Subcellular localization of calcium in sporangiophores of Phycomyces blackesleeanus

The in situ localization of Ca²⁺ in stage I sporangiophores of the fungus Phycomyces blakesleeanus was achieved with the potassium pyroantimonate technique. Precipitates of calcium-antimonate were present in mitochondria, vacuoles, endoplasmic reticulum and adjacent cytoplasm, Golgi-like bodies, and nuclei but not cell walls. Material treated with the calcium chelator EGTA lacked these precipitates. The preferential localization of Ca²⁺ in mitochondria, endoplasmic reticulum and vacuoles suggests that these organelles modulate the level of this cation in sporangiophores of P. blakesleeanus.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N, tetraacetic acid     

Probability of mast cells to mediate anaphylaxis in skeletal muscle

Are there enough mast cells in denervated skeletal muscle to account for autopharmacological mediation of the antigen potentials (APs) elicited by microtaps? Through rough qualitative estimations, some authors have suggested a positive answer to this question. However, in view of measurements performed in this investigation of both the density of mast cells and the diffusion coefficient of antigens, the probability of such mediated effects was found to be relatively low:P=0.016 for egg albumin andP=0.004 for ferritin. Therefore, most APs induced by microtaps should be attributed to the direct effect of antigen over the sensitized muscle fibers. Yet, both the density of mast cells found in this work and the known amount of histamine they are capable of releasing when challenged with antigen, support the hypothesis regarding the involvement of these cells when antigen is massively superfused so as to induce Schultz-Dale reactions in muscle strips. Under this circumstance, the direct and mediated mechanisms may coexist.     

A specific inhibitor of lactate dehydrogenase overcame the resistance toward gemcitabine in hypoxic mesothelioma cells,and modulated the expression of the human equilibrative transporter-1

ABSTRACT

Malignant pleural mesothelioma (MPM) is a very hypoxic malignancy, and hypoxia has been associated with resistance towards gemcitabine. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis. Therefore we investigated the combination of a new LDH-A inhibitor (NHI-1) with gemcitabine in MPM cell lines. Under hypoxia (O₂ tension of 1%) the cell growth inhibitory effects of gemcitabine, were reduced, as demonstrated by a 5- to 10-fold increase in IC₅₀s. However, the simultaneous addition of NHI-1 was synergistic (combination index < 1). Flow cytometry demonstrated that hypoxia caused a G1 arrest, whereas the combination of NHI-1 significantly increased gemcitabine-induced cell death. Finally, the mRNA expression levels of the human equilibrative transporter-1 (hENT1) were significantly down-regulated under hypoxia, but treatment with NHI-1 was associated with a recovery of hENT1 expression. In conclusion, our data show that hypoxia increased MPM resistance to gemcitabine. However, cell death induction and modulation of the key transporter in gemcitabine uptake may contribute to the synergistic interaction of gemcitabine with the LDH-A inhibitor NHI-1 and support further studies for the rational development of this combination.     

Gene Expression Studies in Formalin-Fixed Paraffin-Embedded Samples of Cutaneous Cancer: The Need for Reference Genes

Formalin-fixed paraffin-embedded (FFPE) tumour samples may provide crucial data regarding biomarkers for neoplasm progression. Analysis of gene expression is frequently used for this purpose. Therefore, mRNA expression needs to be normalized through comparison to reference genes. In this study, we establish which of the usually reported reference genes is the most reliable one in cutaneous malignant melanoma (MM) and cutaneous squamous cell carcinoma (CSCC). ACTB, TFRC, HPRT1 and TBP expression was quantified in 123 FFPE samples (74 MM and 49 CSCC biopsies) using qPCR. Expression stability was analysed by NormFinder and Bestkeeper softwares, and the direct comparison method between means and SD. The in-silico analysis with BestKeeper indicated that HPRT1 was more stable than ACTB and TFRC in MM (1.85 vs. 2.15) and CSCC tissues (2.09 vs. 2.33). The best option to NormFinder was ACTB gene (0.56) in MM and TFRC (0.26) in CSCC. The direct comparison method showed lower SD means of ACTB expression in MM (1.17) and TFRC expression in CSCC samples (1.00). When analysing the combination of two reference genes for improving stability, NormFinder indicated HPRT1 and ACTB to be the best for MM samples, and HPRT1 and TFRC genes for CSCC. In conclusion, HPRT1 and ACTB genes in combination are the most appropriate choice for normalization in gene expression studies in MM FFPE tissue, while the combination of HPRT1 and TFRC genes are the best option in analysing CSCC FFPE samples. These may be used consistently in forthcoming studies on gene expression in both tumours.     

