Apoptosis inhibitor of macrophage (AIM) expression in alveolar macrophages in COPD

Background

Marked accumulation of alveolar macrophages (AM) conferred by apoptosis resistance has been implicated in pathogenesis of chronic obstructive pulmonary disease (COPD). Apoptosis inhibitor of macrophage (AIM), has been shown to be produced by mature tissue macrophages and AIM demonstrates anti-apoptotic property against multiple apoptosis-inducing stimuli. Accordingly, we attempt to determine if AIM is expressed in AM and whether AIM is involved in the regulation of apoptosis in the setting of cigarette smoke extract (CSE) exposure.

Methods

Immunohistochemical evaluations of AIM were performed. Immunostaining was assessed by counting total and positively staining AM numbers in each case (n = 5 in control, n = 5 in non-COPD smoker, n = 5 in COPD). AM were isolated from bronchoalveolar lavage fluid (BALF). The changes of AIM expression levels in response to CSE exposure in AM were evaluated. Knock-down of anti-apoptotic Bcl-xL was mediated by siRNA transfection. U937 monocyte-macrophage cell line was used to explore the anti-apoptotic properties of AIM.

Results

The numbers of AM and AIM-positive AM were significantly increased in COPD lungs. AIM expression was demonstrated at both mRNA and protein levels in isolated AM, which was enhanced in response to CSE exposure. AIM significantly increased Bcl-xL expression levels in AM and Bcl-xL was involved in a part of anti-apoptotic mechanisms of AIM in U937 cells in the setting of CSE exposure.

Conclusions

These results suggest that AIM expression in association with cigarette smoking may be involved in accumulation of AM in COPD.     

Dietary intake of the flower extracts of German Chamomile (Matricaria recutita L.) inhibited compound 48/80-induced itch-scratch responses in mice

The antipruritic effects of the diets containing German chamomile on the compound 48/80-induced scratching in ddY mice were examined. Since it is reported that an injection of compound 48/80, but not histamine, induced scratching behaviour due to itch but not to pain in ddY mice (Kuraishi et al., 1995), compound 48/80-induced scratching in ddY mice seems to be a suitable parameter for evaluating antipruritic agents independent of histamine receptor antagonism. In the mice fed the diet containing 1.2 w/w % of the ethyl acetate extract of dried flower of German chamomile (Matricaria recutita L.) for 11 days, the compound 48/80-induced scratching behaviour was significantly suppressed. The ethyl acetate extract of German chamomile dose dependently suppressed compound 48/80-induced scratching without affecting body weight increase. The ethyl acetate fraction of the ethanol extract and the ethanol extract of hot water extraction residue of German chamomile flower also showed strong inhibition on the compound 48/80-induced scratching. The inhibitory effects of the dietary intake of the German chamomile extracts on compound 48/80-induced itch-scratch response were comparable to oxatomide (10 mg/kg, p.o.), an anti-allergic agent.     

Taenia taeniaeformis: fate and proliferation of mucosal cells during gastric hyperplasia in larvae infected rats

Fate and proliferation of gastric mucosal cells during hyperplasia of Taenia taeniaeformis eggs inoculated Wistar rats were investigated using PCNA immunohistochemistry, BrdU labeling and other histopathologic staining techniques. Results revealed marked cell proliferation in gastric corpus and antral mucosa of infected rats as evidenced by increased lengths of proliferative zones and indices of BrdU labeling. The gastropathy in corpus was characterized by massive accumulation of precursors, neck and intermediate cells following significant decreases in numbers of parietal and zymogenic cells. Gastropathy in antrum was described with significant increases in precursors and mucous cells. Our results suggested that T. taeniaeformis-induced gastric hyperplasia was initiated by depletion of parietal cells presumably due to the cestode's ES products. As a result, there was inhibition of zymogenic cell differentiation due to the disruption of normal development pathways of gastric mucosal lineages. These sequences of events were considered to cause the increase in cell proliferation and accumulation of intermediate cells resulting to the hyperplastic lesions.     

Culture of Hypnea musciformis (Rhodophyta,Gigartinales) on artificial substrates attached to linear ropes

Hypnea musciformis is the only species so far exploited in Brazil as raw material for the production of k-carrageenan. Due to the erratic production in space and time, increasing harvest and transportation costs, experiments have been performed in order to assess the viability of H. musciformis mariculture.In nature the species occurs as an epiphyte, and so mariculture using artificial substrates that simulated the natural host of the species was tried. These substrates were attached, at regular intervals, to linear ropes. In the sea, these ropes were stretched between cement blocks.Seeding occurs naturally, by means of spores, or detached pieces of H. musciformis scattered in the water column that get entangled on the ropes. The best yields (0.54 wet kg m^–1 month^–1) were obtained with unthreaded rope substrates maintained in a vertical position by small rafts. Production is highest in the first 18 m off the rocky shore (0–2.1 m deep), at the highest substrate density utilized (10 m^–1), 2–3 months after installing the ropes in seawater. The main factor controlling seasonal production is water movement.     

Neighboring base damage induced by permanganate oxidation of 8-oxoguanine in DNA.

