Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells

Background

Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.

Methods

We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.

Results

We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.

Conclusion

The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.     

Genomic affinities in Arachis section Arachis (Fabaceae): molecular and cytogenetic evidence

Section Arachis is the largest of nine sections in the genus Arachis and includes domesticated peanut, A. hypogaea L. Most species are diploids (x=10) with two tetraploids and a few aneuploids. Three genome types have been recognized in this section (A, B and D), but the genomes are not well characterized and relationships of several newly described species are uncertain. To clarify genomic relationships in section Arachis, cytogenetic information and molecular data from amplified fragment length polymorphism (AFLP) and the trnT-F plastid region were used to provide an additional insight into genome composition and species relationships. Cytogenetic information supports earlier observations on genome types of A. cruziana, A. herzogii, A. kempff-mercadoi and A. kuhlmannii but was inconclusive about the genome composition of A. benensis, A. hoehnei, A. ipaensis, A. palustris, A. praecox and A. williamsii. An AFLP dendrogram resolved species into four major clusters and showed A. hypogaea grouping closely with A. ipaensis and A. williamsii. Sequence data of the trnT-F region provided genome-specific information and showed for the first time that the B and D genomes are more closely related to each other than to the A genome. Integration of information from cytogenetics and biparentally and maternally inherited genomic regions show promise in understanding genome types and relationships in Arachis.     

Degradation of skeletal muscle plasma membrane proteins by calpain

Summary Observations described here provide the first demonstration that calpain (Ca²⁺-dependent cysteine protease) can degrade proteins of skeletal muscle plasma membranes. Frog muscle plasma membrane vesicles were incubated with calpain preparations and alterations of protein composition were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Calpain II (activated by millimolar concentrations of Ca²⁺) was isolated from frog skeletal muscle, but the activity of calpain I (activated by micromolar concentrations of Ca²⁺) was lost during attempts at fractionation. Calpain I obtained from skeletal muscle and erythrocytes of rats was tested instead, and exerted effects similar to those of frog muscle calpain on the membrane proteins. All of the calpain preparations caused striking losses of a major membrane protein of molecular mass of approximately 97 kDa, designated band c, and diminution of a thinner band of approximately 200 kDa. There were concomitant increases in 83-and 77-kDa polypeptides. These effects were absolutely dependent on the presence of free Ca²⁺, and were completely blocked by calpastatin, a specific inhibitor of calpain action. Frog muscle calpain differed only in being relatively more active at 0°C than were the calpains from rat tissues. Experimental observations suggest that calpain acts at the cytoplasmic surface of the plasma membrane.     

Investigation of the protective effect of ellagic acid for preventing kidney injury in rats exposed to nicotine during the fetal period

We investigated the possible protective effects of ellagic acid on rat kidneys exposed to nicotine during the fetal period. Twenty pregnant female rats were divided randomly into four groups: control (C), nicotine (N), ellagic acid (EA) and nicotine + ellagic acid (N + EA). Nicotine and ellagic acid treatments were continued throughout the pregnancies and for 15 days after delivery. On day 15, all neonatal pups were sacrificed and their kidneys were removed for biochemical and histopathological examination. The nicotine treatment significantly decreased body weight, total glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, and increased malondialdehyde (MDA) and nitric oxide (NO) levels in the N group compared to controls. EA treatment ameliorated decreased body weight, GSH, GSH-Px and SOD activities, and increased MDA and NO levels in group N + EA compared to group N (p < 0.05). Nicotine caused kidney damage as shown by incomplete development of glomeruli and Bowman's capsules. Nicotine also caused greater apoptosis in group N compared to group C. Ellagic acid treatment produced histological kidney structure that was closer to normal and it exerted an anti-apoptotic effect in the N + EA group compared to the N group. EA played a protective role against nicotine-induced nephrotoxicity and oxidative stress in rats owing to its antioxidant, radical scavenging and anti-apoptotic effects.     

Action of insulin on the transport of 3-O-methylglucose in frog sartorius muscles

The initial rate of entry of 3-O-methyl-D-glucose into isolated frog sartorius muscles was measured at various concentrations of substrate, at 0, 9, 19, or 29 degrees C, after prior incubation at 19 degrees C with a maximally stimulating concentration of insulin. Control muscles were treated similarly, except for the omission of insulin. A saturable transport system provided for most of the entry of 3-O-methylglucose into muscle cells, but a small amount of penetration occurred by a nonsaturable route. The major effect of insulin was to produce a large increase in activity of the suturable system. The Vmax of entry increased, but there was no significant change in the apparent Km. The ratio of insulin-stimulated to basal Vmax was 10 when transport was measured at 29 degrees C but was 22 at 0 degrees C. These findings support the hypothesis that, although a large part of the effect of insulin on sugar transport can be accounted for by an increase in the number of functional transporters in the plasma membrane, there is a separate hormonal effect that permits a relatively greater activity of transporters at lower temperatures, compared with control rates. An additional effect of insulin was to produce a small but definite increase in the entry of sugar by the nonsaturable transport system.     

Glycogen debranching enzyme in bovine brain

Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-1,6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5,000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-exchange HPLC, and gel-permeation HPLC. The enzyme preparation had no alpha-glucosidase or alpha-amylase activities and degraded phosphorylase limit dextrin of glycogen with phosphorylase. The molecular weight of the enzyme was 190,000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an alpha-1,6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase.     

A thiocyanate hydrolase of Thiobacillus thioparus. A novel enzyme catalyzing the formation of carbonyl sulfide from thiocyanate.

A thiocyanate hydrolase that catalyzes the first step in thiocyanate degradation was purified to homogeneity from Thiobacillus thioparus, an obligate chemolithotrophic eubacterium metabolizing thiocyanate to sulfate as an energy source. The thiocyanate hydrolase was purified 52-fold by steps involving ammonium sulfate precipitation, DEAE-Sephacel column chromatography, and hydroxylapatite column chromatography. The enzyme hydrolyzed 1 mol of thiocyanate to form 1 mol of carbonyl sulfide and 1 mol of ammonia as follows: SCN- + 2H2O----COS + NH3 + OH-. This is the first report describing the hydrolysis of thiocyanate to carbonyl sulfide by an enzyme. The enzyme had a molecular mass of 126 kDa and was composed of three different subunits: alpha (19 kDa), beta (23 kDa), and gamma (32 kDa). The enzyme exhibited optimal activities at pH 7.5-8.0 and at temperatures ranging from 30 to 40 degrees C. The Km value for thiocyanate was approximately 11 mM. Immunoblot analysis with polyclonal antibodies against the purified enzyme suggested that it was induced in T. thioparus cells when the cells were grown with thiocyanate.