Release Of Hydrolytic Enzymes Research Articles

Lyososome Membrane Permeabilization Induces Plasma Membrane Macropores to Activate K +-Efflux-Dependent NLRP3 Inflammasomes in Murine Dendritic Cells

Abstract NLRP3 is a cytosolic sensor protein in myeloid leukocytes that recruits the adaptor protein ASC and activates caspase-1 in functional NLRP3 inflammasomes. Caspase-1 coordinately cleaves pro-IL-1β to bioactive IL-1β and gasdermin D (GSDMD) to induce N-GSDMD macropores in the plasma membrane (PM). The latter mediate efflux of mature IL-1β and initiate progression to pyroptosis, a lytic cell death. NLRP3 activation is triggered by perturbed cellular homeostasis with decreased cytosolic [K +] being the best-characterized signal. The required K +efflux is typically induced by the ionophore nigericin or extracellular ATP as an agonist for P2X7 receptor channels. K +efflux-dependent activation of NLRP3 is also triggered by soluble or particulate agents which accumulate in lysosomes to induce lysosomal membrane permeabilization (LMP) and release of hydrolytic enzymes into the cytosol; particulates include alum widely used as a vaccine adjuvant. The mechanisms by which LMP rapidly triggers changes in plasma membrane permeability remain undefined. We used a model of murine bone marrow derived dendritic cells (BMDC) treated with the soluble LMP agent Leu-Leu-O-methyl ester (LLME) to elicit K+ efflux and NLRP3 activation. Notably, this K+ efflux was coincident with a rapidly induced influx of propidiumiodide (PI), a 668 Da organic salt routinely used to track assembly of plasma membrane macropores such as GSDMD. Equivalent LLME-induced PI influx was observed in wildtype and Gsdmd−/−BMDC and was unaffected by inhibition of NLRP3 or caspase-1. These observations indicate that LMP triggers rapid activation of an endogenous pore-forming protein distinct from GSDMD. Current studies are evaluating gasdermin E or pannexin-1 as candidates. Supported by grants from the NIH (P01-AI141350 and R01-EY014362)

Niclosamide causes lysosome-dependent cell death in endometrial cancer cells and tumors.

Endometrial cancer is the most common female cancer showing continuous rise in its incidence and mortality rate. Despite the extensive research efforts in cancer therapeutics, still there is a lack of effective treatment options and the outcome is poor for patients with advanced and recurrent endometrial cancers. In this study, we aimed to evaluate the efficacy of niclosamide (NIC) against endometrial cancer. NIC is an FDA-approved anti-helminthic drug, which has been recently extensively studied as a potent anti-cancerous agent in several cancers. The anti-cancerous activity of NIC was analyzed in-vitro (ANC3A, Hec1B, and Ishikawa endometrial cancer cell lines) by cell viability-, soft agar-, invasion- and migration- assay. The action mechanism of NIC was demonstrated by western blot analysis and immune-fluorescence imaging and validated by specific inhibitors. The in-vivo efficacy of NIC was studied in the Ishikawa xenograft animal model. NIC effectively suppressed the viability (IC50<1 μM), colony formation ability, migration, and invasion of all endometrial cancer cells tested. We demonstrated that NIC inhibited AKT/mTOR signaling pathway and induced apoptosis and autophagy in endometrial cancer cells. Further study demonstrated that although NIC induced autophagosome formation, it inhibits autolysosome formation. In addition, we observed that NIC induced BAX co-localization with lysosome and inhibited Cathepsin B maturation from pro-cathepsin B, thereby inducing the lysosomal membrane permeability and release of hydrolytic enzymes from the lysosome to cytosol, which eventually contributed cell death. NIC also inhibited tumor weight and volume in the Ishikawa xenograft animal model without having any evidence of toxicity. Due to its potent anti-cancerous activity and safety profile, NIC seems to be a promising agent for human endometrial cancer therapeutics.

Eurotium cristatum produced β-hydroxy acid metabolite of monacolin K and improved bioactive compound contents as well as functional properties in fermented wheat bran

For the first time, wheat bran was fermented with Eurotium cristatum (E. cristatum), and alterations in the structural and functional characteristics were explored. The results showed that the soluble dietary fibre (SDF) content significantly increased in fermented bran, and the water and oil holding capacities improved. The total polyphenols, anthocyanin, and main flavour substance phenylethyl alcohol were significantly increased by E. cristatum fermentation. The ferulic acid content was approximately 12.06 times higher in fermented bran than in unfermented bran. These changes may be due to the release of hydrolytic enzymes during E. cristatum fermentation. E. cristatum fermentation enhanced the antioxidant and anti-obesity activities of bran. Furthermore, this study showing that E. cristatum fermentation produced β-hydroxy acid metabolites of monacolin K (MKA) in wheat bran. Thus, this research suggests that E. cristatum can be a starter culture to produce functional foods.

