In situ TEM observations of fracture in nanolaminated metallic thin films

Fracture of single crystal nanolaminated thin films has been investigated through in situ straining of cross-sectional samples of Cu/Ni nanolaminates grown on Cu (001) single crystal substrates. The earlest stages of deformation exhibits a confined layer slip mechanism. With continued straining, unstable fracture occurs creating a mixed-mode crack that propagates across the nanolaminate, roughly perpendicular to the interfaces. Eventually, stable crack growth with intense plastic deformation ahead of the crack tip occurs over many bilayers in the direction of crack growth. Simultaneously, plasticity was seen to spread only 1 or 2 bilayer distances normal to the crack, creating an extremely localized plastic zone. Transmission electron microscopic (TEM) examination after the test did not reveal the presence of dislocations in the crack wake, except where severe crack deflection was observed. By comparison, the plastic zone size in the substrate was greater by several of orders of magnitude.     

Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: role in restricted conformational mobility and compaction of native state

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193-7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein's surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen-deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein's interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.     

Insect chitinases: molecular biology and potential use as biopesticides

Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents. One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. For several years, our laboratories have been characterizing this enzyme and its gene. Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests. Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined. Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized. Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance. A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity. Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis. Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs. Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review.     

Plasma GABA in children and adolescents with mood, behavior, and comorbid mood and behavior disorders: a preliminary study

Plasma GABA concentrations (pGABA) were measured in 115 inpatients (aged 7-17) with child psychiatric disorders. Group mean pGABAs were compared for 38 patients with mood disorders only (MOOD), 29 with behavior disorders only (BEH), 48 with comorbid mood and behavior disorders (MOOD + BEH), and 14 normal controls (CON, aged 14-17). The BEH group was characterized by (a) high mean pGABAs (157 vs. 133 pmol/ml), (b) lower mean pGABAs in BEH subjects who had been receiving pharmacotherapy with SSRIs or other medications (p < 0.026), and (c) decreased pGABA with increasing age (p = 0.019). These features were not found in controls or in groups of patients with mood disorders (MOOD or MOOD + BEH). Elevated mean pGABA in the BEH group appeared specifically in patients with comorbid CD and ADHD, not in patients with ADHD or CD alone (p = 0.004). No patient in BEH (or CON) had pGABA below 100 pmol/ml, but low pGABAs were found in 15% of MOOD patients (who had no behavior disorder) and in 16% of MOOD + BEH patients. Pharmacotherapy did not change pGABAs in the MOOD or the MOOD + BEH groups. No pGABA differences were found among the anxiety disorders, either alone or with mood or behavior comorbidity. The finding that plasma GABA levels are elevated in nonmedicated behavior disorders that present in the absence of mood disorders, and appear to lower following medication treatments, merits increased attention to the pharmacological study of nonaffective behavior disorders.     

Vasopressin receptor subtypes differentially modulate calcium-activated potassium currents in the horizontal limb of the diagonal band of Broca

Because many testicular toxicants cause damage to specific stages of spermatogenesis, the present study has investigated the utility of a model in which the testis is synchronized to contain only a few closely related spermatogenic stages. The susceptibility of different stages to 1,3-dinitrobenzene (1,3-DNB) toxicity was investigated in rats, the testes of which had been stage synchronized by a vitamin A depletion/repletion (VADR) procedure. 1,3-DNB (25 mg/kg, IP) or vehicle was injected 58, 61, or 78 d after vitamin A readministration, and testicular histopathology was evaluated 48 h later. At the time of sacrifice, testes in the three groups were synchronized to stages I-VI, VII-IX, or X-XIV+I. The data indicated that tubules in all stages of spermatogenesis, in both synchronized and unsynchronized animals, demonstrated histopathologic changes in response to 1,3-DNB. However, the lesion seen in synchronized animals was more severe and less stage specific than that seen in weight-matched, unsynchronized animals. This increase in degree of susceptibility could be partially explained by differences in toxicokinetics. Stage-synchronized testes could provide unique insights into stage-specific cellular and molecular events, especially for in vitro studies where the stage enrichment could be maximally exploited. However, results obtained from in vivo toxicity studies using animals subjected to VADR should be interpreted carefully in light of the confounding physiologic/metabolic perturbations potentially induced by the VADR procedure.     

