Green tea polyphenol (-)-epigallocatechin gallate blocks epithelial barrier dysfunction provoked by IFN-gamma but not by IL-4

A characteristic of many enteropathies is increased epithelial permeability, a potentially pathophysiological event that can be evoked by T helper (Th)-1 (i.e., IFN-gamma) and Th2 (i.e., IL-4) cytokines and bacterial infection [e.g., enteropathogenic Escherichia coli (EPEC)]. The green tea polyphenol (-)-epigallocatechin gallate (EGCG) has immunosuppressive properties, and we hypothesized that it would ameliorate the increased epithelial permeability induced by IFN-gamma, IL-4, and/or EPEC. EGCG, but not the related epigallocatechin, completely prevented the increase in epithelial (i.e., T84 cell monolayer) permeability caused by IFN-gamma exposure as gauged by transepithelial resistance and horseradish peroxidase flux; EGCG did not alleviate the barrier disruption induced by IL-4 or EPEC. IFN-gamma-treated T84 and THP-1 (monocytic cell line) cells displayed STAT1 activation (tyrosine phosphorylation on Western blot analysis, DNA binding on EMSA) and upregulation of interferon response factor-1 mRNA, a STAT1-dependent gene. All three events were inhibited by EGCG pretreatment. Aurintricarboxylic acid also blocked IFN-gamma-induced STAT1 activation, but it did not prevent the increase in epithelial permeability. Additionally, pharmacological blockade of MAPK signaling did not affect IFN-gamma-induced epithelial barrier dysfunction. Thus, as a potential adjunct anti-inflammatory agent, EGCG can block STAT1-dependent events in gut epithelia and monocytes and prevent IFN-gamma-induced increased epithelial permeability. The latter event is both a STAT1- and MAPK-independent event.     

Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are necessary for lipoprotein assembly. ApoB consists of five structural domains, betaalpha(1)-beta(1)-alpha(2)-beta(2)-alpha(3). We propose that MTP contains three structural motifs (N-terminal beta-barrel, central alpha-helix, and C-terminal lipid cavity) and three functional domains (lipid transfer, membrane associating, and apoB binding). MTP's lipid transfer activity is required for the assembly of lipoproteins. This activity renders nascent apoB secretion-competent and may be involved in the import of triglycerides into the lumen of endoplasmic reticulum. In addition, MTP binds to apoB with high affinity involving ionic interactions. MTP interacts at multiple sites in the N-terminal betaalpha(1) structural domain of apoB. A novel antagonist that inhibits apoB-MTP binding decreases apoB secretion. Furthermore, site-directed mutagenesis and deletion analyses that inhibit apoB-MTP binding decrease apoB secretion. Lipids modulate protein-protein interactions between apoB and MTP. Lipids associated with MTP increase apoB-MTP binding whereas lipids associated with apoB decrease this binding. Thus, specific antagonist, site-directed mutagenesis, deletion analyses, and modulation studies support the notion that apoB-MTP binding plays a role in lipoprotein biogenesis. However, specific steps in lipoprotein assembly that require apoB-MTP binding have not been identified. ApoB-MTP binding may be important for the prevention of degradation and lipidation of nascent apoB.     

Multiple,independently regulated pathways of cholesterol transport across the intestinal epithelial cells

The present study provides a new understanding about the mechanisms involved in cholesterol absorption by the intestinal cells. Contrary to general belief, our data show that newly absorbed cholesterol is neither immediately available for secretion with apoB lipoproteins nor exclusively secreted as part of chylomicrons. Based on our data, cholesterol transport by enterocytes can be broadly classified into two independently modulated, apoB-dependent and -independent, pathways. Cholesterol secretion by the apoB-dependent pathway is induced by oleic acid, is repressed by microsomal triglyceride transfer protein inhibitors, and occurs only with larger apoB-containing lipoproteins. ApoB-independent pathways do not require microsomal triglyceride transfer protein and involve efflux mediated by ABCA1, high density lipoprotein assembly, and possibly other unknown mechanisms. There are at least two different metabolic pools of cholesterol. The newly absorbed and pre-absorbed cholesterol are preferentially secreted via apoB-independent and apoB-dependent pathways, respectively. In contrast to compartmentalization for secretion, these two metabolic pools are equally accessible for cellular esterification. The esterified cholesterol is mainly secreted by the apoB-dependent pathway, whereas both the pathways are involved in the secretion of free cholesterol. Thus, enterocytes transport exogenous cholesterol by several independently regulated pathways raising the possibility that targeting of apoB-independent pathways may result in selective inhibition of cholesterol transport without affecting triglyceride transport.     

