Estrogenic regulation of skeletal muscle proteome: a study of premenopausal women and postmenopausal MZ cotwins discordant for hormonal therapy

Female middle age is characterized by a decline in skeletal muscle mass and performance, predisposing women to sarcopenia, functional limitations, and metabolic dysfunction as they age. Menopausal loss of ovarian function leading to low circulating level of 17β‐estradiol has been suggested as a contributing factor to aging‐related muscle deterioration. However, the underlying molecular mechanisms remain largely unknown and thus far androgens have been considered as a major anabolic hormone for skeletal muscle. We utilized muscle samples from 24 pre‐ and postmenopausal women to establish proteome‐wide profiles, associated with the difference in age (30–34 years old vs. 54–62 years old), menopausal status (premenopausal vs. postmenopausal), and use of hormone replacement therapy (HRT; user vs. nonuser). None of the premenopausal women used hormonal medication while the postmenopausal women were monozygotic (MZ) cotwin pairs of whom the other sister was current HRT user or the other had never used HRT. Label‐free proteomic analyses resulted in the quantification of 797 muscle proteins of which 145 proteins were for the first time associated with female aging using proteomics. Furthermore, we identified 17β‐estradiol as a potential upstream regulator of the observed differences in muscle energy pathways. These findings pinpoint the underlying molecular mechanisms of the metabolic dysfunction accruing upon menopause, thus having implications for understanding the complex functional interactions between female reproductive hormones and health.     

Lipid body formation during maturation of human mast cells

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.     

Identification of a novel streptococcal adhesin P (SadP) protein recognizing galactosyl-α1-4-galactose-containing glycoconjugates: convergent evolution of bacterial pathogens to binding of the same host receptor

Bacterial adhesion is often a prerequisite for infection, and host cell surface carbohydrates play a major role as adhesion receptors. Streptococci are a leading cause of infectious diseases. However, only few carbohydrate-specific streptococcal adhesins are known. Streptococcus suis is an important pig pathogen and a zoonotic agent causing meningitis in pigs and humans. In this study, we have identified an adhesin that mediates the binding of S. suis to galactosyl-α1-4-galactose (Galα1-4Gal)-containing host receptors. A functionally unknown S. suis cell wall protein (SSU0253), designated here as SadP (streptococcal adhesin P), was identified using a Galα1-4Gal-containing affinity matrix and LC-ESI mass spectrometry. Although the function of the protein was not previously known, it was recently identified as an immunogenic cell wall protein in a proteomic study. Insertional inactivation of the sadP gene abolished S. suis Galα1-4Gal-dependent binding. The adhesin gene sadP was cloned and expressed in Escherichia coli. Characterization of its binding specificity showed that SadP recognizes Galα1-4Gal-oligosaccharides and binds its natural glycolipid receptor, GbO(3) (CD77). The N terminus of SadP was shown to contain a Galα1-Gal-binding site and not to have apparent sequence similarity to other bacterial adhesins, including the E. coli P fimbrial adhesins, or to E. coli verotoxin or Pseudomonas aeruginosa lectin I also recognizing the same Galα1-4Gal disaccharide. The SadP and E. coli P adhesins represent a unique example of convergent evolution toward binding to the same host receptor structure.     

Kinetics of phase variation between S and type-1 fimbriae of Escherichia coli

Abstract The rate of fimbrial phase variation in Escherichia coli strain 3040 was determined. The strain has type-1 and S fimbriae. The bacterial culture was fractionated into homogeneous subpopulations expressing either one of the fimbrial types only; the subpopulations were inoculated into broth and the fimbriation of individual cells was assayed by immunofluorescence as a function of time. The rate of the shift from S- or type-1-fimbriate cells to non-fimbriate ones was of the order of 10⁻² per cell generation and more rapid than a direct shift from one fimbrial phase to another, although both types of phase variations were observed.     

Fractionation of a bacterial cell population by adsorption to erythrocytes and yeast cells

Abstract Fluorochrome-labeled antibodies specific for either S or type-1 fimbriae of Escherichia coli were used to show that in broth culture the two fimbrial types of strain 3040 mostly occurred on different cells. 12% of the cells were nonfimbriated. A fractionation procedure that involved adsorption of bacterial cells onto erythrocytes and yeast cells was developed to isolate homogeneous subpopulations (S-fimbriated, type-1-fimbriated, and non-fimbriated) of. E. coli. The level of contamination in each isolated subpopulation was 4% at the highest. The method is useful in obtaining homogeneous bacterial populations for adherence studies and for purification of specific fimbrial antigens.     

