Statistical method for characterization of hypertrophic cardiomyopathy by use of morphologic and pathologic measurements in pigs (Sus scrofa domestica).

BACKGROUND AND PURPOSE: Hypertrophic cardiomyopathy (HCM) is characterized by symmetric or asymmetric hypertrophy of the left and/or right ventricle. Morphologic and pathologic indices (MI and PI) of hearts were established for classification of HCM in pigs. METHODS: Fifty on-farm-performance-tested pigs (average body weight, 104.3 kg; age, 224.5 days) were randomly selected. Heart weight, length, width, heart-to-body weight ratio, and thickness of the cranial and middle portions of ventricular septum and left ventricular free wall were measured. Myocyte disorganization and necrosis, myocardial and endocardial fibrosis, and intramural coronary arterial occlusion were scored. Principal component analysis and stepwise regression analysis were used to establish MI and PI. RESULTS: MI was established by using the first principal component as the dependent variable and applying stepwise regression analysis. Hearts were classified as morphologically normal, suspicious, and hypertrophic according to the range of MI. The same statistical method was used to find PI. Hearts were classified as pathologically normal, moderately affected, or seriously affected according to the range of PI. Combining MI and PI, hearts could be classified into five groups: no hypertrophy with minor lesion (normal); hypertrophy but with rare lesion; no hypertrophy but seriously affected; suspicious; and hypertrophy and seriously affected (heart with HCM). Another 119 hearts were collected and classified. The variation of heart measurements was consistent with the original purpose of classification. CONCLUSIONS: Using fewer measurements for identification of HCM objectively in pigs seems to have practical application.     

The insulin A and B chains contain structural information for the formation of the native molecule. Studies with protein disulphide-isomerase.

It has been shown previously [Tang, Wang & Tsou (1988) Biochem. J. 255, 451-455] that, under appropriate conditions, native insulin can be obtained from scrambled insulin or the S-sulphonates of the chains with a yield of 25-30%, together with reaction products containing the separated A and B chains. The native hormone is by far the predominant product among the isomers containing both chains. It is now shown that the presence of added C peptide has no appreciable effect on the yield of native insulin. At higher temperatures the content of the native hormone decreases whereas those of the separated chains increase, and in no case was scrambled insulin containing both chains the predominant product in the absence of denaturants. Both the scrambling and the unscrambling reactions give similar h.p.l.c. profiles for the products. Under similar conditions cross-linked insulin with native disulphide linkages can be obtained from the scrambled molecule or from the S-sulphonate derivative with yields of 50% and 75% respectively at 4 degrees C, and with a dilute solution of the hexa-S-sulphonate yields better than 90% can be obtained. The regenerated product is shown to have the native disulphide bridges by treatment with CNBr to give insulin and by the identity of the h.p.l.c. profile of its peptic hydrolysate with that for cross-linked insulin. It appears that the insulin A and B chains contain sufficient information for the formation of the native molecule and that the role of the connecting C peptide is to bring and to keep the two chains together.     

THE USE OF TETRANITRO-BLUE TETRAZOLIUM FOR THE CYTOCHEMICAL LOCALIZATION OF SUCCINIC DEHYDROGENASE : Cytochemical and Cytological Studies of Sarcoma 37 Ascites Tumor Cells

Succinic dehydrogenase (SDH) was localized in the mitochondria of Sarcoma 37 ascites tumor cells by the use of tetranitro-BT (TNBT) and nitro-BT (NBT) in smear preparations. Results with each tetrazolium salt as electron acceptor were evaluated with respect to: (a) size and shape of the formazan precipitate relative to standard mitochondrial morphology; (b) crystallization phenomena of reduced dye; (c) lipid adsorption of formazan. The association of formazan- or iron hematoxylin-stained mitochondria with lipid droplets within the cells was investigated, as was also the influence of formalin fixation, with and without cold acetone pretreatment, on mitochondrial morphology and enzymatic staining. Data from these studies appear to indicate that TNBT is more suitable than NBT for use as a cytochemical reagent in oxidative and/or dehydrogenase enzyme histochemistry and cytochemistry.     

A potent and orally active HIV-1 integrase inhibitor

A 1,6-naphthyridine inhibitor of HIV-1 integrase has been discovered with excellent inhibitory activity in cells, good pharmacokinetics, and an excellent ability to inhibit virus with mutant enzyme.     

New targeted approaches for the quantification of data‐independent acquisition mass spectrometry

The use of data‐independent acquisition (DIA) approaches for the reproducible and precise quantification of complex protein samples has increased in the last years. The protein information arising from DIA analysis is stored in digital protein maps (DIA maps) that can be interrogated in a targeted way by using ad hoc or publically available peptide spectral libraries generated on the same sample species as for the generation of the DIA maps. The restricted availability of certain difficult‐to‐obtain human tissues (i.e., brain) together with the caveats of using spectral libraries generated under variable experimental conditions limits the potential of DIA. Therefore, DIA workflows would benefit from high‐quality and extended spectral libraries that could be generated without the need of using valuable samples for library production. We describe here two new targeted approaches, using either classical data‐dependent acquisition repositories (not specifically built for DIA) or ad hoc mouse spectral libraries, which enable the profiling of human brain DIA data set. The comparison of our results to both the most extended publically available human spectral library and to a state‐of‐the‐art untargeted method supports the use of these new strategies to improve future DIA profiling efforts.     