Вплив цинку на метаболічні процеси в нормі та при патології

Zinc belongs to the independent human trace elements. Its biological role is taken into account with the activity of attracting endocrine secretion. As a component of some enzymes, zinc affects the processes of reproduction, live and carbohydrate metabolism. Zinc is an important trace element with a variety of biological functions, which is involved in cell growth, apoptosis and metabolism, as well as in the regulation of endocrine, immune and neuronal functions. At the cellular level, zinc stimulates the formation of polysomes, inhibits free radical oxidation, which is catalyzed by iron. The important role of zinc in nucleic metabolism, transcription processes, stabilization of nucleic acids, proteins and especially components of biological membranes. Zinc plays an important role in skeletal development. Zinc accelerates the synthesis of collagen in wounds, has a stabilizing effect on cytoplasmic membranes, and prevents the release of hydrolytic enzymes. In mitochondria, zinc inhibits the transport of electrons in the respiratory chain. This property is realized in sperm, where there is a lot of zinc and there is low respiratory activity of mitochondria. Zinc is necessary for the development of brain tissue. Zincergic neurons are found in the cerebral cortex. Zinc has been shown to play an important role in signal transduction by modulating excitatory and inhibitory cellular receptors and intracellular signaling proteins. Zinc has a special effect on the development and course of many diseases, such as Down syndrome, alcoholism, angina, dysmenorrhea, atherosclerosis and more.

The release of hydrolytic enzymes by the salivary glands and their content in the blood after unilateral nephrectomy

We studied the mechanisms of transformation of some salivary enzymes and established the real contribution of the salivary glands to the enzymatic homeostasis of the body in unilateral nephrectomy.The results were obtained that with unilateral nephrectomy, the content of amylase and pepsinogen in the blood increases, but its lipolytic activity remains unchanged, the volume of basal secretion of the salivary glands, the content and release of amylase by the parotid salivary gland increases. Unilateral nephrectomy stimulates the increment of pepsinogen by the gastric glands, and, accordingly, enhances its recreation from the blood, by the salivary glands. After unilateral nephrectomy, lipolytic activity and its secretion in saliva remain unchanged

Glucosinolate catabolism during postharvest drying determines the ratio of bioactive macamides to deaminated benzenoids in Lepidium meyenii (maca) root flour

Postharvest processing of maca (Lepidium meyenii Walp., Brassicaceae), a traditional high-altitude Andean root crop, involves slow field drying prior to milling into flour. The progressive tissue dehydration and release of hydrolytic enzymes and substrates from cellular compartments results in the slow accumulation of free monosaccharides, fatty acids and amino acids. A more complex, and faster, kinetic profile is that of glucosinolate breakdown. A number of reactive transient and stable accumulation products are generated during drying, some of which have noteworthy bioactive properties. Among these are macamides, inhibitors of endocannabinoid neurotransmitter degradation in mammalian nervous systems. They result from the condensation of benzyl amine, a glucosinolate hydrolysis product, with free fatty acids released from lipid hydrolysis. Recent research has focused on developing drying processes under controlled conditions that can modulate the biochemistry of glucosinolate hydrolysis to optimize the content of bioactive compounds in the root flour. Low temperature (35 °C) oven-drying of shredded maca roots under controlled air flow generates benzyl amine as primary accumulation product, accounting for up to 94% of hydrolyzed glucosinolate in the flour. Kinetic evidence suggests that both deaminated benzenoids and macamides are allocated from the benzylamine pool through amine oxidase activity or condensation with free fatty acids, accounting for the remaining hydrolyzed glucosinolate (<5%). These activities determine the allocation to either one of these pathways. Later stages of dehydration result in shifts in the molar ratios of deaminated benzenoids, the accumulation of benzoic acid esters and benzyl alcohol. We propose that these are the result of changes in the rates of the reductive and oxidative half-reactions of endogenous aldehyde dehydrogenases. It is the ratio of benzylamine deamination to amide formation that determines the eventual yields of macamides in relation to benzenoids and their esters in maca flour.

