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.     

Inositol 1,3,4,5-tetrakisphosphate stimulates calcium release from bovine adrenal microsomes by a mechanism independent of the inositol 1,4,5-trisphosphate receptor.

In bovine adrenal microsomes, Ins(1,4,5)P3 binds to a specific high-affinity receptor site (Kd = 11 nM) with low affinity for two other InsP3 isomers, Ins(1,3,4)P3 and Ins(2,4,5)P3. In the same subcellular fractions Ins(1,4,5)P3 was also the most potent stimulus of Ca2+ release of all the inositol phosphates tested. Of the many inositol phosphates recently identified in angiotensin-II-stimulated adrenal glomerulosa and other cells, Ins(1,3,4,5)P4 has been implicated as an additional second messenger that may act in conjunction with Ins(1,4,5)P3 to elicit Ca2+ mobilization. In the present study, an independent action of Ins(1,3,4,5)P4 was observed in bovine adrenal microsomes. Heparin, a sulphated polysaccharide which binds to Ins(1,4,5)P3 receptors in several tissues, inhibited both the binding of radiolabelled Ins(1,4,5)P3 and its Ca2(+)-releasing activity in adrenal microsomes. In contrast, heparin did not inhibit the mobilization of Ca2+ by Ins(1,3,4,5)P4, even at doses that abolished the Ins(1,4,5)P3 response. Such differential inhibition of the Ins(1,4,5)P3- and Ins(1,3,4,5)P4-induced Ca2+ responses by heparin indicates that Ins(1,3,4,5)P4 stimulates the release of Ca2+ from a discrete intracellular store, and exerts this action via a specific receptor site that is distinct from the Ins(1,4,5)P3 receptor.     

DNA “fingerprints” and paternity ascertainment in chimpanzees (Pan troglodytes)

Highly variable regions of DNA are found in a wide diversity of organisms and are typically composed of alleles consisting of a variable number of tandem repeats (VNTRs) of a short core sequence. DNA fingerprinting probes are VNTR probes that simultaneously detect a large number of similar VNTRs in the target DNA. The highly polymorphic pattern observed in a DNA fingerprint allows resolution of questions concerning individual identification. M13 phage was used to fingerprint captive chimpanzees for paternity ascertainment. Although the probability of band sharing among captive chimps appears to be higher than among some other reported captive and feral animal populations, the probe is highly useful and can be expected to become more widely used in the genetic management of captive populations.     

Comparison of five tandem repeat loci between humans and chimpanzees.

Five tandem repeat loci were studied in humans and chimpanzees using VNTR probes derived from human DNA. Shared alleles were found at three loci and were often the modal allele in one species but never in both. There was no difference in the mean number of alleles per locus. However, these species exhibited substantially different levels of gene diversity, with chimpanzees monomorphic at two loci. Evidence of reduced variability in chimpanzees corroborates earlier comparisons using isozymes and plasma proteins. Molecular mechanisms, population dynamics, or both may be responsible for these differences. Equal numbers of alleles per locus may reflect high mutation rates. By one test, chimpanzees were out of equilibrium at one locus, which may reflect a typing error or population substructure. The long divergence time, and the high probability of backward mutations, precludes accurate estimation of genetic distance between these species.     

Pressure-induced conformational changes in a human Bence-Jones protein (Mcg)

The effect of high static pressures on the internal structure of the immunoglobulin light chain (Bence-Jones) dimer from the patient Mcg was assessed with measurements of intrinsic protein fluorescence polarization and intensity. Depolarization of intrinsic fluorescence was observed at relatively low pressures (less than 2 kbar), with a standard volume change of -93 mL/mol. The significant conformational changes indicated by these observations were not attributable to major protein unfolding, since pressures exceeding 2 kbar were required to alter intrinsic fluorescence emission maxima and yields. Fluorescence intensity and polarization measurements were used to investigate pressure effects on the binding of bis(8-anilino-naphthalene-1-sulfonate) (bis-ANS), rhodamine 123, and bis(N-methylacridinium nitrate) (lucigenin). Below 1.5 kbar the Mcg dimer exhibited a small decrease in affinity for bis-ANS (standard volume change approximately 5.9 mL/mol). At 3 kbar the binding activity increased by greater than 250-fold (volume change -144 mL/mol) and remained 10-fold higher than its starting value after decompression. With rhodamine 123 the binding activity showed an initial linear increase but plateaued at pressures greater than 1.5 kbar (standard volume change -23 mL/mol). These pressure effects were completely reversible. Binding activity with lucigenin increased slightly at low pressures (standard volume change -5.5 mL/mol), but the protein was partially denatured at pressures greater than 2 kbar. Taken in concert with the results of parallel binding studies in crystals of the Mcg dimer, these observations support the concept of a large malleable binding region with broad specificity for aromatic compounds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiation effects on diamine oxidase activities in intestine and plasma of the rat

Diamine oxidase (DAO; EC 1.4.3.6) activity was measured in plasma and in ileal tissue homogenates prepared from male Sprague-Dawley rats euthanized at 1-15 days after acute whole-body irradiation with 14.5-MeV electrons. Animals irradiated with 1 Gy showed no diminution in plasma and ileal DAO activities through Day 13 relative to nonirradiated controls. Animals irradiated with 5, 10, and 12 Gy displayed marked declines in ileal DAO activity, with levels reaching a nadir on Day 3. This was paralleled by a decrease in plasma DAO activity in all three dose groups. Recovery of ileal and plasma DAO levels was later seen as early as Day 4 in animals irradiated with 5- and 10-Gy doses, but animals receiving 12 Gy did not survive beyond Day 3. The relationship between radiation dose and levels of plasma and ileal DAO on Day 3, the time of maximum decrease at all doses, was also investigated. Ileal DAO activity decreased almost linearly between 2 and 8 Gy. Plasma DAO activity closely paralleled the dose dependency of the ileal levels. These data suggest that plasma DAO activity might be useful as a biologic marker of intestinal epithelial injury and recovery after acute radiation exposure.     

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.     

Plasmids and Bacteriocins in Caulobacter Species

A survey of wild-type Caulobacter strains revealed naturally occurring plasmids in three species. Further analysis showed instances of naturally occurring antibiotic resistance and bacteriocin production.