Restoration of natural vegetation in degraded Imperata cylindrica grassland: understorey development in forest plantations

Abstract. Reclamation of former, degraded forest lands occupied by Imperata cylindrica is one of the crucial environmental and forestry issues in the humid tropics, notably Southeast Asia. We suggest that it is possible to gradually restore the original natural forest cover with the help of a sacrifice fallow crop of fast-growing exotic tree species. Recently, a set of suitable fast-growing plantation tree species has been identified and stand establishment methods developed for this purpose. We assessed the regeneration of natural vegetation in stands of different plantation tree species and evaluated the ecological impact of species composition in the plantation understorey. PCA ordination, regression analysis and analysis of covariance were applied at different stages of the study. We found a marked vegetational resemblance between stands dominated by Acacia mangium: they had the highest number of indigenous trees in their understorey, whereas stands of other plantation trees supported more diverse grass and herb vegetation. A high proportion of evergreen woody vegetation reduces the risk of fire and grass competition and enhances secondary succession towards natural forest.     

Effects of mevinolin,an inhibitor of cholesterol synthesis,on the morphology and function of differentiating and differentiated rat adrenocortical cells in primary culture

Summary Mevinolin, an inhibitor of cholesterol synthesis, was used to study the effect of endogenous cholesterol synthesis on the morphology and function of differentiating and differentiated fetal rat adrenocortical cells grown in primary culture. Upon adrenocorticotrophic hormone (ACTH) stimulation under conditions in which endogenous cholesterol synthesis was inhibited but exogenous (lipoprotein) cholesterol was available, the cells differentiated normally from glomerulosa-like to fasciculata-like cells; the steroid hormone secretion was maximally induced. Under conditions in which cholesterol synthesis was maximally inhibited by mevinolin and the cells had no access to exogenous cholesterol, the cells did not differentiate into fasciculata-like cells; the ACTH-induced steroid response was highly suppressed under these conditions. The addition of either human low-density lipoprotein (LDL) or high-density lipoprotein (HDL₃) to the culture medium restored the ACTH-induced differentiation and steroid secretion. Thus, in the absence of exogenous cholesterol, endogenous cholesterol synthesis was a prerequisite for differentiation. In cultures grown in the presence of exogenous cholesterol and ACTH with mevinolin-inhibited cholesterol synthesis and high steroid output, an increase in cytoplasmic lipids was evident, suggesting upregulation of LDL and HDL receptors. The results also demonstrated that induction of phenotypic differentiation from glomerulosalike into fasciculata-like cells can proceed in the presence of a cholesterol synthesis inhibitor like mevinolin; this differentiation in the absence of endogenous cholesterol synthesis is accompanied by the appearance of cytoplasmic cholesterol ester droplets, typical of fasciculata cells.     

Low density lipoprotein degradation by secretory granules of rat mast cells. Sequential degradation of apolipoprotein B by granule chymase and carboxypeptidase A

The secretory granules of rat serosal mast cells are able efficiently to degrade the apolipoprotein B component of low density lipoproteins (LDL) Kokkonen, J. O., and Kovanen, P. T. (1985) J. Biol. Chem. 260, 14756-14763). The granules are known to contain two neutral proteases with complementary specificities: a chymotrypsin-like endopeptidase called chymase, and an exopeptidase, the granule carboxypeptidase A. The role of this enzyme pair in the proteolytic degradation of LDL was studied with the aid of specific enzyme inhibitors. Incubation of LDL with intact granules (both enzymes active) led to the formation of numerous low molecular weight peptides and the liberation of free amino acids, most of which (95%) were aromatic (Phe, Tyr, Trp) or branched-chain aliphatic (Leu, Ile, Val). Selective inhibition of granule carboxypeptidase A (leaving chymase active) blocked the liberation of free amino acids, but left the formation of peptides uninhibited. On the other hand, selective inhibition of granule chymase (leaving carboxypeptidase A active) totally abolished the proteolytic degradation of LDL. The results are consistent with a model according to which the proteolytic degradation of LDL by mast cell granules results from coordinated action of the two granule-bound enzymes, whereby the chymase first cleaves peptides from the apolipoprotein B of LDL, and thereafter the carboxypeptidase A cleaves amino acids from the peptides formed.