Toxicity of argemone oil: Effect on hsp70expression and tissue damage in transgenic Drosophila melanogaster(hsp70-lacZ) Bg 9

The effect of argemone oil on hsp70expression and tissue damage was investigated by studying β-galactosidase activity, Western blotting and hybridization, and trypan blue staining in the larval tissues of transgenic Drosophila melanogaster(hsp70-lacZ)Bg ⁹. Different concentrations of argemone oil were mixed with food and third-instar larvae were allowed to feed on them for different time intervals (2, 4, 24, and 48 h). Argemone oil was found to induce hsp70even in the lowest concentration of the adulterant while maximum tissue damage was observed in the higher two treatment groups. Malpighian tubules and midgut tissue reflected maximum damage as evidenced by both high β-galactosidase activity and trypan blue staining in these tissues. A prior temperature shock treatment to the larvae was enough to protect the larvae from argemone oil-induced tissue damage as evidenced by little or no trypan blue staining. The present study suggests the cytotoxic potential of argemone oil and further strengthens the evidence for the use of hsp70as a biomarker in risk assessment. This revised version was published online in August 2006 with corrections to the Cover Date.     

An approach based on diffusion to study ligand-macromolecule interaction

A new approach has been developed to study binding of a ligand to a macromolecule based on the diffusion process. In terms of the Fick's first law, the concentration of free ligand in the presence of a protein can be determined by the measurement of those ligands which are diffused out. This method is applied to the study of binding of methyl-orange to lysozyme in phosphate buffer of pH 6.2, at 30 degrees C. The binding isotherm was determined initially, followed by application of the Hill equation to the data obtained, then binding constant and binding capacity were estimated.     

Fgf3 and Fgf8 are required together for formation of the otic placode and vesicle

Fgf3 has long been implicated in otic placode induction and early development of the otocyst; however, the results of experiments in mouse and chick embryos to determine its function have proved to be conflicting. In this study, we determined fgf3 expression in relation to otic development in the zebrafish and used antisense morpholino oligonucleotides to inhibit Fgf3 translation. Successful knockdown of Fgf3 protein was demonstrated and this resulted in a reduction of otocyst size together with reduction in expression of early markers of the otic placode. fgf3 is co-expressed with fgf8 in the hindbrain prior to otic induction and, strikingly, when Fgf3 morpholinos were co-injected together with Fgf8 morpholinos, a significant number of embryos failed to form otocysts. These effects were made manifest at early stages of otic development by an absence of early placode markers (pax2.1 and dlx3) but were not accompanied by effects on cell division or death. The temporal requirement for Fgf signalling was established as being between 60% epiboly and tailbud stages using the Fgf receptor inhibitor SU5402. However, the earliest molecular event in induction of the otic territory, pax8 expression, did not require Fgf signalling, indicating an inductive event upstream of signalling by Fgf3 and Fgf8. We propose that Fgf3 and Fgf8 are required together for formation of the otic placode and act during the earliest stages of its induction.     

Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy

Twenty flavonoid compounds of five different subclasses were selected, and the relationship of their structure to the inhibition of low-density lipoprotein (LDL) oxidation in vitro was investigated. The most effective inhibitors, by either copper ion or 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) induction, were flavonols and/or flavonoids with two adjacent hydroxyl groups at ring B. In the presence of the later catechol group, the contribution of the double bond and the carbonyl group at ring C was negligible. Isoflavonoids were more effective inhibitors than other flavonoid subclasses with similar structure. Substituting ring B with hydroxyl group(s) at 2' position resulted in a significantly higher inhibitory effect than by substituting ring A or ring B at other positions. The type of LDL inducer had no effect in flavonoids with catechol structure. Calculated heat of formation data (deltadeltaH(f)) revealed that the donation of a hydrogen atom from position 3 was the most likely result, followed by that of a hydroxyl from ring B. Position 3 was favored only in the presence of conjugated double bonds between ring A to ring B. This study makes it possible to assign the contribution of different functional groups among the flavonoid subclasses to in vitro inhibition of LDL oxidation.     

A new species of hognose pitviper, genus Porthidium, from the southwestern Pacific of Costa Rica (Serpentes: Viperidae)

A new species of terrestrial pitviper, Porthidium porrasi, is described from mesophytic forests of the Península de Osa and surrounding area of the Pacific versant of southwestern Costa Rica. It is most similar to P. nasutum and is characterized by a pattern of bands, persistence of the juvenile tail color in adults, and a high number of dorsal scales. Analysis of mtDNA sequences confirms its distinction from P. nasutum. The existence of this species reinforces the notion of elevated herpetofaunal endemism in southwestern Costa Rica.