A multifrequency electron spin resonance study of T4 lysozyme dynamics.

Electron spin resonance (ESR) spectroscopy at 250 GHz and 9 GHz is utilized to study the dynamics and local structural ordering of a nitroxide-labeled enzyme, T4 lysozyme (EC 3.2.1.17), in aqueous solution from 10 degrees C to 35 degrees C. Two separate derivatives, labeled at sites 44 and 69, were analyzed. The 250-GHz ESR spectra are well described by a microscopic ordering with macroscopic disordering (MOMD) model, which includes the influence of the tether connecting the probe to the protein. In the faster "time scale" of the 250-GHz ESR experiment, the overall rotational diffusion rate of the enzyme is too slow to significantly affect the spectrum, whereas for the 9-GHz ESR spectra, the overall rotational diffusion must be accounted for in the analysis. This is accomplished by using a slowly relaxing local structure model (SRLS) for the dynamics, wherein the tether motion and the overall motion are both included. In this way a simultaneous fit is successfully obtained for both the 250-GHz and 9-GHz ESR spectra. Two distinct motional/ordering modes of the probe are found for both lysozyme derivatives, indicating that the tether exists in two distinct conformations on the ESR time scale. The probe diffuses more rapidly about an axis perpendicular to its tether, which may result from fluctuations of the peptide backbone at the point of attachment of the spin probe.     

Cellular motor protein KIF-4 associates with retroviral Gag

Previously we demonstrated that murine retroviral Gag proteins associate with a cellular motor protein, KIF-4. Using the yeast two-hybrid assay, we also found an association of KIF-4 with Gag proteins of Mason-Pfizer monkey virus (MPMV), simian immunodeficiency virus (SIV), and human immunodeficiency virus type 1 (HIV-1). Studies performed with mammalian cell systems confirmed that the HIV-1 Gag protein associates with KIF-4. Soluble cytoplasmic proteins from cells infected with recombinant vaccinia virus expressing the entire Gag-Pol precursor protein of HIV-1 or transfected with HIV-1 molecular clone pNL4-3 were fractionated by sucrose gradient centrifugation and further separated by size-exclusion and anion-exchange chromatographies. KIF-4 and HIV-1 Gag cofractionated in both chromatographic separations. Immunoprecipitation assays have also verified the KIF-4-Gag association. KIF-4 binds mainly to the Gag precursor (Pr55 Gag) and a matrix-capsid processing intermediate (Pr42) but not to other processed Gag products. The binding of Gag is mediated by a domain of KIF-4 proximal to the C terminus. These results, and our previous studies, raise the possibility that KIF-4 may play an important role in retrovirus Gag protein transport.     

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage

Tissue engineered cartilage can be grown in vitro if the necessary physical and biochemical factors are present in the tissue culture environment. Cell metabolism and tissue composition were studied for engineered cartilage cultured for 5 weeks using bovine articular chondrocytes, polymer scaffolds (5 mm diameter x 2 mm thick fibrous discs), and rotating bioreactors. Medium pH and concentrations of oxygen, carbon dioxide, glucose, lactate, ammonia, and glycosoaminoglycan (GAG) were varied by altering the exchange rates of gas and medium in the bioreactors. Cell-polymer constructs were assessed with respect to histomorphology, biochemical composition and metabolic activity. Low oxygen tension ( approximately 40 mmHg) and low pH ( approximately 6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension ( approximately 80 mmHg) and higher pH ( approximately 7.0) were associated with more aerobic cell metabolism (YL/G of 1.65-1.79 mol/mol). Under conditions of infrequent medium replacement (50% once per week), cells utilized more economical pathways such that glucose consumption and lactate production both decreased, cell metabolism remained relatively aerobic (YL/G of 1.67 mol/mol) and the resulting constructs were cartilaginous. More aerobic conditions generally resulted in larger constructs containing higher amounts of cartilaginous tissue components, while anaerobic conditions suppressed chondrogenesis in 3D tissue constructs.     

