Differential role of CD18 integrins in mediating lung neutrophil sequestration and increased microvascular permeability induced by Escherichia coli in mice

The in vivo contributions of CD18 integrin-dependent and -independent mechanisms in mediating the increases in lung neutrophil (polymorphonuclear leukocyte; PMN) sequestration and microvascular permeability are not well understood. We determined the time course of these responses to Gram-negative sepsis in the mouse lung and addressed the specific contributions of CD18 integrins and ICAM-1. PMN sequestration in the lung was assessed by morphometric analysis, and transalveolar PMN migration was assessed by bronchoalveolar lavage. Lung tissue PMN number increased by 6-fold within 1 h after i.p. Escherichia coli challenge; this value peaked at 3 h (7-fold above control) and decreased at 12 h (3.5-fold above control). PMN migration into the airspace was delayed; the value peaked at 6 h and remained elevated up to 12 h. Saturating concentrations of anti-CD18 and anti-ICAM-1 mAbs reduced lung tissue PMN sequestration and migration; however, peak responses at 3 and 6 h were inhibited by 40%, indicating that only a small component of PMN sequestration and migration was CD18 dependent at these times. In contrast to the time-dependent decreased role of CD18 integrins in mediating PMN sequestration and migration, CD18 and ICAM-1 blockade prevented the increase in lung microvascular permeability and edema formation at all times after E. coli challenge. Thus, Gram-negative sepsis engages CD18/ICAM-1-independent mechanisms capable of the time-dependent amplification of lung PMN sequestration and migration. The increased pulmonary microvascular permeability induced by E. coli is solely the result of engagement of CD18 integrins even when PMN accumulation and migration responses are significantly CD18 independent.     

The enzymatic hydrolysis rate of cellulose decreases with irreversible adsorption of cellobiohydrolase I

Protein adsorption onto solid substrates usually takes place in an irreversible fashion and this irreversible adsorption also occurs in some enzymatic reactions. In this work the adsorption behavior of intact β-1, 4-glucan-cellobiohydrolase (CBH I) from Trichoderma reesei onto microcrystalline cellulose was monitored by surface plasmon resonance and UV-spectral method. It was found that there existed reversible binding and irreversible binding of CBH I during its interaction with cellulose substrate. To evaluate the influence of adsorption on cellulose enzymatic hydrolysis, the reaction dynamics on pure cellulose were determined. A plot of the hydrolysis rate against the surface density of irreversibly adsorbed CBH I, revealed an inverse relationship in which an apparent decrease in the hydrolysis rate was observed with increasing surface density. Taken together, results presented here should be useful for modifying the binding characteristics of CBH I and making them more effective in cellulose hydrolysis.     

Characterization and evolution of a single-copy sequence from the human Y chromosome.

To study the evolution and organization of DNA from the human Y chromosome, we constructed a recombinant library of human Y DNA by using a somatic cell hybrid in which the only cytologically detectable human chromosome is the Y. One recombinant (4B2) contained a 3.3-kilobase EcoRI single-copy fragment which was localized to the proximal portion of the Y long arm. Sequences homologous to this human DNA are present in male gorilla, chimpanzee, and orangutan DNAs but not in female ape DNAs. Under stringent hybridization conditions, the homologous sequence is either a single-copy or a low-order repeat in humans and in the apes. With relaxed hybridization conditions, this human Y probe detected several homologous DNA fragments which are all derived from the Y in that they occur in male DNAs from humans and the apes but not in female DNAs. In contrast, this probe hybridized to highly repeated sequences in both male and female DNAs from old world monkeys. Thus, sequences homologous to this probe underwent a change in copy number and chromosomal distribution during primate evolution.     

Distribution of <Emphasis Type="Italic">S</Emphasis> haplotypes and its relationship with restorer–maintainers of self-incompatibility in cultivated <Emphasis Type="Italic">Brassica napus</Emphasis>

