Determination of cellular strains by combined atomic force microscopy and finite element modeling

Many organs adapt to their mechanical environment as a result of physiological change or disease. Cells are both the detectors and effectors of this process. Though many studies have been performed in vitro to investigate the mechanisms of detection and adaptation to mechanical strains, the cellular strains remain unknown and results from different stimulation techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by atomic force microscopy with finite element modeling and computational fluid dynamics, we report the cellular strain distributions exerted by common whole-cell straining techniques and from micromanipulation techniques, hence enabling their comparison. Using data from our own analyses and experiments performed by others, we examine the threshold of activation for different signal transduction processes and the strain components that they may detect. We show that modulating cell elasticity, by increasing the F-actin content of the cytoskeleton, or cellular Poisson ratio are good strategies to resist fluid shear or hydrostatic pressure. We report that stray fluid flow in some substrate-stretch systems elicits significant cellular strains. In conclusion, this technique shows promise in furthering our understanding of the interplay among mechanical forces, strain detection, gene expression, and cellular adaptation in physiology and disease.     

Carbon Isotope Fractionation during Aerobic Biodegradation of Trichloroethene by Burkholderia cepacia G4: a Tool To Map Degradation Mechanisms

The strain Burkholderia cepacia G4 aerobically mineralized trichloroethene (TCE) to CO₂ over a time period of ~20 h. Three biodegradation experiments were conducted with different bacterial optical densities at 540 nm (OD₅₄₀s) in order to test whether isotope fractionation was consistent. The resulting TCE degradation was 93, 83.8, and 57.2% (i.e., 7.0, 16.2, and 42.8% TCE remaining) at OD₅₄₀s of 2.0, 1.1, and 0.6, respectively. ODs also correlated linearly with zero-order degradation rates (1.99, 1.11, and 0.64 μmol h⁻¹). While initial nonequilibrium mass losses of TCE produced only minor carbon isotope shifts (expressed in per mille δ¹³C_VPDB), they were 57.2, 39.6, and 17.0‰ between the initial and final TCE levels for the three experiments, in decreasing order of their OD₅₄₀s. Despite these strong isotope shifts, we found a largely uniform isotope fractionation. The latter is expressed with a Rayleigh enrichment factor, , and was −18.2 when all experiments were grouped to a common point of 42.8% TCE remaining. Although, decreases of to −20.7 were observed near complete degradation, our enrichment factors were significantly more negative than those reported for anaerobic dehalogenation of TCE. This indicates typical isotope fractionation for specific enzymatic mechanisms that can help to differentiate between degradation pathways.     

Transcriptional programs activated by exposure of human prostate cancer cells to androgen

Background  

Androgens are required for both normal prostate development and prostate carcinogenesis. We used DNA microarrays, representing approximately 18,000 genes, to examine the temporal program of gene expression following treatment of the human prostate cancer cell line LNCaP with a synthetic androgen.     

Motors and switches: AAA+ machines within the replisome

Clamp loaders are required to load the ring-shaped clamps that tether replicative DNA polymerases onto DNA. Recently solved crystal structures, along with a series of biochemical studies, have provided a detailed understanding of the clamp loading reaction. In particular, studies of the Escherichia coli clamp loader--an AAA+ machine--have provided insights into the architecture of clamp loaders from eukaryotes, bacteriophage T4 and archaea. Other AAA+ proteins are also involved in the initiation of DNA replication, and studies of the E. coli clamp loader indicate mechanisms by which these proteins might function.     

Thylakoid‐bound and stromal antioxidative enzymes in wheat treated with excess copper

Copper is a catalyst in the formation of reactive free radicals and its toxicity may be due, at least in part, to oxidative damage. The response of thylakoid‐bound and stromal antioxidative enzymes against the generation of superoxide radical was investigated in seedlings of wheat ( Triticum durum L. cv. Adamello) grown in hydroponic culture for 10 days and subjected to 10 and 50 µ M copper treatments. Electron spin resonance of roots evidenced a spectrum of copper, the intensity of which increased with the treatment, whereas the carbon‐centered free radical spectrum detected in the control leaves was not seen anymore in the treated samples. As well as thylakoids, photosystem II (PSII) particles were able to produce the superoxide radical. Increased superoxide production both by thylakoids and PSII was observed in the sample treated with 50 µ M Cu. Induction of thylakoid‐bound and stromal antioxidative enzymes, with the exception of dehydroascorbate reductase, was also detected in leaves treated with the highest copper concentration. No Mn‐superoxide dismutase (SOD, EC 1.15.1.1) was detected in thylakoids of wheat. Both stromal and thylakoid‐bound SOD were CuZn‐SOD with 16.2‐kDa subunits. Both western blotting and immuno‐electron microscopy showed that the SOD subunit was recognized by a polyclonal antibody against glyoxisomal CuZn‐SOD from watermelon cotyledon. In the stroma of wheat, ascorbate peroxidase showed at least three well‐resolved bands differently induced by copper treatments.     