The interplay between increased tooth crown‐height and chewing efficiency,and implications for Cervidae evolution

Mammals of numerous lineages have evolved high‐crowned (hypsodont) teeth particularly during the last 20 million years. This major phenotypic change is one of the most widely studied evolutionary phenomena in a broad range of disciplines, though the mechanisms underlying its transformation remain unresolved. Here, we present the first Finite Element Analysis (FEA) to investigate the alternative hypothesis that there is a biomechanical link between increased hypsodonty and a more effective mastication in deer. Our FE experiments compared patterns of stress and strain within and between different fossil and living species under different loading conditions, and found that more hypsodont teeth are suited for restricting stresses to those areas where chewing loading occurs. This mechanical improvement is consequence of specific and pronounced variations in tooth geometry and morphology of the occlusal surface that are strongly related to crown growth in the vertical plane. We demonstrate that hypsodonty enables selenodont‐teeth to adopt a mechanically improved design that increases the pressure whilst shearing foods. As ruminants are physiologically limited by both the quantity of food consumed and the time spent in the mastication and digestion, hypsodonty is highly advantageous when feeding on mechanically resistant, tough and fibrous foods. Consequently, it allows grass‐eaters to spend less time chewing, thereby increasing the volume of food ingested and/or providing more time for digestion. This study provides a promising line of evidences in support of biomechanical effectiveness, in addition to or instead of increased wear resistance, as a factor in explaining the evolutionary origins of the hypsodont phenotype.     

Extensive Phylogenetic Analysis of a Soil Bacterial Community Illustrates Extreme Taxon Evenness and the Effects of Amplicon Length,Degree of Coverage,and DNA Fractionation on Classification and Ecological Parameters

To thoroughly investigate the bacterial community diversity present in a single composite sample from an agricultural soil and to examine potential biases resulting from data acquisition and analytical approaches, we examined the effects of percent G+C DNA fractionation, sequence length, and degree of coverage of bacterial diversity on several commonly used ecological parameters (species estimation, diversity indices, and evenness). We also examined variation in phylogenetic placement based on multiple commonly used approaches (ARB alignments and multiple RDP tools). The results demonstrate that this soil bacterial community is highly diverse, with 1,714 operational taxonomic units demonstrated and 3,555 estimated (based on the Chao1 richness estimation) at 97% sequence similarity using the 16S rRNA gene. The results also demonstrate a fundamental lack of dominance (i.e., a high degree of evenness), with 82% of phylotypes being encountered three times or less. The data also indicate that generally accepted cutoff values for phylum-level taxonomic classification might not be as applicable or as general as previously assumed and that such values likely vary between prokaryotic phyla or groups.Efforts to describe bacterial species richness and diversity have long been hampered by the inability to cultivate the vast majority of bacteria from natural environments. New methods to study bacterial diversity have been developed in the last two decades (32), many of which rely on PCR-based procedures and phylogenetic comparison of 16S rRNA gene sequences. However, PCR using complex mixtures of templates (as in the case of total microbial community DNA) is presumed to preferentially amplify certain templates in the mixture (23) based on their primary sequence, percent G+C (hereafter GC) content, or other factors, resulting in so-called PCR bias. Moreover, the amplification of template sequences depends on their initial concentration and tends to skew detection toward the most abundant members of the community (23). To further complicate matters, subsequent random cloning steps on amplicon mixtures are destined to result in the detection of numerically dominant sequences, especially where relative abundance can vary over orders of magnitude. Indeed, any analysis based on random encounter is destined to primarily detect numerically dominant populations. This is especially of concern where limited sampling is performed on highly complex microbial communities exhibiting mostly even distribution of populations with only a few showing any degree of dominance, as typically perceived for soils (17). These artifacts and sampling limitations represent major hurdles in bacterial community diversity analysis, since the vast majority of bacterial diversity probably lies in “underrepresented minority” populations (24, 30). This is important because taxa that are present only in low abundance may still perform important ecosystem functions (e.g., ammonia-oxidizing bacteria). Of special concern is that biases in detection might invalidate hypothesis testing on complex communities where limited sampling is performed (5).Recently, there has been a concerted effort toward addressing problems impeding comprehensive bacterial diversity studies (7, 13, 24, 26, 28). In recent years, studies have increased sequencing efforts, with targeted 16S rRNA gene sequence libraries approaching 2,000 clones (11) and high-throughput DNA-sequencing efforts (e.g., via 454 pyrosequencing and newer-generation high-throughput approaches) of up to 149,000 templates from one or a few samples (25, 30). These technological advances have come as researchers recognize that massive sequencing efforts are required to accurately assess the diversity of populations that comprise complex microbial communities (29, 30). Alternatively, where fully aligned sequence comparisons need to be made, novel experimental strategies that allow more-comprehensive detection of underrepresented bacterial taxa can be applied. One such approach involves the application of prefractionation of total bacterial community genomic DNA based on its GC content (hereafter GC fractionation) prior to subsequent molecular manipulations of total community DNA (14). This strategy has been successfully applied in combination with denaturing gradient gel electrophoresis (13) and 16S rRNA gene cloning (2, 21) to study microbial communities. This approach separates community genomic DNA, prior to any PCR, into fractions of similar percent GC content, effectively reducing the overall complexity of the total community DNA mixture by physical separation into multiple fractions. This facilitates PCR amplification, cloning, and detection of sequences in fractions with relatively low abundance in the community, thereby enhancing the detection of minority populations (13). Collectively, this strategy reduces the biases introduced by PCR amplification and random cloning of the extremely complex mixtures of templates of different GC content, primary sequence, and relative abundance present in total environmental genomic DNA.Any large molecular survey that relies on sequencing further requires the analysis of large amounts of data that must be catalogued into phylogenetically relevant groups. This is usually done using high-throughput methods like RDP Classifier or Sequence Match (6) or a tree-based method like Greengenes (8) or ARB (18). Two major pitfalls that are encountered using these former approaches are the presence of huge numbers of unclassified sequences in databases and the lack of representative sequences from all phyla. This leads to most surveys having large portions of their phylotypes designated as unclassified. The latter tree-based approaches, although better suited for classification schemes, are also dependent on having a comprehensive database with well-classified sequences for reproducible results. This reproducibility becomes especially important when trying to compare data across different studies, especially those that utilize different approaches and study systems.In the current study, we analyzed an extensive (∼5,000 clones) partial 16S rRNA gene library from a single soil sample that was generated using very general primers and GC-fractionated DNA. Total DNA was extracted from soil at a cultivated treatment plot at the National Science Foundation Long Term Ecological Research (NSF-LTER) site at the Kellogg Biological Station (KBS) in mid-Michigan (http://www.kbs.msu.edu/lter). To test the effect of GC fractionation on recovery of 16S rRNA gene sequences, we conducted a direct comparison with a nonfractionated library generated from the same soil sample. Using the GC-fractionated library, we also calculated several measures of bacterial diversity and examined the effects of sampling size and sequence length on Shannon-Weaver diversity index, Simpson''s reciprocal index (1/D, where D is the probability that two randomly selected individuals from a sample belong to the same species), evenness, and Chao1 richness estimation. The results show that GC fractionation is a powerful tool to help mitigate limitations of random PCR- and cloning-based analyses of total microbial community diversity, resulting in the recovery of underrepresented taxa and, in turn, reducing the sampling size needed for accurate estimations of bacterial richness. The results also provided evidence for the need to expand the typical scale of sequence-based survey efforts, particularly in environments where evenness abounds or where minority bacterial populations may have important effects on community function and processes. We suggest that there is a need for the establishment of standardized approaches for the analysis of sequence data from community diversity studies in order to maximize data comparisons across independent studies and show examples of software programs developed to facilitate comparative analysis of large sequence datasets.     