The catalytic core of RNase P

A deletion mutant of the catalytic RNA component of Escherichia coli RNase P missing residues 87-241 retains the ability to interact with the protein component to form a functional catalyst. The deletion of this phylogenetically conserved region significantly increases the Km, indicating that the deleted structures may be important for binding to the precursor tRNA substrate but not for the cleavage reaction. Under some reaction conditions, this RNase P deletion mutant can become a relatively non-specific nuclease, indicating that this RNA's catalytic center may be more exposed. The catalytic core of the RNase P is formed by less than one third of the 377 residues of the RNase P RNA.     

Effects of Na+ and Mg2+ on the structures of supercoiled DNAs: comparison of simulations with experiments

Recent cryo-electron microscopy (cryo-EM) results suggest that sufficient NaCl concentration (> or approximately 0.1 M) and superhelix density (> or approximately-0.05) cause circular DNAs to adopt highly extended, tightly interwound configurations, in which the strands are laterally contiguous along almost their entire length. Millimolar levels of MgCl2 reportedly act synergistically with NaCl to produce similar conformations. However, Monte Carlo simulations with purely repulsive interduplex forces failed to reproduce such structures. In the present work, solution measurements of particular physical properties were performed both to characterize the effects of Na+ and Mg2+ on DNA structure and to provide quantitative tests of Monte Carlo simulations of circular DNAs. Supercoiled p30 delta DNAs in 10 mM Tris plus 0, 0.122, and 0.1 M NaCl, and 0.1 M NaCl plus 4 mM Mg2+ were examined by static and dynamic light scattering (LS and DLS), time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium, and circular dichroism (CD) spectroscopy. Upon addition of 0.122 M NaCl, the radius of gyration (Rg) decreased substantially, which indicates that p30 delta adopts a more compact structure. This contradicts the cryo-EM studies, where molecular extension and Rg both increase upon adding 0.1 M NaCl. In 0.1 M NaCl, the torsion constant measured by FPA is practically invariant to superhelix density, and the plateau diffusion coefficient at large scattering vector (Dplat) is likewise nearly the same at both relaxed and native superhelix densities. Such invariance is difficult to reconcile with any transition from relaxed circles to tightly interwound structures with laterally contiguous strands. Metropolis Monte Carlo simulations were performed to generate canonically distributed sets of structures, from which average Do values and scattered intensity ratios, [symbol: see text]I (zero) [symbol: see text]/[symbol: see text] l(k) [symbol: see text], were calculated. Agreement between simulations and experiments in regard to [symbol: see text] I(O) [symbol: see text] /[symbol: see text] I(k) [symbol: see text], D(zero) and the supercoiling free energy, delta Gsc (delta l), is remarkably good for the most extensively studied p30 delta samples. The simulated structures exhibit no sign of very tight interwinding with extensive lateral contacts, but instead exhibit most probable superhelix diameters of 85 to 90 A. When 4 mM Mg2+ was added to native supercoiled p30 delta in 0.1 M NaCl, Rg decreased, D(zero) increased, and the longest internal relaxation rate (1/tau 2(zero)) increased, all of which indicate a further overall contraction of the molecular envelope. The torsion constant exhibited a slight increase that is hardly statistically significant. In this case, agreement between the simulations and experiments was only semi-quantitative for most samples investigated, although the predicted contraction was exhibited by all five samples of p30 delta and one of pBR322 DNA. The simulated structures in 0.1 M NaCl plus 4 mM Mg2+ again showed no sign of extensive lateral contacts. A plausible explanation is proposed for the highly extended, tightly interwound structures seen in cryo-EM, and explicitly tested by Monte Carlo simulations of a 1000 bp circular DNA at +25 and -50 degrees C. Structures identical to those seen in cryo-EM are in fact the equilibrium structures in the simulations at -50 degrees C, and the estimated time for equilibration (2.3 x 10(-6) second) is much smaller than the estimated time for vitrification (1 x 10(-4) second).