Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.     

Secretory cell activity in the hamster seminal vesicle following castration. A morphometric ultrastructural study

The ultrastructure of hamster seminal vesicle epithelium was studied 7, 14, 21 and 28 days after castration using a stereological approach. The results show that castration promotes epithelial reorganization, mainly characterized by reduced epithelial cell size and number, decreased rough endoplasmic reticulum and Golgi complex, increased lysosomes and lipid droplets, increased apical secretory granule size and number, and increased intracellular secretory products per average epithelial cell. It is concluded that after testosterone withdrawal the secretory activity of hamster seminal vesicle epithelial cells, although reduced, is not abolished, and that exocytosis is relatively more reduced than secretory protein production. We suggest that an extracellular androgen source is responsible for secretory activity not being lost in the epithelial cells of castrated hamster seminal vesicle.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Purification of Tissue Forms (Amastigotes) of Trypanosoma cruzi by Immunoaffinity Chromatography1

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Scpharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.     

2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties

2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Ascaris metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 degrees C. The molecular weight of the native protein (Mr = 64,000 by exclusion chromatography) and the size of the subunit (Mr = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.     

13C NMR Analysis of some simple tetrahydroisoquinolines

¹³C NMR resonances of 15 simple tetrahydroisoquinolines have been assigned on the basis of chemical shift theory, ¹³C-¹H coupling constants     

Fecal Microflora in Healthy Persons in a Preindustrial Region

Procedures for quantitating the fecal microflora of man were described. Special attention was given to criteria for characterizing the culturable aerobic, Micro-aerophilic, and anaerobic bacteria. Three groups of healthy persons were studied: wholly breast-fed infants (2 to 4 month-olds), weanlings (1 to 2 year-olds), and adults. In breast-fed children, bifidobacteria predominate and outnumber by one or several logs all other culturable bacteria. The fecal flora of wholly breast-fed infants is "simpler" and more numerous [10(11) to 10(12) per g (wet weight) of feces than that of the adult 10(2) to 10(11) per g]. In the adult, gram-negative anaerobic bacilli (bacteroides) outnumber all other groups by a factor of 1 log or more. Weanlings receiving an adult-type diet, but still breast-fed, showed a flora intermediate between that of the wholly breast-fed infant and that of the adult, but more similar to the latter. Anaerobes always constitute the predominant component of the culturable flora of children and adults and are always found in large numbers under conditions of health. The aerobes are significantly less numerous, and vary widely in their number and in the frequency with which they appear in feces.     

Cryptophytes: An emerging algal group in the rapidly changing Antarctic Peninsula marine environments

The western Antarctic Peninsula (WAP) is a climatically sensitive region where foundational changes at the basis of the food web have been recorded; cryptophytes are gradually outgrowing diatoms together with a decreased size spectrum of the phytoplankton community. Based on a 11-year (2008–2018) in-situ dataset, we demonstrate a strong coupling between biomass accumulation of cryptophytes, summer upper ocean stability, and the mixed layer depth. Our results shed light on the environmental conditions favoring the cryptophyte success in coastal regions of the WAP, especially during situations of shallower mixed layers associated with lower diatom biomass, which evidences a clear competition or niche segregation between diatoms and cryptophytes. We also unravel the cryptophyte photo-physiological niche by exploring its capacity to thrive under high light stress normally found in confined stratified upper layers. Such conditions are becoming more frequent in the Antarctic coastal waters and will likely have significant future implications at various levels of the marine food web. The competitive advantage of cryptophytes in environments with significant light level fluctuations was supported by laboratory experiments that revealed a high flexibility of cryptophytes to grow in different light conditions driven by a fast photo-regulating response. All tested physiological parameters support the hypothesis that cryptophytes are highly flexible regarding their growing light conditions and extremely efficient in rapidly photo-regulating changes to environmental light levels. This plasticity would give them a competitive advantage in exploiting an ecological niche where light levels fluctuate quickly. These findings provide new insights on niche separation between diatoms and cryptophytes, which is vital for a thorough understanding of the WAP marine ecosystem.     

The legacy of forest logging on organic matter inputs and storage in tropical streams

Riparian forests play an important role in stream ecosystems, as they support biodiversity, reduce water erosion, and provide litter that fuels aquatic biota. However, they are affected by great array of anthropogenic threats (e.g., fire, logging, and organic pollution), which alter species composition and their physical structure. Although forest recovery after disturbance such as logging can take decades, the legacy of forest clear-cut logging on key processes in tropical riparian ecosystems is mostly unknown. Here, we investigated how litter inputs (leaves, twigs, and reproductive parts) and storage, key processes for carbon and nutrient recycling and for forest and stream biota, are influenced by riparian vegetation undergoing succession (after 28 years from logging) through the comparison of reference and logged forest sites in the Cerrado biome. Litterfall was overall similar between forest types, but litterfall of twigs was twofold higher at logged than reference sites. Similarly, litter inputs from the bank to the stream (i.e., lateral inputs) and streambed storage were 50–60% higher at logged than reference sites. The higher litterfall observed in logged forests could be related to higher proportion of tree species that are characteristic of primary and secondary successional stages, including fast-growing and liana species, which often are more productive and common in anthropogenic areas. Our results showed that the legacy impact of clear-cut logging, even if residual woody vegetation is maintained in riparian buffers, can shift the type, quantity, and seasonality of litter subsidies to tropical streams. This knowledge should be considered within the context of management and conservation of communities and ecosystem processes in the forest-stream interfaces.