Ultrastructure of the corpus luteum of antarctic seals during pregnancy

Summary The fine structure of granulosa lutein cells from three crabeater seals, Lobodon carcinophagus, and two leopard seals, Hydrurga leptonyx, has been studied from early through mid-pregnancy. Analysis of the arrangement and modifications of the cytoplasmic organelles and inclusions has revealed three types of lutein cells throughout the corpus. Type I cell typically possesses a central nucleus and cytoplasm containing very large amounts of smooth and/or fenestrated endoplasmic cisternae which frequently extend from the juxta-nuclear to the periphery of the cell. Type II cell contains a central or eccentric nucleus, moderate amounts of peripheral, smooth and fenestrated cisternae which often form large and concentric membranous whorls, numerous mitochondria and small lipid droplets. Frequently these cells show polarity in the arrangement of the cytoplasmic organelles and inclusions. Type III cell contains predominant large lipid droplets, many mitochondria, and small amounts of smooth and fenestrated cisternae. In light microscopy the type I cell is evenly granular, while the type III cell is highly vacuolated. Type II cells have both granular and vacuolated conditions. Ultrastructural features of type I and II cells suggest that they probably secrete most of the steroids, whereas the primary role of the type III cells appear to be lipid storage.This research was supported by National Science Foundation, Grant No. 1325 from the Office of Antarctic Biology.     

Liquid-ordered microdomains in lipid rafts and plasma membrane of U-87 MG cells: a time-resolved fluorescence study

Lipid rafts, the functional microdomains in the cell membrane, are believed to exist as liquid-ordered (Lo) phase domains along with the liquid-disordered (Ld) phase of the bulk of the cell membranes. We have examined the lipid order in model and natural membranes by time-resolved fluorescence of trimethylammonium-1,6-diphenylhexatriene incorporated into the membranes. The lipid phases were discerned by the limiting anisotropy, rotational diffusion rate and distribution of the fluorescence lifetime. In dipalmitoylphosphatidylcholine (DPPC)-cholesterol mixtures the gel phase exhibited higher anisotropy and a two-fold slower rotational diffusion rate of the probe as compared to the Ld phase. On the other hand, the Lo phase exhibited higher limiting anisotropy but a rotational diffusion rate comparable to the Ld phase. The Ld and Lo phases elicited unimodal distribution of lifetimes with distinct mean values and their co-existence in phospholipid-cholesterol mixtures was reflected as a biphasic change in the width of the lifetime distribution. Global analysis of the lifetimes yielded a best fit with two lifetimes which were identical to those observed in single Lo or Ld phases, but their fractional contribution varied with cholesterol concentration. Attributing the shorter and longer lifetime components to the Ld and Lo phases, respectively, the extent of the Lo/Ld phase domains in the membranes was estimated by their fractional contribution to the fluorescence decay. In ternary mixtures of egg PC-gangliosides-cholesterol, the gangliosides induced heterogeneity in the membrane but the Ld phase prevailed. The Lo phase properties were observed only in the presence of cholesterol. Results obtained in the plasma membrane and detergent-resistant membrane fractions (DRMs) isolated from U-87 MG cells revealed that DRMs mainly possess the Lo phase; however, a substantially large proportion of plasma membrane also exists in the Lo phase. Our data show that, besides cholesterol, the membrane proteins play a significant role in the organization of lipid rafts and, furthermore, a considerable amount of heterogeneity is present among the lipid rafts.     

Staphylococcus aureus alpha-toxin-induced cell death: predominant necrosis despite apoptotic caspase activation

Recent data suggest that alpha-toxin, the major hemolysin of Staphylococcus aureus, induces cell death via the classical apoptotic pathway. Here we demonstrate, however, that although zVAD-fmk or overexpression of Bcl-2 completely abrogated caspase activation and internucleosomal DNA fragmentation, they did not significantly affect alpha-toxin-induced death of Jurkat T or MCF-7 breast carcinoma cells. Caspase inhibition had also no effect on alpha-toxin-induced lactate dehydrogenase release and ATP depletion. Furthermore, whereas early assessment of apoptosis induction by CD95 resulted solely in the generation of cells positive for active caspases that were, however, not yet permeable for propidium iodide, a substantial proportion of alpha-toxin-treated cells were positive for both active caspases and PI. Finally, electron microscopy demonstrated that even in the presence of active caspases, alpha-toxin-treated cells displayed a necrotic morphology characterized by cell swelling and cytoplasmic vacuolation. Together, our data suggest that alpha-toxin-induced cell death proceeds even in the presence of activated caspases, at least partially, in a caspase-independent, necrotic-like manner.     

