Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture

Recombinant retroviruses are effective vectors able to integrate transgenes into the target cell's genome to achieve longer‐term expression. This study investigates the effect of cell lysis products, a common cell culture by‐product, on the transduction of suspension cells by gammaretroviral vectors. Cell lysates derived from human and murine suspension cell lines significantly increased the transduction of human TF‐1 and K‐562 cell lines by gibbon ape leukemia virus‐pseudotyped retroviral vectors without altering tropism. The transduction efficiency of TF‐1 cells increased as a function of lysate concentration and decreased with increasing target cell concentrations. This was adequately predicted using a saturation equation based on the lysed‐to‐target cell concentration ratio, R, where: Lysate completely masked the effects of fibronectin when the two were added in combination. With protamine sulfate, the transduction efficiency was increased by lysate to 58% from 20% for protamine sulfate alone. Overall, the presence of cell lysate significantly influenced the outcome of the transduction process, either alone or in the presence of protamine sulfate or fibronectin. Biotechnol. Bioeng. 2010;105: 1168–1177. © 2009 Wiley Periodicals, Inc.     

Conservation and management implications of fine-scale genetic structure of Gulf sturgeon in the Pascagoula River, Mississippi

The anadromous Gulf sturgeon occurs along the north central coast of the Gulf of Mexico and is federally listed as threatened. We analyzed fine‐scale patterns of Gulf sturgeon population structure, focusing on the Pascagoula River drainage of Mississippi, in reference to movement patterns as determined via telemetry and capture data. We genotyped 361 Gulf sturgeon using eight microsatellite loci including samples from the Pascagoula, Pearl, Escambia, Yellow, Choctawhatchee, and Apalachicola river drainages. Pairwise F_ST estimates indicated that genetic structure occurs at least at the drainage level. The Pascagoula and Pearl rivers form a western group, demonstrating 100% bootstrap support for a division with drainages to the east. Assignment tests detected non‐natal genotypes occurring in all drainages. According to assignment tests, the Pascagoula supports an admixture of individuals, containing minimal influence from drainages to the east (2%) and substantial interaction with the Pearl River (14.1%). The occurrence of Pascagoula River fish in the Pearl was non‐reciprocal, observed at 1.1%. After accounting for non‐natal genetic diversity within the Pascagoula, there remained a disparity between a pooled Pascagoula group and the only documented spawning site within the drainage located in the Bouie River. We interpret this as an indication of a second genetic stock within the Pascagoula River drainage. Radio telemetry data suggest that spawning likely occurs in the Chickasawhay River, in areas isolated from the Bouie River spawning site by about 350 river kilometers. We emphasize the utility of integrating field and molecular approaches when delineating fine‐scale patterns of population structure in anadromous fishes.     

Two novel Venturia inaequalis genes induced upon morphogenetic differentiation during infection and in vitro growth on cellophane

Venturia inaequalis is a hemibiotrophic ascomycete that causes apple scab. Germ tubes, from conidia or ascospores, penetrate the leaf or fruit surface directly via appressoria-like swellings; subsequently the hyphae divide laterally to form a stroma between the cuticle and the outer wall of the epidermal cells. This morphological switch can be mimicked by growing the fungus in vitro on cellophane discs. The aim of this work was to identify genes upregulated in planta using growth on cellophane as a model. Four cDNA clones were found to be induced by growth on cellophane, and qRT-PCR showed two of these genes were up-regulated over a thousand fold in infected apple leaves compared to liquid culture. The predicted proteins for both genes possess putative signal peptides for secretion but have no similarity to sequences in publicly available databases. Both genes encode proteins with novel, imperfect repeat domain structures, the number of which vary in an isolate-specific fashion. Cin1 has seven or eight repeats of about 60 amino acids with four conserved cysteine residues per repeat, while Cin3 has four or five repeats of 32 amino acids with no cysteines. Both proteins appear to have evolved through internal duplication. Cin3, in particular, shows considerable between-strain variation in domain structure, indicating a high degree of recombination at this locus and revealing that the repeat structure has most likely arisen by unequal crossing-over. Results of this study support the hypothesis that cellophane-grown V. inaequalis mimics aspects of biotrophic infection and provide the first insights into novel fungal genes expressed during apple scab infection and their mechanisms of evolution.     