Investigations into sequence and conformational dependence of backbone entropy,inter-basin dynamics and the Flory isolated-pair hypothesis for peptides

The populations and transitions between Ramachandran basins are studied for combinations of the standard 20 amino acids in monomers, dimers and trimers using an implicit solvent Langevin dynamics algorithm and employing seven commonly used force-fields. Both the basin populations and inter-conversion rates are influenced by the nearest neighbor's conformation and identity, contrary to the Flory isolated-pair hypothesis. This conclusion is robust to the choice of force-field, even though the use of different force-fields produces large variations in the populations and inter-conversion rates between the dominant helical, extended beta, and polyproline II basins. The computed variation of conformational and dynamical properties with different force-fields exceeds the difference between explicit and implicit solvent calculations using the same force-field. For all force-fields, the inter-basin transitions exhibit a directional dependence, with most transitions going through extended beta conformation, even when it is the least populated basin. The implications of these results are discussed in the context of estimates for the backbone entropy of single residues, and for the ability of all-atom simulations to reproduce experimental protein folding data.     

The pesticide rotenone induces caspase-3-mediated apoptosis in ventral mesencephalic dopaminergic neurons

In vivo, the pesticide rotenone induces degeneration of dopamine neurons and parkinsonian-like pathology in adult rats. In the current study, we utilized primary ventral mesencephalic (VM) cultures from E15 rats as an in vitro model to examine the mechanism underlying rotenone-induced death of dopamine neurons. After 11 h of exposure to 30 nm rotenone, the number of dopamine neurons identified by tyrosine hydroxylase (TH) immunostaining declined rapidly with only 23% of the neurons surviving. By contrast, 73% of total cells survived rotenone treatment, indicating that TH+ neurons are more sensitive to rotenone. Examination of the role of apoptosis in TH+ neuron death, revealed that 10 and 30 nm rotenone significantly increased the number of apoptotic TH+ neurons from 7% under control conditions to 38 and 55%, respectively. The increase in apoptotic TH+ neurons correlated with an increase in immunoreactivity for active caspase-3 in TH+ neurons. The caspase-3 inhibitor, DEVD, rescued a significant number of TH+ neurons from rotenone-induced death. Furthermore, this protective effect lasted for at least 32 h post-rotenone and DEVD exposure, indicating lasting neuroprotection achieved with an intervention prior to the death commitment point. Our results show for the first time in primary dopamine neurons that, at low nanomolar concentrations, rotenone induces caspase-3-mediated apoptosis. Understanding the mechanism of rotenone-induced apoptosis in dopamine neurons may contribute to the development of new neuroprotective strategies against Parkinson's disease.     

Transduction of the mammary epithelium with adenovirus vectors in vivo

Because the mammary parenchyma is accessible from the exterior of an animal through the mammary duct, adenovirus transduction holds promise for the short-term delivery of genes to the mammary epithelium for both research and therapeutic purposes. To optimize the procedure and evaluate its efficacy, an adenovirus vector (human adenovirus type 5) encoding a green fluorescent protein (GFP) reporter and deleted of E1 and E3 was injected intraductally into the mouse mammary gland. We evaluated induction of inflammation (by intraductal injection of [(14)C]sucrose and histological examination), efficiency of transduction, and maintenance of normal function in transduced cells. We found that transduction of the total epithelium in the proximal portion of the third mammary gland varied from 7% to 25% at a dose of 2 x 10(6) PFU of adenovirus injected into day 17 pregnant mice. Transduction was maintained for at least 7 days with minimal inflammatory response; however, significant mastitis was observed 12 days after transduction. Adenovirus transduction could also be used in the virgin animal with little mastitis 3 days after transduction. Transduced mammary epithelial cells maintained normal morphology and function. Our results demonstrate that intraductal injection of adenovirus vectors provides a versatile and noninvasive method of investigating genes of interest in mouse mammary epithelial cells.     

