Characterization of the lectin from females of Phlebotomus duboscqi sand flies.

Lectin from females of the important sand fly vector, Phlebotomus duboscqi (Diptera: Psychodidae), was isolated by immunoaffinity chromatography using a minicolumn with immobilized anti-lectin immunoglobulins. Carbohydrate-binding specificity of active fractions corresponded to that of midgut and salivary gland lysates. Haemagglutination was inhibited by d-glucosamine, d-galactosamine and d-mannosamine. The homogeneity and molecular mass of the purified lectin was examined by SDS/PAGE in both reducing and nonreducing conditions. The active fractions showed one band strongly stained by Coomassie blue or silver nitrate; the molecular mass of the lectin was 42 kDa under nonreducing and 44 kDa under reducing conditions. SDS/PAGE of active fractions from the gel filtration revealed four to six protein bands, but the 42/44-kDa protein present in all active fractions was the only component reacting with specific antibodies in Western blots. Localization of the lectin in the gut of females was studied using indirect immunofluorescence on sections. The positive reaction of specific antibodies was localized in the lumen and along the microvillar surfaces of epithelial cells. The lectin was partially sequenced and characterized by MS. Peptide maps were obtained by MALDI-TOF MS, and several sequence tags were identified from tandem mass spectra on an ion trap. These sequences displayed high similarity to salivary protein precursors previously identified in a cDNA library of the sand flies Phlebotomus papatasi and Lutzomyia longipalpis. Two main hypotheses on the role of female lectin in Leishmania development are discussed.     

Study on the ophthalmic diseases in ICR mice and BALB/c mice.

In pharmaceutical companies and research institutes, many toxicity tests are performed with laboratory animals. This study was performed to produce reference data for eye toxicity tests and to investigate the ophthalmic diseases of 408 ICR mice and 119 BALB/c mice, which are commonly used as subjects in toxicity tests. The experimental animals without clinical disorders were selected regardless of sex. The ophthalmic diseases were examined by using special ophthalmic instruments: direct ophthalmoscope, indirect ophthalmoscope, slit-lamp biomicroscope and focal illuminator. The most prevalent ocular variation within normal limits was hyaloid vessel remnant (ICR mice, 28.2%; BALB/c mice, 31.9%) and the incidence gradually decreased with age. The ocular diseases found in ICR mice were retinal degeneration (9.8%), corneal scar (4.2%), focal cataract (2.2%), anisocoria (1.2%), corneal ulcer (0.2%) and uveitis (0.2%). In BALB/c mice, corneal scar (9.2%), focal cataract (1.7%) and corneal ulcer (0.8%) were the ocular diseases found.     

Local skin sensory changes after acupuncture

Forty normal subjects were assessed for local skin sensory changes of the knees after acupuncture needle insertion. Correct insertion sites were used on one knee and incorrect sites on the other knee. No significant change of sensation was found over the L3 and L5 dermatomes when incorrect sites were used. Pain sensation was markedly diminished on the side where correct sites were used; minimal transient diminished sensation of heat and vibration was noted.     

COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits.We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant.Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.     

Mechanisms of Gasdermin Family Members in Inflammasome Signaling and Cell Death

The Gasdermin (GSDM) family consists of Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C (GSDMC), Gasdermin D (GSDMD), Gasdermin E (GSDME) and Pejvakin (PJVK). GSDMD is activated by inflammasome-associated inflammatory caspases. Cleavage of GSDMD by human or mouse caspase-1, human caspase-4, human caspase-5, and mouse caspase-11 liberates the N-terminal effector domain from the C-terminal inhibitory domain. The N-terminal domain oligomerizes in the cell membrane and forms a pore of 10–16?nm in diameter, through which substrates of a smaller diameter, such as interleukin-1β and interleukin-18, are secreted. The increasing abundance of membrane pores ultimately leads to membrane rupture and pyroptosis, releasing the entire cellular content. Other than GSDMD, the N-terminal domain of all GSDMs, with the exception of PJVK, have the ability to form pores. There is evidence to suggest that GSDMB and GSDME are cleaved by apoptotic caspases. Here, we review the mechanistic functions of GSDM proteins with respect to their expression and signaling profile in the cell, with more focused discussions on inflammasome activation and cell death.     

Organogold(III) compounds as experimental anticancer agents: chemical and biological profiles

In the last few years gold(III) complexes have attracted growing attention in the medicinal chemistry community as candidate anticancer agents. In particular some organogold(III) compounds manifested quite attractive pharmacological behaviors in preclinical studies. Here we compare the chemical and biological properties of the novel organogold(III) complex [Au(bipy^dmb?H)(NH(CO)CH₃)][PF₆] (Aubipy^aa) with those of its parent compounds [Au(bipy^dmb?H)(OH)][PF₆] (Aubipy^c) and [Au₂(bipy^dmb?H)₂)(μ?O)][PF₆]₂ (Au2bipy^c), previously synthesized and characterized. The three study compounds were comparatively assessed for their antiproliferative actions against HCT-116 cancer cells, revealing moderate cytotoxic effects. Proapoptotic and cell cycle effects were also monitored. Afterward, to gain additional mechanistic insight, the three gold compounds were challenged against the model proteins HEWL, RNase A and cytochrome c and reactions investigated through UV–Vis and ESI–MS analysis. A peculiar and roughly invariant protein metalation profile emerges in the three cases consisting of protein binding of {Au(bipy^dmb?H)} moieties. The implications of these results are discussed in the frame of current knowledge on anticancer gold compounds.     

The presence of serotonin in cigarette smoke – a possible mechanistic link to 5-HT-induced airway inflammation

We previously reported the involvement of serotonin (5-HT) metabolism in cigarette smoke-induced oxidative stress in rat lung in vivo. Here, we report cigarette smoke as a source of serotonin (5-HT) to the airways and aim at investigating the effects of 5-HT on oxidative stress and inflammation in human bronchial epithelial cells (BEAS-2B). A 5-HT analog was identified to be present in aqueous phase cigarette smoke using the LC-MS/MS approach, which was later confirmed by a 5-HT enzyme-linked immune assay (EIA). Furthermore, exposure to 5-HT caused a time-dependent elevation of intracellular ROS level, which was blocked in the presence of apocynin (a NOX inhibitor). In support, the immunoblot analysis indicated that there was an increase in the expression of NOX2 time-dependently. 5-HT-induced elevation of IL-8 at both mRNA and protein levels was observed, which was inhibited by TEMPOL (a free radical scavenger), and inhibitors for p38 MAPK (SB203580) and ERK (U0126), in line with the time-dependent phosphorylation of p38 MAPK and ERK. In conclusion, our findings suggest that 5-HT presented in bronchial epithelium of smokers may be involved in cigarette smoke-induced oxidative stress and inflammation via activation of p38 MAPK and ERK pathway after the formation of free radicals.