The 'Ethereal' nature of TLR4 agonism and antagonism in the AGP class of lipid A mimetics

To overcome the chemical and metabolic instability of the secondary fatty acyl residues in the AGP class of lipid A mimetics, the secondary ether lipid analogs of the potent TLR4 agonist CRX-527 (2) and TLR4 antagonist CRX-526 (3) were synthesized and evaluated along with their ester counterparts for agonist/antagonist activity in both in vitro and in vivo models. Like CRX-527, the secondary ether lipid 4 showed potent agonist activity in both murine and human models. Ether lipid 5, on the other hand, showed potent TLR4 antagonist activity similar to CRX-526 in human cell assays, but did not display any antagonist activity in murine models and, in fact, was weakly agonistic. Glycolipids 2, 4, and 5 were synthesized via a new highly convergent method utilizing a common advanced intermediate strategy. A new method for preparing (R)-3-alkyloxytetradecanoic acids, a key component of ether lipids 4 and 5, is also described.     

Binding free energy calculation for duocarmycin/DNA complex based on the QPLD-derived partial charge model

The 3ns unrestrained MD simulations were carried out on the DNA/duocarmycin complex based on (1) the classic RESP charge model, and (2) the QM-polarized ligand docking (QPLD)-based charge model. The RMSDs of the trajectories and the DeltaG(bind) of the QPLD model perform much better than the RESP model, with the DeltaG(bind) estimation for QPLD model (-16.11 kcal/mol) versus DeltaG(bind) estimation for RESP model (-10.05 kcal/mol).     

Immunohistochemical localization of collagen type XI alpha1 and alpha2 chains in human colon tissue.

In previous studies, collagen XI mRNA has been detected in colon cancer, but its location in human colon tissue has not been determined. The heterotrimeric collagen XI consists of three alpha chains. While it is known that collagen XI plays a regulatory role in collagen fibril formation, its function in the colon is unknown. The characterization of normal human colon tissue will allow a better understanding of the variance of collagen XI in abnormal tissues. Grossly normal and malignant human colon tissue was obtained from pathology archives. Immunohistochemical staining with a 58K Golgi marker and alpha1(XI) and alpha2(XI) antisera was used to specifically locate their presence in normal colon tissue. A comparative bright field microscopic analysis showed the presence of collagen XI in human colon. The juxtanuclear, dot-like collagen XI staining in the Golgi apparatus of goblet cells in normal tissue paralleled the staining of the 58K Golgi marker. Ultra light microscopy verified these results. Staining was also confirmed in malignant colon tissue. This study is the first to show that collagen XI is present in the Golgi apparatus of normal human colon goblet cells and localizes collagen XI in both normal and malignant tissue. Although the function of collagen XI in the colon is unknown, our immunohistochemical characterization provides the foundation for future immunohistopathology studies of the colon.     

Colocalization of cellular nanostructure using confocal fluorescence and partial wave spectroscopy

A new multimodal confocal microscope has been developed, which includes a parallel Partial Wave Spectroscopic (PWS) microscopy path. This combination of modalities allows molecular‐specific sensing of nanoscale intracellular structure using fluorescent labels. Combining molecular specificity and sensitivity to nanoscale structure allows localization of nanostructural intracellular changes, which is critical for understanding the mechanisms of diseases such as cancer. To demonstrate the capabilities of this multimodal instrument, we imaged HeLa cells treated with valinomycin, a potassium ionophore that uncouples oxidative phosphorylation. Colocalization of fluorescence images of the nuclei (Hoechst 33342) and mitochondria (anti‐mitochondria conjugated to Alexa Fluor 488) with PWS measurements allowed us to detect a significant decrease in nuclear nanoscale heterogeneity (Σ), while no significant change in Σ was observed at mitochondrial sites. In addition, application of the new multimodal imaging approach was demonstrated on human buccal samples prepared using a cancer screening protocol. These images demonstrate that nanoscale intracellular structure can be studied in healthy and diseased cells at molecular‐specific sites.