Hydration, structure, and molecular interactions in the headgroup region of dioleoylphosphatidylcholine bilayers: an electron spin resonance study

The relationship between bilayer hydration and the dynamic structure of headgroups and interbilayer water in multilamellar vesicles is investigated by electron spin resonance methods. Temperature variations of the order parameter of a headgroup spin label DPP-Tempo in DOPC in excess water and partially dehydrated (10 wt % water) show a cusp-like pattern around the main phase transition, Tc. This pattern is similar to those of temperature variations of the quadrupolar splitting of interbilayer D2O in PC and PE bilayers previously measured by 2H NMR, indicating that the ordering of the headgroup and the interbilayer water are correlated. The cusp-like pattern of these and other physical properties around Tc are suggestive of quasicritical fluctuations. Also, an increase (a decrease) in ordering of DPP-Tempo is correlated with water moving out of (into) interbilayer region into (from) the bulk water phase near the freezing point, Tf. Addition of cholesterol lowers Tf, which remains the point of increasing headgroup ordering. Using the small water-soluble spin probe 4-PT, it is shown that the ordering of interbilayer water increases with bilayer dehydration. It is suggested that increased ordering in the interbilayer region, implying a lowering of entropy, will itself lead to further dehydration of the interbilayer region until its lowered pressure resists further flow, i.e., an osmotic phenomenon.     

Lipid-gramicidin interactions: dynamic structure of the boundary lipid by 2D-ELDOR

The use of 2D-electron-electron double resonance (2D-ELDOR) for the characterization of the boundary lipid in membrane vesicles of DPPC and gramicidin A' (GA) is reported. We show that 2D-ELDOR, with its enhanced spectral resolution to dynamic structure as compared with continuous-wave electron spin resonance, provides a reliable and useful way of studying lipid-protein interactions. The 2D-ELDOR spectra of the end-chain spin label 16-PC in DPPC/GA vesicles is composed of two components, which are assigned to the bulk lipids (with sharp auto peaks and crosspeaks) and to the boundary lipids (with broad auto peaks). Their distinction is clearest for higher temperatures and higher GA concentrations. The quantitative analysis of these spectra shows relatively faster motions and very low ordering for the end chain of the bulk lipids, whereas the boundary lipids show very high "y-ordering" and slower motions. The y-ordering represents a dynamic bending at the end of the boundary lipid acyl chain, which can then coat the GA molecules. These results are consistent with the previous studies by Ge and Freed (1999) using continuous-wave electron spin resonance, thereby supporting their model for GA aggregation and H(II) phase formation for high GA concentrations. Improved instrumental and simulation methods have been employed.     

Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation

Bovine calf articular chondrocytes, either primary or expanded in monolayers (2D) with or without 5 ng/ml fibroblast growth factor-2 (FGF-2), were cultured on three-dimensional (3D) biodegradable polyglycolic acid (PGA) scaffolds with or without 10 ng/ml bone morphogenetic protein-2 (BMP-2). Chondrocytes expanded without FGF-2 exhibited high intensity immunostaining for smooth muscle alpha-actin (SMA) and collagen type I and induced shrinkage of the PGA scaffold, thus resembling contractile fibroblasts. Chondrocytes expanded in the presence of FGF-2 and cultured 6 weeks on PGA scaffolds yielded engineered cartilage with 3.7-fold higher cell number, 4.2-fold higher wet weight, and 2.8-fold higher wet weight glycosaminoglycan (GAG) fraction than chondrocytes expanded without FGF-2. Chondrocytes expanded with FGF-2 and cultured on PGA scaffolds in the presence of BMP-2 for 6 weeks yielded engineered cartilage with similar cellularity and size, 1.5-fold higher wet weight GAG fraction, and more homogenous GAG distribution than the corresponding engineered cartilage cultured without BMP-2. The presence of BMP-2 during 3D culture had no apparent effect on primary chondrocytes or those expanded without FGF-2. In summary, the presence of FGF-2 during 2D expansion reduced chondrocyte expression of fibroblastic molecules and induced responsiveness to BMP-2 during 3D cultivation on PGA scaffolds.