Comparative analyses of the conformational stability of a hyperthermophilic protein and its mesophilic counterpart.

Comparison of the conformational stability of an O(6)-methylguanine-DNA methyltransferase (MGMT) from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk-MGMT), and its mesophilic counterpart C-terminal Ada protein from Escherichia coli (Ec-AdaC) was performed in order to obtain information about the relationship between thermal stability and other factors, such as thermodynamic parameters, thermodynamic stability and other unfolding conditions. Tk-MGMT unfolded at Tm = 98.6 degrees C, which was 54.8 degrees C higher than the unfolding temperature of Ec-AdaC. The maximum free energy (DeltaG(max)) of the proteins were different; the value of Tk-MGMT (42.9 kJ.mol-1 at 29.5 degrees C) was 2.6 times higher than that of Ec-AdaC (16.6 kJ.mol-1 at 7.4 degrees C). The high conformational stability of Tk-MGMT was attributed to a 1.6-fold higher enthalpy value than that of Ec-AdaC. In addition, the DeltaG(max) temperature of Tk-MGMT was considerably higher (by 22.1 degrees C). The apparent heat capacity of denaturation (DeltaC(p)) of Tk-MGMT was 0.7-fold lower than that of Ec-AdaC. These three synergistic effects, increasing DeltaGmax, shifted DeltaG vs. temperature curve, and low DeltaC(p), give Tk-MGMT its thermal stability. Unfolding profiles of the two proteins, tested with four alcohols and three denaturants, showed that Tk-MGMT possessed higher stability than Ec-AdaC in all conditions studied. These results indicate that the high stability of Tk-MGMT gives resistance to chemical unfolding, in addition to thermal unfolding.     

Crystal structures of modified myoglobins. I. Heme orientation and structural changes around heme in myoglobins reconstituted with isopemptoheme, pemptoheme, 2-ethyldeuteroheme, and 4-ethyldeuteroheme

The crystal structures of sperm whale metmyoglobins reconstituted with four modified hemes, isopemptoheme, pemptoheme, 2-ethyldeuteroheme, and 4-ethyldeuteroheme, have been determined and refined at 2.2 A resolution to R = 0.217, 0.218, 0.213, and 0.222, respectively. All the crystals of these myoglobins are isomorphous with that of native metmyoglobin. The structural changes of the modified myoglobin from the native myoglobin were examined on difference Fourier maps; the orientation of 4-ethyldeuteroheme in the heme pocket is such that the heme is rotated by 180 degrees about an axis through the alpha-gamma-meso carbons, whereas the orientations of the other three hemes are the same as that of the protoheme in the native myoglobin. The changes of the structures around the heme become greater in the order of isopemptoheme, 2-ethyldeuteroheme less than pemptoheme less than 4-ethyldeuteroheme. The magnitudes of the changes seem to be related to the oxygen affinities of these four reconstituted myoglobins.     

Increased Insect Virulence in Beauveria bassiana Strains Overexpressing an Engineered Chitinase

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.     

A flow-injection chemiluminescence method for the determination of human serum albumin, using the reaction of fluorescein-human serum albumin-sodium hypochlorite by the enhancement effect of the cationic surfactant cetyltrimethylammonium bromide.

The interaction between surfactant and fluorescein was studied, using a fluorescence spectroscopy and flow-injection (FI) chemiluminescence (CL) method. It was found that the cationic surfactant cetyltrimethylammonium bromide (CTAB) could cause the structural transformation of fluorescein from the quinone to the spirolactone form, and greatly enhance the CL intensity of the fluorescein-human serum albumin (HSA) complex. Based on this finding, a rapid and sensitive FI-CL method was developed for the determination of HSA. Under the optimum conditions, the proposed method has a linear range of 0.05-24.0 microg/mL, with a detection limit of 0.03 microg/mL for HSA (3sigma). The relative standard deviation (RSD) of 1.2 microg/mL HSA (n = 8) is 0.8%. The method was applied to the determination of protein content in urine samples, with satisfactory results. Density functional theory was used to study the mechanism of surfactant-enhanced CL intensity of the fluorescein-HSA complex.     

Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis

The “cysteine string protein” (CSP) genes of higher eukaryotes code for a novel family of proteins characterized by a “J” domain and an unusual cysteine-rich region. Previous studies had localized the proteins in neuropil and synaptic terminals of larval and adult Drosophila and linked the temperature-sensitive paralysis of the mutants described here to conditional failure of synaptic transmission. We now use the null mutants as negative controls in order to reliably detect even low concentrations of CSPs by immunohistochemistry, employing three monoclonal antibodies. In wild-type flies high levels of cysteine string proteins are found not only in apparently all synaptic terminals of the embryonic, larval, and adult nervous systems, but also in the “tall cells” of the cardia, in the follicle cells of the ovary, in specific structures of the female spermatheca, and in the male testis and ejaculatory bulb. In addition, low levels of CSPs appear to be present in all tissues examined, including neuronal perikarya, axons, muscles, Malpighian tubules, and salivary glands. Western blots of isolated tissues demonstrate that of the four isoforms expressed in heads only the largest is found in non-neural organs. The wide expression of CSPs suggests that at least some of the various phenotypes of the null mutants observed at permissive temperatures, such as delayed development, short adult lifespan, modified electroretinogram, and optomotor behavior, may be caused by the lack of CSPs outside synaptic terminals.     

THE MOLECULAR WEIGHT AND ISOELECTRIC POINT OF THYROGLOBULIN

1. The sedimentation constant of hog thyroglobulin is 19.2ċ10^–13. That of human thyroglobulin is essentially the same. 2. The specific volume of hog thyroglobulin is 0.72. 3. The isoelectric point of native hog thyroglobulin is at pH 4.58, that of denatured thyroglobulin at pH 5.0. 4. The molecular weight of hog thyroglobulin is, in round numbers, 700,000, as calculated from the sedimentation and diffusion constants, or 650,000, as calculated from the sedimentation equilibrium data. 5. The thyroglobulin molecule deviates markedly from the spherical.     

Ischemic flap survival improvement by composition-selective fat grafting with novel adipose tissue derived product - stromal vascular fraction gel

Background

Flap necrosis due to insufficient blood supply is a common postoperative complication in random pattern flaps. Stem cell therapies have emerged as promising biologics for tissue ischemia. A novel fat derived product, stromal vascular fraction gel (SVF-gel), can be prepared with lipoaspirate through simple mechanical processing, removing only the lipid content. SVF-gel enriches adipose-derived stem cells and potentially beneficial for flap necrosis.