Brassica napus (AACC, 2n = 38) is a self-compatible amphidiploid plant that arose from the interspecies hybridization of two self-incompatible species, B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18). Self-incompatibility (S) haplotypes in one self-incompatible line and 124 cultivated B. napus lines were detected using S-locus-specific primers, and their relationships with restorer-maintainers were investigated. Two class I (S-I ( SLG ) a and S-I ( SLG ) b) and four class II (S-II ( SLG ) a, S-II ( SLG ) b, S-II ( SP11 ) a and S-II ( SP11 ) b) S haplotypes were observed, of which S-II ( SP11 ) b was newly identified. The nucleotide sequence of SP11 showed little similarity to the reported SP11 alleles. The lines were found to express a total of eleven S genotypes. The self-incompatible line had a specific genotype consisting of S-II ( SP11 ) a, similar to B. rapa S-60, and S-II ( SLG ) a, similar to B. oleracea S-15. Restorers expressed six genotypes: the most common genotype contained S-I ( SLG ) a, similar to B. rapa S-47, and S-II ( SLG ) b, similar to B. oleracea S-15. Maintainers expressed nine genotypes: the predominant genotype was homozygous for two S haplotypes, S-II ( SLG ) a and S-II ( SP11 ) b. One genotype was specific to restorers and four genotypes were specific to maintainers, whereas five genotypes were expressed in both restorers and maintainers. This suggests that there is no definitive correlation between the distribution of S genotypes and restorer-maintainers of self-incompatibility. The finding that restorers and maintainers express unique genotypes, and share some common genotypes, would be valuable for detecting the interaction of S haplotypes in inter- or intra-genomes as well as for developing markers-assisted selection in self-incompatibility hybrid breeding.     

Cellulase elicitor induced accumulation of capsidiol in Capsicum annumm L. suspension cultures

When growth-phase cell suspension cultures of Capsicum annuum were treated with cellulase-elicitor preparation at 3 μg/ml, the level of capsidiol was transiently increased in the culture media rather than in the cells reaching its maximum approx 24 h after treatment. With methyl jasmonate it took 18 h. Elicitor treatment doubled phospholiphase A₂ (PLA₂) activity but simultaneous treatment with aristolochic acid, a PLA₂ inhibitor, inhibited sesquiterpenoid accumulation as well as PLA₂ activity. Mastoparan, a G protein activator, treatment also increased PLA₂ activity and capsidiol production. Taken together, the present study shows that induction of capsidiol production in the C. annuum is mediated by PLA₂ activation.     

TGF-β binding in human Wharton’s jelly

Our previous study reported that TGF-β may be isolated from human Wharton’s jelly (WJ) in a form of soluble, high molecular complex(es). We decided to study the effect of extracellular matrix degradation and reduction of disulphide bridges reduction on the release of TGF-β from WJ. The WJ prepared from the umbilical cords of newborns delivered at term by healthy mothers was homogenised and treated with hyaluronidase, collagenase, heparinase, chondroitinase and β-mercaptoethanol, the resulting extracts were then submitted to TGF-β immunoassay and SDS/PAGE followed by Western immunoblotting. The effect of metalloproteinase activation on TGF-β was also studied. Pre-treatment of WJ homogenates with hyaluronidase or collagenase markedly increased the extractability of TGF-β, but did not dissociate the complexes. In contrast, the action of β-mercaptoethanol resulted in the release of free TGF-β; but activation of metalloproteinases resulted in the disappearance of this factor. We conclude that TGF-β1 is bound through disulphide bonds to an extracellular matrix component of WJ. The large amount of collagen fibrils and hyaluronate molecules which surround the cells scattered in WJ may prevent the access of extracting solution to TGF-β causing a low extractability of this factor. Although hyaluronate and collagen do not bind TGF-β directly, they may present a barrier that prevents the diffusion of TGF-β in WJ and results in its concentration around the cells thereby facilitating its interaction with membrane receptors and subsequent stimulation of cell division and synthesis of extracellular matrix components.     

Enhanced production of triterpenoid in submerged cultures of Antrodia cinnamomea with the addition of citrus peel extract

In recent years, Antrodia cinnamomea has become a well-known medicinal mushroom in Taiwan. Triterpenoids are considered one of the most biologically active components found in A. cinnamomea. The aim of this research is to investigate the feasibility of enhancing triterpenoid production in shake flask cultures of A. cinnamomea by adding citrus peel extract. As a result of its containing essential oils, citrus peel extract is inhibitory to mycelial growth. In the experiments, the appropriate adding time is determined to be on day 7. Of the various citrus peel extracts tested, tangerine proves to be the most effective in enhancing polyphenol and triterpenoid production. With an addition of 2 % (v/v), the content and production of total polyphenols rises from 5.95 mg/g DW of the control and 56.73 mg/L to 23.52 mg/g DW and 224.39 mg/L, respectively, on day 28. The production of triterpenoids also increases from 99.93 to 1,028.02 mg/L, for more than a tenfold increase. An optimal level of tangerine peel additive is determined to be around 4 %. Furthermore, when compared with the mycelia of the control culture, the profiles of the HPLC analysis show that the mycelia cultured with the tangerine-peel addition contain more kinds of triterpenoids. This study demonstrates that the addition of citrus peel extract effectively enhances the production of bioactive metabolites in the submerged cultures of A. cinnamomea.