High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B,Type I,PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI''s C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.     

Diversity and convergence in the mechanisms establishing L/R asymmetry in metazoa

Differentiating left and right hand sides during embryogenesis represents a major event in body patterning. Left–Right (L/R) asymmetry in bilateria is essential for handed positioning, morphogenesis and ultimately the function of organs (including the brain), with defective L/R asymmetry leading to severe pathologies in human. How and when symmetry is initially broken during embryogenesis remains debated and is a major focus in the field. Work done over the past 20 years, in both vertebrate and invertebrate models, has revealed a number of distinct pathways and mechanisms important for establishing L/R asymmetry and for spreading it to tissues and organs. In this review, we summarize our current knowledge and discuss the diversity of L/R patterning from cells to organs during evolution.     

Comparative Study of 17-AAG and NVP-AUY922 in Pancreatic and Colorectal Cancer Cells: Are There Common Determinants of Sensitivity?

The use of heat shock protein 90 (Hsp90) inhibitors is an attractive antineoplastic therapy. We wanted to compare the effects of the benzoquinone 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) and the novel isoxazole resorcinol–based Hsp90 inhibitor NVP-AUY922 in a panel of pancreatic and colorectal carcinoma cell lines and in colorectal primary cultures derived from tumors excised to patients. PANC-1, CFPAC-1, and Caco-2 cells were intrinsically resistant to 17-AAG but sensitive to NVP-AUY922. Other cellular models were sensitive to both inhibitors. Human epidermal growth factor receptor receptors and their downstream signaling pathways were downregulated in susceptible cellular models, and concurrently, Hsp70 was induced. Intrinsic resistance to 17-AAG did not correlate with expression of ATP-binding cassette transporters involved in multidrug resistance. Some 17-AAG-resistant, NVP-AUY922–sensitive cell lines lacked NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme and activity. However, colorectal LoVo cells still responded to both drugs in spite of having undetectable levels and activity of NQO1. Pharmacological and biologic inhibition of NQO1 did not confer resistance to 17-AAG in sensitive cell lines. Therefore, even though 17-AAG sensitivity is related to NQO1 protein levels and enzymatic activity, the absence of NQO1 does not necessarily convey resistance to 17-AAG in these cellular models. Moreover, NVP-AUY922 does not require NQO1 for its action and is a more potent inhibitor than 17-AAG in these cells. More importantly, we show in this report that NVP-AUY922 potentiates the inhibitory effects of chemotherapeutic agents, such as gemcitabine or oxaliplatin, and other drugs that are currently being evaluated in clinical trials as antitumor agents.