     

DNA methyltransferase homologue TRDMT1 in Plasmodium falciparum specifically methylates endogenous aspartic acid tRNA

In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite.     

Effects of Exercise on Metabolic Risk Variables in Overweight Postmenopausal Women: A Randomized Clinical Trial

Objective: This study examined the effects of exercise on metabolic risk variables insulin, leptin, glucose, and triglycerides in overweight/obese postmenopausal women. Research Methods and Procedures: Sedentary women (n = 173) who were overweight or obese (BMI ≥ 25 kg/m² or ≥24 kg/m² with ≥33% body fat), 50 to 75 years of age, were randomized to 12 months of exercise (≥45 minutes of moderate‐intensity aerobic activity 5 d/wk) or to a stretching control group. Body composition (DXA) and visceral adiposity (computed tomography) were measured at baseline and 12 months. Insulin, glucose, triglycerides, and leptin were measured at baseline and 3 and 12 months. Insulin resistance was evaluated by the homeostasis model assessment formula. Differences from baseline to follow‐up were calculated and compared across groups. Results: Exercisers had a 4% decrease and controls had a 12% increase in insulin concentrations from baseline to 12 months (p = 0.0002). Over the same 12‐month period, leptin concentrations decreased by 7% among exercisers compared with remaining constant among controls (p = 0.03). Homeostasis model assessment scores decreased by 2% among exercisers and increased 14% among controls from baseline to 12 months (p = 0.0005). The exercise effect on insulin was modified by changes in total fat mass (trend, p = 0.03), such that the exercise intervention abolished increases in insulin concentrations associated with gains in total fat mass. Discussion: Regular moderate‐intensity exercise can be used to improve metabolic risk variables such as insulin and leptin in overweight/obese postmenopausal women. These results are promising for health care providers providing advice to postmenopausal women for lifestyle changes to reduce risk of insulin resistance, coronary heart disease, and diabetes.     

Multiple regulators of Ty1 transposition in Saccharomyces cerevisiae have conserved roles in genome maintenance.

Most Ty1 retrotransposons in the genome of Saccharomyces cerevisiae are transpositionally competent but rarely transpose. We screened yeast mutagenized by insertion of the mTn3-lacZ/LEU2 transposon for mutations that result in elevated Ty1 cDNA-mediated mobility, which occurs by cDNA integration or recombination. Here, we describe the characterization of mTn3 insertions in 21 RTT (regulation of Ty1 transposition) genes that result in 5- to 111-fold increases in Ty1 mobility. These 21 RTT genes are EST2, RRM3, NUT2, RAD57, RRD2, RAD50, SGS1, TEL1, SAE2, MED1, MRE11, SCH9, KAP122, and 8 previously uncharacterized genes. Disruption of RTT genes did not significantly increase Ty1 RNA levels but did enhance Ty1 cDNA levels, suggesting that most RTT gene products act at a step after mRNA accumulation but before cDNA integration. The rtt mutations had widely varying effects on integration of Ty1 at preferred target sites. Mutations in RTT101 and NUT2 dramatically stimulated Ty1 integration upstream of tRNA genes. In contrast, a mutation in RRM3 increased Ty1 mobility >100-fold without increasing integration upstream of tRNA genes. The regulation of Ty1 transposition by components of fundamental pathways required for genome maintenance suggests that Ty1 and yeast have coevolved to link transpositional dormancy to the integrity of the genome.