Activity change during unfolding of bovine pancreatic ribonuclease A in guanidine

Bovine pancreatic ribonuclease A loses almost completely its activity in 2-3 M guanidine, whereas only very slight conformational changes can be detected when following its unfolding by changes in its intrinsic fluorescence at 305 nm and ultraviolet absorbance at 287 nm. Reactivation on diluting out the denaturant is a time-dependent process, indicating that the inactivation is not due to inhibition by a reversible association of the enzyme with guanidine. The kinetic method of following the substrate reaction, in the presence of the denaturant previously proposed for use in the study of rapid inactivation reactions (Tian, W.X. and Tsou, C.-L. (1982) Biochemistry 21, 1028-1032), is applied to examine the inactivation rates of this enzyme during guanidine denaturation, and these have been compared with the unfolding rates as followed by fluorescence and absorbance changes. It is shown that during the unfolding of this enzyme in guanidine, the inactivation of the enzyme occurs within the dead time of mixing in a stopped-flow apparatus and is at least several orders of magnitude faster than the unfolding reaction as detected by the optical parameters. It appears that, as in the case of creatine kinase reported previously, the active site of a small enzyme stabilized by multiple disulfide linkages, such as ribonuclease A, is also situated in a region which is much more liable to being perturbed by denaturants than is the molecule as a whole.     

58-kDa microspherule protein (MSP58) is novel Brahma-related gene 1 (BRG1)-associated protein that modulates p53/p21 senescence pathway

The nucleolar 58-kDa microspherule protein (MSP58) protein is a candidate oncogene implicated in modulating cellular proliferation and malignant transformation. In this study, we show that knocking down MSP58 expression caused aneuploidy and led to apoptosis, whereas ectopic expression of MSP58 regulated cell proliferation in a context-dependent manner. Specifically, ectopic expression of MSP58 in normal human IMR90 and Hs68 diploid fibroblasts, the H184B5F5/M10 mammary epithelial cell line, HT1080 fibrosarcoma cells, primary mouse embryonic fibroblasts, and immortalized NIH3T3 fibroblasts resulted in induction of premature senescence, an enlarged and flattened cellular morphology, and increased senescence-associated β-galactosidase activity. MSP58-driven senescence was strictly dependent on the presence of functional p53 as revealed by the fact that normal cells with p53 knockdown by specific shRNA or cells with a mutated or functionally impaired p53 pathway were effective in bypassing MSP58-induced senescence. At least two senescence mechanisms are induced by MSP58. First, MSP58 activates the DNA damage response and p53/p21 signaling pathways. Second, MSP58, p53, and the SWI/SNF chromatin-remodeling subunit Brahma-related gene 1 (BRG1) form a ternary complex on the p21 promoter and collaborate to activate p21. Additionally, MSP58 protein levels increased in cells undergoing replicative senescence and stress-induced senescence. Notably, the results of analyzing expression levels of MSP58 between tumors and matched normal tissues showed significant changes (both up- and down-regulation) in its expression in various types of tumors. Our findings highlight new aspects of MSP58 in modulating cellular senescence and suggest that MSP58 has both oncogenic and tumor-suppressive properties.     

Preliminary crystallographic studies of lobster D-glyceraldehyde-3-phosphate dehydrogenase and the modified enzyme carrying the fluorescent derivative

When the active-site carboxymethylated D-glyceraldehyde-3-phosphate dehydrogenase is irradiated with ultraviolet light in the presence of NAD+, a fluorescent NAD derivative that is covalently linked to the enzyme is obtained. A preliminary crystallographic study of this fluorescent derivative, as well as of the native and the carboxymethylated enzymes from Palinurus versicolor, showed that they are isomorphous and belong to space group C2 as reported for the native enzyme from Palinurus vulgaris. The three forms of the enzyme, although they have identical unit cell parameters, differ considerably in their diffraction patterns, indicating marked differences in conformation in spite of the fact that they differ chemically only in a restricted region around the active site.     

Comparison of activity and conformation changes during refolding of urea-denatured creatine kinase

The course of the recovery of the enzymatic activity and the native conformation during the renaturation of urea-denatured creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) has been studied. Under suitable conditions, an activity recovery of 95% can be obtained and the reactivation follows a triphasic course. The initial two phases are relatively fast, whereas the slow phase takes some 24 h to reach completion. The recovery of the native conformation has been followed by changes in fluorescence, ultraviolet absorption and in exposed SH groups and has been shown to be a biphasic process. Both the reactivation and the refolding processes are independent of protein concentrations within a certain range, showing that the dimerization of the enzyme molecule is not rate-limiting. A comparison of the rate constants for the refolding of the molecule with those for the recovery of its catalytic activity shows that these are not synchronized and the activity recovery approaches completion after the refolding and dimerization of the subunits so far as can be detected by the methods employed. The final stage of refolding with complete activity recovery probably involves subtle conformational changes of the dimeric enzyme molecule not detectable by the physiochemical methods used in the present study.