Effect of organic solvents on the structure and activity of moderately halophilic Bacillus sp. EMB9 protease

Halophilic enzymes have been manifested for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation in presence of high temperature, pH, presence of organic solvents and chaotropic agents. The present study aims at understanding the stability and activity of a halophilic Bacillus sp. EMB9 protease in organic solvents. The protease was uniquely stable in polar solvents. A clear correlation was evident between the protease function and conformational transitions, validated by CD and fluorescence spectral studies. The study affirms that preservation of protein structure, possibly due to charge screening of the protein surface by Ca²⁺ and Na⁺ ions provides stability against organic solvents and averts denaturation. Salt was also found to exert a protective effect on dialyzed protease against chaotropism of solvents. Presence of 1 % (w/v) NaCl restored the activity in the dialyzed protease and prevented denaturation in methanol, toluene and n-decane. The work will have further implication on discerning protein folding in saline as well as non-aqueous environments.     

Basic trypsin-subtilisin inhibitor from marine turtle egg white: Hydrodynamic and inhibitory properties

A basic trypsin-subtilisin inhibitor has been isolated from the egg white of marine turtle (Caretta caretta Linn.) and purified to homogeneity by gel filtration followed by ion-exchange chromatography. It has a single polypeptide chain of 117 amino acid residues, having a molecular weight of 13,600. It lacks methionine and tryptophan. Its isoelectric point is atpH 10.0 and the sedimentation coefficient (s_20,w) value of 1.62 S is independent of protein concentration. It has a Stokes radius of 18.8 Å, an intrinsic viscosity of 0.048 dl g^–1 and a diffusion coefficient of 10.17×10^–7 cm² sec^–1. Its fluorescence emission spectrum is similar to that of free tyrosine and the bimolecular quencing rate constant of its tyrosine residues with acrylamide is 3.15×10⁹ M^–1 sec^–1. The inhibitor strongly inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio of unity. The nature of inhibition toward both enzymes is not temporary. It has independent binding sites for inhibition of trypsin and subtilisin. Chemical modification with tetranitromethane suggests the presence of three tyrosine residues on the surface of the inhibitor molecule.     

Significant Expression Levels of Transgenic PPP1CC2 in Testis and Sperm Are Required to Overcome the Male Infertility Phenotype of Ppp1cc Null Mice

PPP1CC2, one of four isoforms of the ser/thr protein phosphatase PP1, is a mammalian-specific splice variant of the Ppp1cc gene, and the only isoform whose expression is confined almost completely to spermatogenic cells. Additionally, PPP1CC2 is the sole isoform found in mammalian spermatozoa. Although PPP1CC1, the other Ppp1cc product, is expressed in many tissues including testis, the only phenotype resulting from deletion of Ppp1cc gene is male infertility. To determine which of the products of Ppp1cc is essential for male fertility, we created two PPP1CC2 transgenes, eTg-G2 and pTg-G2, where Ppp1cc2 expression was driven by the putative endogenous promoter of Ppp1cc or by the testis specific human Pgk2 promoter, respectively. Our results demonstrate that the 2.6-kb genomic region directly upstream of the Ppp1cc structural gene can drive expression of Ppp1cc2, and recapitulate the wild-type tissue specificity of PPP1CC2 in transgenic mice. More importantly, we show that expression of PPP1CC2 alone, via either promoter, is able not only to restore normal spermatogenesis, but the fertility of Ppp1cc null mice as well, provided that transgenic PPP1CC2 expression in testis reaches at least a lower threshold level equivalent to approximately 50% of its expression by a Ppp1cc +/− male. We conclude that the endogenous Ppp1cc promoter normally functions in the testis to maintain a sufficient level of PPP1CC2 expression for normal spermatogenesis to occur, and that production of spermatozoa capable of fertilization in vivo can take place in the complete absence of PPP1CC1 expression.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Conformational flexibility of BECN1: Essential to its key role in autophagy and beyond

BECN1 (Beclin 1), a highly conserved eukaryotic protein, is a key regulator of autophagy, a cellular homeostasis pathway, and also participates in vacuolar protein sorting, endocytic trafficking, and apoptosis. BECN1 is important for embryonic development, the innate immune response, tumor suppression, and protection against neurodegenerative disorders, diabetes, and heart disease. BECN1 mediates autophagy as a core component of the class III phosphatidylinositol 3‐kinase complexes. However, the exact mechanism by which it regulates the activity of these complexes, or mediates its other diverse functions is unclear. BECN1 interacts with several diverse protein partners, perhaps serving as a scaffold or interaction hub for autophagy. Based on extensive structural, biophysical and bioinformatics analyses, BECN1 consists of an intrinsically disordered region (IDR), which includes a BH3 homology domain (BH3D); a flexible helical domain (FHD); a coiled‐coil domain (CCD); and a β‐α‐repeated autophagy‐specific domain (BARAD). Each of these BECN1 domains mediates multiple diverse interactions that involve concomitant conformational changes. Thus, BECN1 conformational flexibility likely plays a key role in facilitating diverse protein interactions. Further, BECN1 conformation and interactions are also modulated by numerous post‐translational modifications. A better structure‐based understanding of the interplay between different BECN1 conformational and binding states, and the impact of post‐translational modifications will be essential to elucidating the mechanism of its multiple biological roles.