Actin amino-acid sequences. Comparison of actins from calf thymus, bovine brain, and SV40-transformed mouse 3T3 cells with rabbit skeletal muscle actin.

Actin was purified from calf thymus, bovine brain and SV40-transformed mouse 3T3 cells grown in tissue culture. Isoelectric focusing analysis showed the presence of the two actin polypeptides beta and gamma typical for non-muscle actins in all three actins. Tryptic and thermolytic peptides accounting for the complete amino-acid sequence of the cytoplasmic actins were separated and isolated by preparative fingerprint techniques. All peptides were characterized by amino-acid analysis and compared with the corresponding peptides from rabbit skeletal muscle actin. Peptides which differed in amino-acid composition from the corresponding skeletal muscle actin peptides were subjected to sequence analysis in order to localize the amino-acid replacement. The results obtained show that all three mammalian cytoplasmic actins studied contain the same amino-acid exchanges indicating that mammalian cytoplasmic actins are very similar if not identical in amino-acid sequence. The presence of two different isoelectric species beta and gamma in cytoplasmic actins from higher vertebrates is acccounted for by the isolation of two very similar but not identical amino-terminal peptides in all three actin preparations. The nature of the amino-acid replacements in these two peptides not only accounts for the different isoelectric forms but also shows that beta and gamma cytoplasmic actins are the products of two different structural genes expressed in the same cell. The total number of amino-acid replacements so far detected in the comparison of these cytoplasmic actins and skeletal muscle actin is 25 for the beta chain and 24 for the gamma chain. With the exception of the amino-terminal three or four residues, which are responsible for the isoelectric differences, the replacements do not involve charged amino acids. The exchanges are not randomly distributed. No replacements were detected in regions 18--75 and 299--356 while the regions between residues 2--17 and 259--298 show a high number of replacements. In addition documentation for a few minor revisions of the amino acid sequence of rabbit skeletal muscle actin is provided.     

Both CD34+38+ and CD34+38- cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion

Human hemopoietic stem cells (HSC) have been shown to engraft, differentiate, and proliferate in the hemopoietic tissues of sublethally irradiated NOD/LtSZ scid/scid (NOD/SCID) mice. We used this model to study homing, survival, and expansion of human HSC populations from different sources or phenotype. We observed that CD34+ cells homed specifically to bone marrow (BM) and spleen, but by 3 days after injection, survived only in the BM. These BM-homed CD34+ cells proliferated intensively and gave rise to a 12-fold, 5.5-fold, and 4-fold expansion in 3 days for umbilical cord blood, adult mobilized peripheral blood, and adult BM-derived cells, respectively. By injection of purified subpopulations, it was demonstrated that both CD34+38+ and CD34+38- umbilical cord blood HSC homed to the BM and expanded. Importantly, kinetics of expansion were different: CD34+38+ cells started to increase in cell number from day 3 onwards, and by 4 wk after injection, virtually all CD34+ cells had disappeared. In contrast, CD34+38- cells remained quiescent during the first week and started to expand intensively from the third week on. In this paper, we have shown that homing, survival, and expansion of stem cells are three independent phenomena important in the early phase of BM engraftment and that kinetics of engraftment differ between CD34+38+ and CD34+38- cells.     

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.     

GMF-Knockout Mice are Unable to Induce Brain-Derived Neurotrophic Factor after Exercise

We earlier reported that overexpression of glia maturation factor (GMF) in cultured astrocytes enhances the production of brain-derived neurotrophic factor (BDNF). The current study was conducted to find out whether BDNF production is impaired in animals devoid of GMF. To this end GMF-knockout (KO) mice were subjected to exercise and the neurotrophin mRNAs were determined by real-time RT-PCR. Compared to wild-type (WT) mice, there is a decrease in exercise-induced BDNF in the KO mice. The observation was correlated with the finding that, in WT mice, exercise increases GMF expression. The results are consistent with the hypothesis that GMF is necessary for exercise-induction of BDNF, and that GMF may promote neuroprotection through BDNF production.     

Host plant quality,selection history and trade-offs shape the immune responses of Manduca sexta

Immune defences are an important component of fitness. Yet susceptibility to pathogens is common, suggesting the presence of ecological and evolutionary limitations on immune defences. Here, we use structural equation modelling to quantify the direct effects of resource quality and selection history, and their indirect effects mediated via body condition prior to an immune challenge on encapsulation and melanization immune defences in the tobacco hornworm, Manduca sexta. We also investigate allocation trade-offs among immune defences and growth rate following an immune challenge. We found considerable variation in the magnitude and direction of the direct effects of resource quality and selection history on immune defences and their indirect effects mediated via body condition and allocation trade-offs. Greater resource quality and evolutionary exposure to pathogens had positive direct effects on encapsulation and melanization. The indirect effect of resource quality on encapsulation mediated via body condition was substantial, whereas indirect effects on melanization were negligible. Individuals in better condition prior to the immune challenge had greater encapsulation; however, following the immune challenge, greater encapsulation traded off with slower growth rate. Our study demonstrates the importance of experimentally and analytically disentangling the relative contributions of direct and indirect effects to understand variation in immune defences.     

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.     

Depth gradients in food‐web processes linking habitats in large lakes: Lake Superior as an exemplar ecosystem

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