We found that single-stranded DNA oligomers containing a 7, 8-dihydro-8-oxoguanine (8-oxo-G) residue have high reactivity toward KMnO4; the oxidation of 8-oxo-G induces damage to the neighboring nucleotide residues. This paper describes the novel reaction in detail, including experiments that demonstrate the mechanism involved in the induction of DNA damage. The results using DNAs of various base compositions indicated that damaged G, T and C (but not A) sites caused strand scissions after hot piperidine treatment and that the damage around the 8-oxo-G occurred at G sites in both single and double strands with high frequency. The latter substrates were less sensitive to damage. Further, kinetic studies of the KMnO4reaction of single-stranded oligomers suggested that thereactivity of the DNA bases at the site 5'-adjacent to the 8-oxo-G was in the order G >A >T, C. This preference correlates with the electron donating abilities of the bases. In addition, we found that the DNA damage at the G site, which was connected with the 8-oxo-G by a long abasic chain, was inhibited in the above order by the addition of dG, dA or dC. On the other hand, the damage reactions proceeded even after the addition of scavengers for active oxygen species. This study suggests the involvement of a redox process in the unique DNA damage initiated by the oxidation of the 8-oxo-G.     

Roles of PsbI and PsbM in photosystem II dimer formation and stability studied by deletion mutagenesis and X-ray crystallography

PsbM and PsbI are two low molecular weight subunits of photosystem II (PSII), with PsbM being located in the center, and PsbI in the periphery, of the PSII dimer. In order to study the functions of these two subunits from a structural point of view, we crystallized and analyzed the crystal structure of PSII dimers from two mutants lacking either PsbM or PsbI. Our results confirmed the location of these two subunits in the current crystal structure, as well as their absence in the respective mutants. The relative contents of PSII dimers were found to be decreased in both mutants, with a concomitant increase in the amount of PSII monomers, suggesting a destabilization of PSII dimers in both of the mutants. On the other hand, the accumulation level of the overall PSII complexes in the two mutants was similar to that in the wild-type strain. Treatment of purified PSII dimers with lauryldimethylamine N-oxide at an elevated temperature preferentially disintegrated the dimers from the PsbM deletion mutant into monomers and CP43-less monomers, whereas no significant degradation of the dimers was observed from the PsbI deletion mutant. These results indicate that although both PsbM and PsbI are required for the efficient formation and stability of PSII dimers in vivo, they have different roles, namely, PsbM is required directly for the formation of dimers and its absence led to the instability of the dimers accumulated. On the other hand, PsbI is required in the assembly process of PSII dimers in vivo; once the dimers are formed, PsbI was no longer required for its stability.     

Signal transduction in blue light‐induced oxygen uptake in Chlorella cells

In DCMU‐poisoned wild‐type and in non‐photosynthetic pigment mutant cells of Chlorella kessleri , grown heterotrophically with glucose as a carbon source and with nitrate as sole nitrogen source, the known blue light‐enhanced uptake of oxygen and breakdown of starch were reduced by staurosporine and K252a, both potent inhibitors of protein kinase C. This corresponded to sensitivity to these inhibitors of blue light‐induced uptake of nitrate of such organisms. Cells grown with ammonia as sole nitrogen source responded to short wavelength visible irradiation with an increase in oxygen uptake, and this, too, was inhibited by staurosporine and K252a. However, these cells did not show any blue light‐enhanced uptake of nitrate. From these results, enhanced consumption of oxygen under blue light cannot be a consequence of blue light‐induced protein phosphorylation involved in the light‐dependent uptake of nitrate. However, existence of a specific protein phosphorylation within the process of enhancement of oxygen uptake under blue light is not yet proven by the data. There might be a master reaction that induces both processes independently, or there may be influences of other light‐induced processes which lead to enhanced starch breakdown, thereby supplying the glucose for oxidative degradation.     

Differential expression of dentin matrix protein 1, type I collagen and osteocalcin genes in rat developing mandibular bone

The expression of dentin matrix protein 1 (Dmp1) mRNA has been compared with that of type I collagen and osteocalcin mRNAs during bone formation in the rat mandible, using in situ hybridization. At embryonic day 15 (E15), type I collagen and osteocalcin mRNAs were expressed by the majority of newly-differentiated osteoblasts attached to unmineralized bone matrices, whereas Dmp1 mRNA expression was confined to only a few osteoblasts. Expression of these genes increased as the number of osteoblasts increased in specimens from E16 to E18. At E20, expression of Dmp1, type I collagen and osteocalcin was also observed in osteocytes. Dmp1 expression continued in osteocytes as they matured up to the 90-day-old specimens, whereas type I collagen and osteocalcin expression in osteocytes almost disappeared at 30 days of postnatal life. In contrast, osteoblasts continued to express type I collagen and osteocalcin in 90-day-old rats, but transiently expressed Dmp1 mRNA, which was seen in the minority of osteoblasts at 14 days of postnatal life. These data show that the developmental expression patterns of Dmp1 in osteogenic differentiation differ from those of type I collagen and osteocalcin, and Dmp1 appears to be expressed by osteocytes throughout ossification in the skeleton. These observations indicate that Dmp1 may serve unique biological functions in osteocyte and bone metabolism.     

Rational design of a protein-based molecular device consisting of blue fluorescent protein and zinc protoporphyrin IX incorporated into a cytochrome b 562 scaffold

To make a single molecular photo-device, it is essential to control the exact orientation of two types of proteins. We made a chimeric protein in which cytochrome b562 was linked to the N-terminus of enhanced green fluorescent protein, cytb562-EGFP. Within cytb562-EGFP, the excitation energy of EGFP was transferred to the cytochrome b562 cofactor fixed proximally to EGFP. Cytb562-EGFP was engineered so that iron protoporphyrin IX was substituted by zinc protoporphyrin IX to make it a suitable cofactor for photo-induced electron transfer. The photosensitizer pigment was optimized and the EGFP was replaced by a blue fluorescent mutant that gave 15% higher energy transfer efficiency.