Mitotic Centromere–associated Kinesin Is Important for Anaphase Chromosome Segregation

Mitotic centromere–associated kinesin (MCAK) is recruited to the centromere at prophase and remains centromere associated until after telophase. MCAK is a homodimer that is encoded by a single gene and has no associated subunits. A motorless version of MCAK that binds centromeres but not microtubules disrupts chromosome segregation during anaphase. Antisense-induced depletion of MCAK results in the same defect. MCAK overexpression induces centromere-independent bundling and eventual loss of spindle microtubule polymer suggesting that centromere-associated bundling and/or depolymerization activity is required for anaphase. Live cell imaging indicates that MCAK may be required to coordinate the onset of sister centromere separation.     

Timing and duration of moult in three populations of Purple Sandpipers Calidris maritima with different moult/migration patterns

Timing and duration of primary moult in three populations of Purple Sandpipers Calidris maritima were described and discussed in relation to the birds’ need to complete moult before the onset of winter, when resources are required for survival. We predicted that moult would be completed earlier by birds wintering at higher latitudes. The south Norwegian breeding population, which moults and winters along the coast of east Britain (54–57°N) had a mean starting date of 21 July for primary moult (16 July for females and 24 July for males), a mean duration of 61 days, and completed on 20 September. Resident Icelandic (64–65°N) birds had a mean starting date of 22 July for primary moult (17 July for females and 25 July for males), a mean duration of 51 days, and completed on 11 September. Birds moulting in north Norway (70°N) arrived in north Norway in suspended primary moult or without having started moult, and completed it there. They had a mean completion date of 2 November for primary moult (31 October for females and 3 November for males). Starting date and duration could not be estimated because some suspended moult for an undetermined period, but it was thought that they started in late August. It is likely that most originated from Russia. The onset of moult appears to be set by the end of breeding and there is little overlap in these two events. The earlier start of moult by females in all three populations may be because they abandon the males when the chicks hatch, leaving the males to attend the chicks. Although the duration of primary moult followed the expected trend, being fastest in north Norway and slowest in Britain, the onset of moult was so late in north Norway that they had an unexpectedly late completion date, despite their rapid moult. The late completion of primary moult in north Norway suggests that wintering in the far north may not pose the energetic constraints on Purple Sandpipers that had previously been supposed.     

Thermal stability: a means to assure tertiary structure in therapeutic proteins.

To be both safe and effective, a therapeutic product must have the correct chemical structure and be free of harmful contaminants. Structure in protein therapeutic products, however, implies not only the correct sequence of amino acids (primary structure) but also the proper folding of that amino acid chain in three-dimensional space (tertiary structure). This work is part of a general strategy to develop a battery of physico-chemical methods that could give assurances of structure (and hence function) in formulated therapeutic proteins in the absence of in vivo data. It focuses on recombinant human growth hormone (rhGH), a well-characterized therapeutic protein, and examines the utility of thermodynamic parameters in assessing its tertiary structure. Resistance of solutions of formulated rhGH to thermal denaturation was followed using Fourier Transform Infrared Spectroscopy (FTIR) by observing decreases in total helicity and increases in intermolecular beta-sheet formation. Under conditions known to induce changes in the intra-molecular ionic and H-bonding patterns stabilizing the tertiary structure but not affecting the protein's secondary structure or global fold, we have observed upwards of a 12 degrees C shift in the melting temperature of the protein. Furthermore, our results indicated that the T(m) of unfolding of rhGH was sensitive to much more subtle changes in the protein structure. Thus, resistance to thermal denaturation may well be a useful means to measure structure in formulations of well-characterized therapeutic proteins.