The DNA-damaging effects of five DNA-reactive mutagens and clastogens (benzo[a]pyrene, cyclophosphamide, formaldehyde, 4-nitroquinoline-N-oxide (4NQO) and N-nitrosopiperidine) was determined and compared with the effects of one non-DNA-reactive mutagen (5-hydroxyurea), and one non-mutagenic agent (ethanol). The alkylating and/or DNA-adduct forming agents N-nitrosopiperidine, cyclophosphamide, 4-nitroquinoline-N-oxide and benzo[a]pyrene increased the DNA migration in a dose-dependent manner. In contrast, the DNA/protein-crosslinking agent formaldehyde decreased the migration of DNA during the electrophoresis. The lowest observed effect levels (LOELs) under the experimental conditions used in the present study, were: 0.0001 mM (4-nitroquinoline-N-oxide without S9), 0.05 mM (benzo[a]pyrene with S9), 0.1mM (formaldehyde without S9), 0.25 mM (cyclophosphamide with S9), and 0.5mM (N-nitrosopiperidine with S9), respectively. The antimetabolite 5-hydroxyurea was also found to increase the tail moment, but only in cells that had been exposed to rather high concentrations (> or =10mM) of the compound. Ethanol did not affect the tail moment, not even in cells that had been exposed to an apparently cytotoxic concentration (500 mM). The results of the present study are in qualitative agreement with those obtained using other cells in the alkaline comet assay and it is therefore concluded that extended-term cultures of human T-lymphocytes and the alkaline version of the single-cell gel electrophoresis assay is a useful combination when testing for the potential genotoxicity of chemicals.     

Phylogeny of the lichen genus Placopsis and its allies based on Bayesian analyses of nuclear and mitochondrial sequences

The phylogenetic relationships of the lichen genus Placopsis and related genera in the Agyriales were analyzed using molecular data. We obtained a total of 66 new sequences from the nuclear ITS, LSU and the mitochondrial SSU rDNA. Phylogenetic analyses were conducted in a Bayesian and a maximum-parsimony framework. Our analyses show that Placopsis is paraphyletic with members of Orceolina nesting within the genus. A morphological character supporting the Placopsis-Orceolina clade is the non-amyloid ascus. The section Aspiciliopsis as defined by sunken fruiting bodies is not supported, but the type species of Aspiciliopsis is more closely related to Orceolina. This clade shares apothecia with reduced amphithecia as apomorphic character. We suggest resurrecting the generic name Aspiciliopsis. Trapelia is the sister genus to Placopsis and Aspiciliopsis/Orceolina.     

Rhizopogon spore bank communities within and among California pine forests

In this study we examine the distribution of Rhizopogon species in spore banks from five California pine forests. Four of the forest sites were discontinuous populations of Pinus muricata and a fifth was a Pinus ponderosa stand in Sierra National Forest. Rhizopogon species were retrieved by bioassaying the soils with pine seedlings followed by isolation of axenic cultures from individual root tips with typical Rhizopogon ectomycorrhizal morphology. The cultures were screened by ITS-RFLP and all unique patterns were sequenced. These sequences then were compared with those derived from identified sporocarp material. Bioassaying proved to be an efficient way to bring Rhizopogon species into culture. Approximately 50% of the pots contained ectomycorrhizal tips with Rhizopogon-like morphology, and axenic Rhizopogon cultures were obtained from half these pots. Our results showed that Rhizopogon spores usually are well distributed within local forest areas, while there is significant structuring of species at the regional scale. Spore longevity and homogenization by soil and water movement might explain their distribution within local forest areas, while the regional pattern might be explained by limited long distance dispersal or climatic and edaphic differences.     

PCR-RFLP is used to investigate relations among species in the entomopathogenic genera Eryniopsis and Entomophaga

The shape and nucleation of primary conidia are important characters in the classification of the Entomophthoraceae (Zygomycetes). The five species in the genus Eryniopsis vary in the shapes of primary conidia, although within most genera in the order Entomophthorales species have the same shapes of primary conidia. Using PCR-RFLP, we investigated two species in Eryniopsis, Ery. caroliniana with oblong-ovoid primary conidia and Ery. ptychopterae with pear-shaped primary conidia, with five species of Entomophaga, all having pear-shaped conidia. Molecular results merged with morphological data indicate that Ery. ptychopterae belongs in the genus Entomophaga while Ery. caroliniana clearly differs from Entomophaga. Ery. ptychopterae and Ery. transitans are transferred to the genus Entomophaga. Our results support the idea that morphology of primary conidia is of major importance in defining entomophthoralean genera. These results also show that such studies can be conducted with species that have not been isolated, if fungal-filled cadavers can be obtained.     

Mechanisms behind positive diversity effects on ecosystem functioning: testing the facilitation and interference hypotheses

Little is known about the mechanisms behind positive effects of species richness on ecosystem functioning. In a previous study that showed a positive effect of aquatic detritivore species richness on leaf litter breakdown (process) rates, we proposed that facilitation and release from intra-specific interference were the two most likely mechanisms. To test the interference hypothesis, we performed an experiment using three densities of each of three detritivore species and found varying effects on leaf breakdown rates across species: one species showed no effect, one a positive, marginally insignificant, effect, and a third species showed a significant, positive effect of decreasing density. The density (interference) effect thus partly explained the results from our previous study. The facilitation hypothesis was tested by sequentially introducing and removing two species. We predicted that, if this hypothesis were true, facilitation would be expressed in higher process rates than when replacing with individuals of the same species. We found that process rate per unit biomass did increase when one species was introduced after the other species, while the opposite sequence did not show any increase. Hence, this result was also confirmative of our previous results. Therefore, we conclude that both intra-specific interference and inter-specific facilitation may explain the positive effect of species richness observed in our system. Since many species exhibit intra-specific interference that inhibits foraging efficiency, this may be a general mechanism generating effects of species richness per se. If facilitation is unidirectional, or if it involves few species, it is more likely to be species specific with species identities being more important than species richness per se. We conclude that species loss may be expected to have negative consequences on ecosystem functioning if anyspecies is lost, with additional effects in the event of losing "facilitator" species.     

Role of maxilla 2 and its setae during feeding in the shrimp Palaemon adspersus (Crustacea: Decapoda)

The movements of the basis of maxilla 2 in Palaemon adspersus were examined using macro-video recordings, and the morphology of its setae was examined using both scanning and transmission electron microscopy. The basis of maxilla 2 performs stereotypical movements in the latero-medial plane and gently touches the food with a frequency of 3-5 Hz. The medial rim of the basis of maxilla 2 carries three types of seta. Type 1 is serrate, type 2 and 3 are serrulate, and type 2 has a prominent terminal pore. Type 2 is innervated by 18-25 sensory cells whose cilia protrude through the terminal pore and are in direct contact with the external environment. The structure of type 2 setae indicates that they are mainly gustatory, although still bimodal due to their innervation by presumed chemosensory and mechanosensory neurons. Distally, the three types of setae have a complex arrangement of the cuticle involving water-filled canals, which may serve to improve flexibility. Type 1 and 3 setae have fewer sensory cells (4-9) but probably also have a bimodal sensory function. The function of type 1 setae is probably to protect type 2 setae, while type 3 setae might serve to groom the ventral side of the basis of maxilla 1.