Using a Model Lysosome Membrane to Study Nanomaterial-Membrane Interactions

Some engineered nanomaterials (ENM), phagocytosed by cells and transported to lysosomes can cause the release of hydrolytic enzymes into the cytosol of the cell, resulting in an inflammatory response and cytotoxicity. We designed model lipid systems to study interactions between ENM and lipid membranes to assess ENM-lipid interaction. Liposomes (100 nm) were generated from lipids found in lysosomes: bis(monooleoylglycero)phosphate (BMP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), in a ratio of 2:3:3. Tris-buffered saline (TBS, pH 7.4) and acetate-buffered saline (ABS, pH 4.5) was used to mimic the pH of early and late lysosomes. Liposome structure was evaluated using transmission electron microscopy; all preparations were ∼100 nm in diameter and unilamellar. Liposomes were exposure to two types of titanium dioxide (TiO2) ENM, nanospheres (TNS) and nanobelts (TNB), or zinc oxide (ZnO) nanospheres for 2 hours at 25, 100 or 200 µg/ml. Time-resolved fluorescence anisotropy was performed using the fluorescence probe di-4-ANEPPDHQ and a custom-built time-resolved fluorometer to measure changes in membrane fluidity (reported in cone angle). Liposomes (BMP:DOPC:DOPE) exposed to TNS (100 µg/ml) showed no difference in cone angle compared to control in either buffer. However, TNB exposure produced significant decreases in cone angle in TBS at 200 µg/ml (36.42±0.31° to 32.84±0.33°) and in ABS at both 100 and 200 µg/ml (34.92±0.52° to 34.30±0.83° and 30.88±0.12°). Even though the surface area of TNS was greater than TNB the lack of effect supported that shape was important. ZnO ENM exposed liposomes composed of BMP:DOPC:DOPE showed no significant change in cone angle (100 µg/ml). However, BMP:DOPC:DOPS liposome had a significant decrease in cone angle (37.60±0.35° to 34.12±0.30°), indicating lipid headgroup can influence interactions. Funding: NIH R01ES023209, 1F32ES027324, P20GM103546, P30GM103338 and J Murdock Charitable Trust.

In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets

Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets.The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperature-dependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.

Occurrence of autophagy during pioneer root and stem development in Populus trichocarpa

Main conclusionAutophagy is involved in developmentally programmed cell death and is identified during the early development of phloem, as well as xylem with a dual role, as both an inducer and executioner of cell death.The regulation of primary and secondary development of roots and stems is important for the establishment of root systems and for the overall survival of trees. The molecular and cellular basis of the autophagic processes, which are used at distinct moments during the growth of both organs, is crucial to understand the regulation of their development. To address this, we use Populus trichocarpa seedlings grown in a rhizotron system to examine the autophagy processes involved in root and stem development. To monitor the visual aspects of autophagy, transmission electron microscopy (TEM) and immunolocalization of AuTophaGy-related protein (ATG8) enabled observations of the phenomenon at a structural level. To gain further insight into the autophagy process at the protein and molecular level, we evaluated the expression of ATG gene transcripts and ATG protein levels. Alternations in the expression level of specific ATG genes and localization of ATG8 proteins were observed during the course of root or stem primary and secondary development. Specifically, ATG8 was present in the cells exhibiting autophagy, during the differentiation and early development of xylem and phloem tissues, including both xylary and extraxylary fibers. Ultrastructural observations revealed tonoplast invagination with the formation of autophagic-like bodies. Additionally, the accumulation of autophagosomes was identifiable during the differentiation of xylem in both organs, long before the commencement of cell death. Taken together, these results provide evidence in support of the dual role of autophagy in developmental PCD. A specific role of the controller of cell death, which is a committed step with the release of hydrolytic enzymes from the vacuole and final digestion of protoplast, from which there is no return once initiated, is only attributed to mega-autophagy.

English

Trichoderma, is a useful, filamentous fungi and they are cosmopolitan in nature which have attracted the attention because of their multi prong action against various plant pathogens. There are several biocontrol mechanism employed by Trichoderma against plant pathogens, these include release of hydrolytic enzymes that degrade cell wall of phytopathogenic fungi, competition for nutrients, parasitism, and antibiotics. Trichoderma spp. generally grows in its natural habitat on plant root surface and therefore it controls root diseases in particular. Key words: Trichoderma, anatagonism, phytopathogens.

