John Ding-E Young

Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity

A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.

Ionophore-induced apoptosis: Role of DNA fragmentation and calcium fluxes

Two ionophores specific for K+, valinomycin and beauvericin, induce a type of cell death very similar to apoptosis due to tumor necrosis factor (TNF alpha). Both ionophores cause cytolysis accompanied by internucleosomal DNA fragmentation of the dying cell into units of 200 base pairs. Morphologically, the cell death appears to consist of a mixture of nuclear apoptotic changes and cytoplasmic necrotic changes. As in the case for TNF alpha-mediated death, metabolic inhibitors have no effect on the course of cell death, but DNA fragmentation and cytolysis are decreased by the endonuclease inhibitor, zinc. Beauvericin and valinomycin trigger an increase in the cytoplasmic calcium concentration, most likely due to release of calcium from intracellular stores, and chelation of cytoplasmic calcium with quin-2 inhibits DNA fragmentation. Thus, these ionophores set off apoptosis through a calcium-activatable endonuclease, suggesting that other nonphysiological toxins might also cause apoptosis through their ability to indirectly elevate the cytoplasmic calcium concentration, without the need to invoke specific surface receptors.

Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota

Obesity is associated with low-grade chronic inflammation and intestinal dysbiosis. Ganoderma lucidum is a medicinal mushroom used in traditional Chinese medicine with putative anti-diabetic effects. Here, we show that a water extract of Ganoderma lucidum mycelium (WEGL) reduces body weight, inflammation and insulin resistance in mice fed a high-fat diet (HFD). Our data indicate that WEGL not only reverses HFD-induced gut dysbiosis—as indicated by the decreased Firmicutes-to-Bacteroidetes ratios and endotoxin-bearing Proteobacteria levels—but also maintains intestinal barrier integrity and reduces metabolic endotoxemia. The anti-obesity and microbiota-modulating effects are transmissible via horizontal faeces transfer from WEGL-treated mice to HFD-fed mice. We further show that high molecular weight polysaccharides (>300 kDa) isolated from the WEGL extract produce similar anti-obesity and microbiota-modulating effects. Our results indicate that G. lucidum and its high molecular weight polysaccharides may be used as prebiotic agents to prevent gut dysbiosis and obesity-related metabolic disorders in obese individuals.

Isolation and characterization of a serine esterase from cytolytic T cell granules

Cytotoxic T lymphocytes and lymphocytes with NK-like activity contain a serine esterase activity which has been localized to their cytoplasmic granules by cytochemistry and subcellular fractionation studies. The serine esterase-specific inhibitor 3H-DFP labels two protein species in the granules. The two proteins, referred to as serine esterases 1 and 2 (SE 1 and SE 2), migrate with Mr of 34-36 kd and 28-30 kd, respectively, under reducing conditions. SE 1 shows trypsin-like activity and has been purified to apparent homogeneity. Under nonreducing conditions, SE 1 has an Mr of 60-66 kd, suggesting that it may consist of two disulfide-linked subunits of 34-36 kd each. SE 1 cleaves fibrin and casein, has a pl greater than 10, and optimal activity at pH 8. The substrate specificity of SE 2 is not known. The serine esterase activity is secreted by lymphocytes that have been stimulated with the calcium ionophore A23187. The serine esterases described here could play an active role in cell-mediated killing.

Purported nanobacteria in human blood as calcium carbonate nanoparticles

Recent evidence suggests a role for nanobacteria in a growing number of human diseases, including renal stone formation, cardiovascular diseases, and cancer. This large body of research studies promotes the view that nanobacteria are not only alive but that they are associated with disease pathogenesis. However, it is still unclear whether they represent novel life forms, overlooked nanometer-size bacteria, or some other primitive self-replicating microorganisms. Here, we report that CaCO3 precipitates prepared in vitro are remarkably similar to purported nanobacteria in terms of their uniformly sized, membrane-delineated vesicular shapes, with cellular division-like formations and aggregations in the form of colonies. The gradual appearance of nanobacteria-like particles in incubated human serum as well as the changes seen with their size and shape can be influenced and explained by introducing varying levels of CO2 and NaHCO3 as well as other conditions known to influence the precipitation of CaCO3. Western blotting reveals that the monoclonal antibodies, claimed to be specific for nanobacteria, react in fact with serum albumin. Furthermore, nanobacteria-like particles obtained from human blood are able to withstand high doses of γ-irradiation up to 30 kGy, and no bacterial DNA is found by performing broad-range PCR amplifications. Collectively, our results provide a more plausible abiotic explanation for the unusual properties of purported nanobacteria.

Functional assembly of gap junction conductance in lipid bilayers: demonstration that the major 27 kd protein forms the junctional channel.

Gap junctions isolated from rat liver were incorporated into planar lipid bilayers. A channel activity that was directly dependent on voltage was recorded. Changes of pH and (Ca2+) had no direct effect on channel activity; however, they modulated the voltage-dependent gating of the gap junction channels differently. Single-channel fluctuations showed large scatter with peak amplitudes of 140 and 280 picoSiemmens in 0.1 M NaCl. The major protein of gap junctions (Mr of 27 kd) was also reconstituted into bilayers, giving channel properties similar to those of intact gap junctions. Polyclonal antibodies specific for this protein caused inhibition of the junctional conductance in bilayers. These data provide direct evidence that the 27 kd protein is the molecular species responsible for gap junction communication between cells.

Identification, isolation, and characterization of a novel cytotoxin in murine cytolytic lymphocytes

Murine cytotoxic T lymphocytes contain, in addition to the cytotoxic pore-forming protein perforin, another cytolytic factor localized in both cytoplasm and granules. Like perforin, this CTL cytotoxin lyses a variety of tumor cells; unlike perforin, it is stable in the presence of calcium, requires several hours to induce maximal lytic activity, and is antigenically related to the previously described tumor necrosis factor (TNF) and lymphotoxin (LT). However, it differs from TNF and LT in a number of biochemical and functional properties. TNF- and LT-specific cDNA probes did not hybridize with any CTL-specific message, indicating that the CTL cytotoxin is distinct from those two factors. It has an apparent Mr of 50 and 70 kd under reducing and nonreducing conditions, respectively, is secreted by secretagogue-stimulated CTLs, and causes DNA fragmentation in several targets, a phenomenon previously attributed to target cell damage by CTLs. These results suggest that killing by lymphocytes may encompass multiple mechanisms and polypeptides.

Cellular and Humoral Mechanisms of Cytotoxicity: Structural and Functional Analogies

Publisher Summary Target cell (TC) killing, however, is not restricted to immune cells and has been documented for various cell types, including bacteria, fungi, yeast, plant cells, and protozoan parasites. Perhaps one denominator common to all these different cytotoxic cell types and their killing machineries is the involvement of soluble cytotoxic mediators that are secreted by the killer cell and used to lyse the target. The increased interest in these soluble mediators of cell killing, generally referred to as “cytotoxins,” has been greatly stimulated in part by the feasibility of their isolation in the laboratory in high yields and their further characterization by conventional biochemical techniques. This chapter focuses on the molecular mechanisms of membrane. The surprising functional analogies between the cytotoxin released by lymphocytes and the better known toxins found in certain bacteria and insects, for example, should greatly stimulate and aid future studies on the cytotoxic reaction mediated by immune cells.

Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

Putative living entities called nanobacteria (NB) are unusual for their small sizes (50–500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described earlier as NB may thus represent remnants and by-products of physiological mechanisms used for calcium homeostasis, a concept which explains the vast body of NB literature as well as explains the true origin of NB as lifeless protein-mineralo entities with questionable role in pathogenesis.

Perforin‐Dependent and ‐Independent Pathways of Cytotoxicity Mediated by Lymphocytes

There is little doubt at the present time that both perforin-dependent and -independent pathways are important in mediating the cytotoxicity associated with lymphocytes. The cell distribution of perforin, initially thought to include both CTL and NK cells, now must be viewed with caution because all previous biochemical studies on CTL have been conducted with cell lines propagated in long-term cultures in the presence of T cell growth factors (IL-2 and perhaps some still undefined factors). Under these conditions, CTL are known to assume a broader, NK-like specificity in target cell killing and may thus differ significantly from primary CTL generated in the body. Accordingly, perforin does not seem to be present in primary CTL activated directly through mixed lymphocyte reactions. It remains to be shown how primary CTL lyse target cells in vivo. Initial studies conducted in several laboratories have already provided some clues. It now seems that even in cultured, perforin-containing CTL, the perforin pathway is not an obligatory mechanism required for target cell killing. Other pathways, possibly involving TNF/lymphotoxin-like molecules, may play a direct role in this type of cytotoxicity. Other still unidentified factors now also need to be sought, including membrane polypeptides that may develop cytotoxicity directly upon cell contact and binding. Although from the studies reviewed here it is clear now that perforin has a more limited role in cell killing than originally proposed, it is still intriguing that it should share structural and functional homologies with complement proteins, drawing paradoxical analogies between two systems (the cellular and the humoral immune systems) which have evolved to become specialized to carry out separate immunological tasks. The cloning of the genes for perforin and for all the C proteins that comprise the MAC should reveal important information on how these genes originated and then diverged during evolution. The cellular distribution of other granule products, such as serine esterases, also must be viewed with caution. A serine esterase activity was initially thought to be CTL-specific. This information stimulated an intensive research activity in many laboratories that resulted in both the purification of a serine esterase family and the cloning of several serine esterase transcripts. It is becoming clear from recent evidence that this group of enzymes is not truly CTL-specific and therefore would not be expected to develop any function rendered absolutely necessary for cytolysis.(ABSTRACT TRUNCATED AT 400 WORDS)