Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

C-cell follicles of canine thyroid glands studied by PAS reaction and electron microscopy

Summary Small follicles composed solely of C cells were occasionally observed in large C cell groups of dog thyroid glands. The lumina of C-cell follicles were filled with, or contained peripheral depositions of PAS-positive amorphous material, which was similar in ultrastructural features to thyroglobulin-containing colloid in typical thyroid follicles. This indicates that C cells, in addition to secreting calcitonin, produce a glycoprotein that can be stored in the lumina of the follicles.     

α-Guanidinoglutaric Acid in Cobalt-Induced Epileptogenic Cerebral Cortex of Cats

: Guanidino compounds in the cobalt-induced epileptogenic cerebral cortex of cats were fluorometrically analysed by a JASCO G-520 guanidino compounds analyser, and an unknown high peak was observed in the chromatogram that was identical to the peak of authentic α-guanidinoglutaric acid. In another experiment, the substance was extracted from the cobalt focus tissue, converted into dimethylpyrimidyl derivative-butylester, and analysed by a GC/MS technique. The mass spectrum of the substance was identical to the dimethylpyrimidyl derivative of α-guanidinoglutaric acid butylester (M⁺= 365).     

Early development of the cerebral vesicles in man.

Two human embryos which were immediately before and soon after the appearance of the cerebral vesicle were cut into complete serial sections in almost the same direction and studied comparatively. The findings on these two sets of serial sections indicate that the rostral continuations of subthalamus and ventral thalamus along the ventral thalamic sulcus continuing from the sulcus limitans of the midbrain are destined to give rise to the formation of the cerebral vesicle. The rostral continuation of the subthalamus becomes the floor of the interventricular foramen, and that of the ventral thalamus the pallium. The rostral end of the continuation of the subthalamic matrix is not only the site of the beginning of evagination of the cerebral vesicle, but also the site of elevation of the ganglionic hill or striatum. As the evagination of the cerebral vesicle proceeds exceedingly dorsocaudolaterally from the rostral end of the continuation of the ventral thalamus, the elevation of the striatum occurs also in the same direction, getting in touch with the folding of the hemispheric stalk of Kuhlenbeck to enclose the interventricular foramen. Tt is noticed that, at the beginning of evagination, the grade of depression of the nasal pit was always parallel with that of evagniation of the cerebral vesicle.     

Anatomy-based prediction method for determining ipsilateral lung doses in postoperative breast radiation therapy assisted by diagnostic computed tomography images

BackgroundThis study aimed to investigate whether ipsilateral lung doses (ILDs) could be predicted by anatomical indexes measured using diagnostic computed tomography (CT) prior to the planning stage of breast radiation therapy (RT).Materials and methodsThe thoracic diameters and the length of lines drawn manually were measured on diagnostic CT images. The parameters of interest were the skin maximum lung distance (sMLD), central lung distance (CLD), Haller index (HI), and body mass index (BMI). Lung dose-volume histograms were created with conformal planning, and the lung volumes receiving 5–40 Gy (V5–V40) were calculated. Linear regression models were used to investigate the correlations between the anatomical indexes and dose differences and to estimate the slope and 95% confidence intervals (CIs).ResultsA total of 160 patients who had undergone three-dimensional conformal RT after breast-conserving surgery were included. Univariable analysis revealed that the sMLD (p < 0.001), CLD (p < 0.001), HI (p = 0.002), and BMI (p < 0.001) were significantly correlated with the V20. However, multivariable analysis revealed that only the sMLD (slope: 0.147, p = 0.001, 95% CI: 0.162–0.306) and CLD (0.157, p = 0.005, 0.048–0.266) were strongly correlated with the V20. The p-value for the sMLD was the lowest among the p-values for all indexes, thereby indicating that the sMLD had the best predictive power for ILD.ConclusionssMLD and CLD are anatomical markers that can be used to predict ILD in whole breast RT. An sMLD > 20.5 mm or a CLD > 24.3 mm positively correlated with a high ILD.     

Xylanase Produced by Alkalophilic Bacillus No. C-59-2

Bacillus No. C–59–2 isolated from soil produced a xylanase in alkaline media. The characteristic point of this bacteria was especially good growth in alkaline media, and no growth was observed in neutral media such as nutrient broth. The xylanase of this bacteria was purified by CM-celluIose, hydroxyl apatite and Sephadex G–75 columns. The enzyme was most active at pH 5.5~9 which was much broader and higher than those of other xylanases. The sedimentation constant was about 3.5 S and isoelectric point was pH 6.3. The enzyme was most stable at pH 7 and calcium ion was effective to stabilize the enzyme. The enzyme activity was inhibited by Hg²⁺, Ag²⁺ and Cd^{2 +} Maximum hydrolysis rate of xylan by the enzyme was about 40%. The enzyme split xylan and yielded xylobiose and higher oligosaccharides. Therefore, this enzyme is considered to be a type of endo-xylanase.     

Solar radiation and functional traits explain the decline of forest primary productivity along a tropical elevation gradient

One of the major challenges in ecology is to understand how ecosystems respond to changes in environmental conditions, and how taxonomic and functional diversity mediate these changes. In this study, we use a trait‐spectra and individual‐based model, to analyse variation in forest primary productivity along a 3.3 km elevation gradient in the Amazon‐Andes. The model accurately predicted the magnitude and trends in forest productivity with elevation, with solar radiation and plant functional traits (leaf dry mass per area, leaf nitrogen and phosphorus concentration, and wood density) collectively accounting for productivity variation. Remarkably, explicit representation of temperature variation with elevation was not required to achieve accurate predictions of forest productivity, as trait variation driven by species turnover appears to capture the effect of temperature. Our semi‐mechanistic model suggests that spatial variation in traits can potentially be used to estimate spatial variation in productivity at the landscape scale.     

Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.     

Polyglutamine diseases and molecular chaperones

The polyglutamine diseases, a group of diseases currently thought to consist of nine inherited neurodegenerative diseases, are caused by the expansion of unstable CAG trinucleotide repeats that code for polyglutamine tracts in the responsible genes. These diseases are now recognized as being of a type with conformationally abnormal or amyloid-related proteins, and thus are called 'conformational diseases'. Recently, many studies using cell cultures and model organisms have suggested that the two major machineries for protein quality control (the molecular chaperone and the protein degradation machineries) play important roles in the pathogenesis of the polyglutamine diseases. Interestingly, molecular chaperones have been shown to behave in totally different ways in these studies, namely in suppressing as well as enhancing neurodegeneration or cell death. These apparently opposite actions of molecular chaperones suggest that a certain balance between the activities of molecular chaperones and the expression level of polyglutamine is an important determinant of the pathogenesis. In this review, we summarize recent findings on such ambiguous effects of molecular chaperones on polyglutamine diseases, and discuss possible mechanisms by which molecular chaperones, especially VCP, are involved in the pathogenesis.