Responses of soil microeukaryotic communities to short-term fumigation-incubation revealed by MiSeq amplicon sequencing.

In soil microbiology, there is a “paradox” of soil organic carbon (SOC) mineralization, which is that even though chloroform fumigation destroys majority of the soil microbial biomass, SOC mineralization continues at the same rate as in the non-fumigated soil during the incubation period. Soil microeukaryotes as important SOC decomposers, however, their community-level responses to chloroform fumigation are not well understood. Using the 18S rRNA gene amplicon sequencing, we analyzed the composition, diversity, and C-metabolic functions of a grassland soil and an arable soil microeukaryotic community in response to fumigation followed by a 30-day incubation. The grassland and arable soil microeukaryotic communities were dominated by the fungal Ascomycota (80.5–93.1% of the fungal sequences), followed by the protistan Cercozoa and Apicomplexa. In the arable soil fungal community, the predominance of the class Sordariomycetes was replaced by the class Eurotiomycetes after fumigation at days 7 and 30 of the incubation. Fumigation changed the microeukaryotic α-diversity in the grassland soil at days 0 and 7, and β-diversity in the arable soil at days 7 and 30. Network analysis indicated that after fumigation fungi were important groups closely related to other taxa. Most phylotypes (especially Sordariomycetes, Dothideomycetes, Coccidia, and uncultured Chytridiomycota) were inhibited, and only a few were positively stimulated by fumigation. Despite the inhibited Sordariomycetes, the fumigated communities mainly consisted of Eurotiomycetes and Sordariomycetes (21.9 and 36.5% relative frequency, respectively), which are able to produce hydrolytic enzymes associated with SOC mineralization. Our study suggests that fumigation not only decreases biomass size, but modulates the composition and diversity of the soil microeukaryotic communities, which are capable of driving SOC mineralization by release of hydrolytic enzymes during short-term fumigation-incubation.

Lysosomotropic agents: impact on lysosomal membrane permeabilization and cell death.

Lysosomes are acidic organelles essential for degradation, signalling and cell homoeostasis. In addition, they play a key role in cell death. Permeabilization of the lysosomal membrane and release of hydrolytic enzymes to the cytosol accompanies apoptosis signalling in several systems. The regulatory mechanism of lysosomal stability is, however, poorly understood. Lipophilic or amphiphilic compounds with a basic moiety will become protonated and trapped within lysosomes, and such lysosomotropic behaviour is also found in many pharmacological drugs. The natural sphingolipid sphingosine exhibits lysosomotropic detergent ability and is an endogenous candidate for controlling lysosomal membrane permeabilization. The lysosomotropic properties of certain detergents might be of use in lysosome-targeting anticancer drugs and drug delivery system in the future. The present review summarizes the current knowledge on the targeting and permeabilizing properties of lysosomotropic detergents from a cellular and physicochemical perspective.

Cancer Drugs Inhibit Germ Tube Elongation in Candida albicans

Aim: Hyphal forms of human fungal pathogen Candida albicans play a major role in infection process. During mucosal infections, they invade epithelial and endothelial cells and cause damage probably through the release of hydrolytic enzymes. Targeting yeast to hyphal form morphogenetic switching may be useful against invasive candidiasis. In this study, we report the germ tube elongation inhibitory properties of thirty cancer drugs from 12 different classes. Methodology: Candida albicans, ATCC 90028 was used throughout the study and Candida filamentation assay was performed on polystyrene coated 96-well microtitre plates. Results: Most of the drugs inhibited elongation at concentrations lower than their minimum Inhibitory Concentration. These concentrations were much lower than prescribed doses. The drugs were found to inhibit Candida growth at higher concentrations, suggesting that chemotherapy with anticancer drugs may not favor invasive candidiasis caused by C. albicans. Conclusion: Our study indicates the possibility of repositioning cancer drugs as antimorphogenetic agents in C. albicans. Research Article Routh et al.; JSRR, Article no. JSRR.2014.003 98

Concurrent Cryptosporidium and Candida infections in a buffalo calf (Bubalus bubalis)

Cryptosporidiosis and candidiasis are opportunistic infections of neonates. An investigation was contemplated to study the enteric lesions associated with concurrent infections by the pathogens in a male bubaline calf, aged 8 weeks, which died on March 23, 2010. The gross lesions were comprised of hyperplastic changes and erosion of the microvillus surface of the enteric epithelium. The histological sections revealed invasion of the mucosal cells by two types (Cryptosporidium spp. and Candida spp.) of pathogens. The deeper layers of the intestine did not reveal appreciable architectural changes, except for the presence of inflammatory cellular elements in the lamina propria. The sections stained with Ziehl–Neelsen revealed bright red, oval to spherical bodies (3–5 μm), and those stained with Gomori's Methanamine silver nitrate revealed black-colored fungal spores and pseudohyphae. Based on morphological and in vitro cultural characteristics, the pathogens were identified as Cryptosporidium spp. and Candida spp., respectively. The lesions seem to be consequential events to synergistic invasion of the enterocytes by the pathogens. Complex changes in the microenvironment of the gut, incidental to in situ release of hydrolytic enzymes, toxins, and products of host–parasite interaction liberated at the site of infection by the pathogens, have been reported and discussed.

Can we teach an old drug new tricks?

Although resistance to chloroquine (CQ) has relegated it from modern chemotherapeutic strategies to treat Plasmodium falciparum malaria, new evidence suggests that higher doses of the drug may exert a different killing mechanism and offers this drug a new lease of life. Whereas the established antimalarial mechanisms of CQ are usually associated with nanomolar levels of the drug, micromolar levels of CQ trigger a distinct cell death pathway involving the permeabilization of the digestive vacuole of the parasite and a release of hydrolytic enzymes. In this paper, we propose that this pathway is a promising antimalarial strategy and suggest that revising the CQ treatment regimen may elevate blood drug levels to trigger this pathway without increasing the incidence of adverse reactions.

Ultrastructural aspects and programmed cell death in the tapetal cells ofLathyrus undulatusBoiss

Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporogenous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage.

Human PrP90-231-induced cell death is associated with intracellular accumulation of insoluble and protease-resistant macroaggregates and lysosomal dysfunction

To define the mechanisms by which hPrP90-231 induces cell death, we analyzed its interaction with living cells and monitored its intracellular fate. Treatment of SH-SY5Y cells with fluorescein-5-isothiocyanate (FITC)-conjugated hPrP90-231 caused the accumulation of cytosolic aggregates of the prion protein fragment that increased in number and size in a time-dependent manner. The formation of large intracellular hPrP90-231 aggregates correlated with the activation of apoptosis. hPrP90-231 aggregates occurred within lysotracker-positive vesicles and induced the formation of activated cathepsin D (CD), indicating that hPrP90-231 is partitioned into the endosomal–lysosomal system structures, activating the proteolytic machinery. Remarkably, the inhibition of CD activity significantly reduced hPrP-90-231-dependent apoptosis. Internalized hPrP90-231 forms detergent-insoluble and SDS-stable aggregates, displaying partial resistance to proteolysis. By confocal microscopy analysis of lucifer yellow (LY) intracellular partition, we show that hPrP90-231 accumulation induces lysosome destabilization and loss of lysosomal membrane impermeability. In fact, although control cells evidenced a vesicular pattern of LY fluorescence (index of healthy lysosomes), hPrP90-231-treated cells showed diffuse cytosolic fluorescence, indicating LY diffusion through damaged lysosomes. In conclusion, these data indicate that exogenously added hPrP90-231 forms intralysosomal deposits having features of insoluble, protease-resistant aggregates and could trigger a lysosome-mediated apoptosis by inducing lysosome membrane permeabilization, followed by the release of hydrolytic enzymes.

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O 2 − and HO − and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O 2 − or HO −. Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.

Very Long-chain Polyunsaturated Fatty Acids Are the Major Acyl Groups of Sphingomyelins and Ceramides in the Head of Mammalian Spermatozoa

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups ( approximately 70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 degrees C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.

Surfactant disaturated-phosphatidylcholine kinetics in acute respiratory distress syndrome by stable isotopes and a two compartment model

BackgroundIn patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes.MethodsWe studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of 13C-dipalmitoyl-phosphatidylcholine, we measured the 13C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5–100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds.ResultsIn ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls (0.16 ± 0.04 vs. 1.31 ± 0.40 mg/kg, p < 0.05). Fluxes between tissue and alveoli and de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 ± 3.5 in ARDS and 31.9 ± 7.3 in controls (p = 0.08).ConclusionIn ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered.