Calcium bridges are not load-bearing cell-wall bonds in Avena coleoptiles

I examined the ability of frozen-thawed Avena sativa L. coleoptile sections under applied load to extend in response to the calcium chelators ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) and 2-[(20bis-[carboxymethyl] amino-5-methylphenoxy)methyl]-6-methoxy-8-bis [carboxymethyl]aminoquinoline (Quin II). Addition of 5 mM EGTA to weakly buffered (0.1 mM, pH 6.2) solutions of 2(N-morpholino) ethanesulfonic acid (Mes) initiated rapid extension and wall acidification. When the buffer strength was increased (e.g. from 20 to 100 mM Mes, pH 6.2) EGTA did not initiate extension nor did it cause wall acidification. At 5 mM Quin II failed to stimulate cell extension or wall acidification at all buffer molarities tested (0.1 to 100 mM Mes). Both chelators rapidly and effectively removed Ca²⁺ from Avena sections. These data indicate that Ca²⁺ chelation per se does not result in loosening of Avena cells walls. Rather, EGTA promotes wall extension indirectly via wall acidification.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - Quin II 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis(carboxymethyl)aminoquinoline - Mes 2(N-morpholino)ethanesulfonic acid     

Calcium and the mechanical properties of soybean hypocotyl cell walls: Possible role of calcium and protons in cell-wall loosening

The role of calcium in the mechanical strength of isolated cell walls of soybean (Glycine max (L.) Merr. cv. Wayne) hypocotyls has been investigated, using the Instron technique to measure the plastic extensibility (PEx) of methanol-boiled, bisected hypocotyl sections and epidermal strips, and atomic absorption spectroscopy to measure wall calcium. Plastic extensibility was closely correlated with the growth rate of intact soybean hypocotyls. Removal of calcium from isolated cell walls by ethylene glycol-bis(2-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or low pH increased PEx, while addition of calcium decreased PEx; both effects were reversible. The amount of calcium removed and the increase in PEx at pH 4.5 were strongly dependent upon the chelating ability of the buffer anion. There was a direct correlation between the amount of calcium removed from the wall by EGTA or acid and the increase in PEx. Removal of up to 60% of the calcium increased PEx of half-section up to two fold, but further loss of calcium caused a much greater increase in PEx. With epidermal strips, PEx increased only when calcium was reduced below a threshold. At pH 3.5, there was an additional increase in PEx after a lag of about 2 h; this additional increase may be the result of acid-induced cleavage of a different set of load-bearing bonds. We conclude that calcium bridges are part of the load-bearing bonds in soybean hypocotyl cell walls, and that breakage of these crosslinks by apoplastic acid participates in wall loosening. Acid-induced solubilization of wall calcium may be one mechanism involved in wall loosening of dicotyledonous stems.Abbreviations EGTA ethylene glycol-bis(2-aminoethyl ether)-N,N,N,N-tetraacetic acid - PEx Instron plastic extensibility     

Effect of micellar lipids on rabbit intestinal brush-border membrane phospholipid bilayer integrity studied by31P NMR

Summary The effect of biliary salts and fatty acids on the bilayer structure of rabbit intestinal brush-border membranes was studied using the nonperturbing probe³¹P NMR. The broad. asymmetric lineshape of the³¹P NMR spectrum of isolated brush-border vesicles demostrates that their component phospholipids are organized in extended bilayers. These membranes are not significantly perturbed by incubation with physiological concentrations of biliary salts (3, 9, 18mm), demonstrating that the vesicles are highly stable, corresponding to their biological function. However, the emergence of a narrow peak superimposed on the broad lineshape indicates that a small proportion of the membrane phospholipids has reached isotropic motion, which may correspond to external or internal micellar structures. Incubation with mixed micelles of fatty acids and taurochlorate show that long-chain fatty acids enhance the membrane-perturbing effect of taurocholate while short-chain, watersoluble fatty acids do not, suggesting a difference in the absorption mechanisms.     

Immuno-isolation and culture of biliary epithelial cells from normal human liver

Summary Biliary epithelial cells (BEC) lining the intra-hepatic biliary ducts are the site of damage in several immunologically mediated liver diseases. BEC are difficult to isolate since they represent only 5% of the total cell number in normal liver. In this communication, a novel method for their isolation from normal liver is presented using a monoclonal antibody (HEA125) with specificity for an epithelial cell surface glyco-protein reported to be expressed in liver only by biliary epithelium. By combining differential density centrifugation and immuno-magnetic separation using HEA125 pure BEC (10⁵ cells/g fresh tissue) were prepared routinely. These cells were maintained in culture for up to 4 weeks with significant increases in cell numbers. The ability to prepare BEC from human liver offers an opportunity to develop In Vitro models to investigate the aetiology of diseases in intra-hepatic biliary epithelium. EDITOR’S STATEMENT This is a novel application to purification of specific liver cell types directly from tissue. It is well-suited for rapid communication because of its novelty and potential utility to investigators.     

Quantitative Detection of 15 Serum Bile Acid Metabolic Products by LC/MS/MS in the Diagnosis of Primary Biliary Cholangitis

To determine 15 bile acid metabolic products in human serum by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and value their diagnostic outcome in primary biliary cholangitis (PBC). Serum from 20 healthy controls and 26 patients with PBC were collected and went LC/MS/MS analysis of 15 bile acid metabolic products. The test results were analyzed by bile acid metabolomics, and the potential biomarkers were screened and their diagnostic performance was judged by statistical methods such as principal component and partial least squares discriminant analysis and area under curve (AUC). 8 differential metabolites can be screened out: Deoxycholic acid (DCA), Glycine deoxycholic acid (GDCA), Lithocholic acid (LCA), Glycine ursodeoxycholic acid (GUDCA), Taurolithocholic acid (TLCA), Tauroursodeoxycholic acid (TUDCA), Taurodeoxycholic acid (TDCA), Glycine chenodeoxycholic acid (GCDCA). The performance of the biomarkers was evaluated by the AUC, specificity and sensitivity. In conclusion, DCA, GDCA, LCA, GUDCA, TLCA, TUDCA, TDCA and GCDCA were identified as eight potential biomarkers to distinguish between healthy people and PBC patients by multivariate statistical analysis, which provided reliable experimental basis for clinical practice.     

Mucin-associated T antigens in benign and malignant epithelium of the gall bladder,extrahepatic bile ducts and ampulla of Vater

The mucin-associated antigens Tn, sialosyl-Tn (STn), T and sialosyl-T (STAg) antigens accumulate through aberrant and incomplete glycosylation in malignant epithelial cells. Their diagnostic and prognostic significance in tumours of the colon and cervix has been described, and a possible role for Tn antigen in cell-to-cell adhesion has been suggested. These antigens have been demonstrated through peanut agglutinin (PNA) lectin binding and more recently using specific monoclonal antisera. Differences between the two methods have been described, which may be due to fixation schedules and/or specificity. We have investigated the effect of fixation on the binding of biotinylated PNA lectin and compared its reactivity with the immunoreactivity of monoclonal antisera to Tn, STn, T and STAg antigens in benign and malignant epithelium of the gall bladder, extrahepatic bile ducts and ampulla of Vater. We found that short-term fixation in formol sublimate resulted in poor PNA binding. All other tested fixation schedules showed strong perinuclear binding, similar to that found on cryostat sections. When compared with monoclonal antisera, PNA binding demonstrated the lowest specificity in benign epithelium. In both benign and malignant epithelium, the two methods cannot substitute for each other. STn and STAg antigens were found to be oncodevelopmental throughout the extrahepatic biliary tract. When used in a panel, they are useful as diagnostic markers of malignancy in gall bladder epithelium.     

Molecular distribution of zinc in biliary and pancreatic secretions

Rat bile and pancreatic fluid were examined for the presence of low molecular weight zinc complexes. Fluids were collected separately by cannulation, and zinc distribution in collected samples was analyzed by gel filtration on Sephadex G-50. Most of the zinc in bile was associated with low molecular weight zinc complexes; only a small amount of zinc was present in the high molecular weight fraction. In contrast, pancreatic secretions did not contain low molecular weight zinc complexes, but there were considerable amounts of zinc bound to high molecular weight compounds. The addition of zinc to bile resulted in an increased amount of zinc in the low molecular weight fraction, while the addition of zinc to pancreatic fluid resulted primarily in an increase in zinc bound to the high molecular weight components. Like pancreatic fluid, homogenates of pancreatic tissue had no low molecular weight zinc complex. In rats whose bile and pancreatic fluid were removed and not returned into the intestine, the amount of zinc bound to low molecular weight complexes in intestinal homogenates was reduced. This alteration of the molecular distribution of zinc in intestinal homogenates by removal of bile and pancreatic fluid suggests the potential importance of low molecular weight zinc complexes for zinc homeostasis.     

Penetration of soil aggregates of finite size

Summary Maximum penetrometer pressure was measured on artificial soil aggregates of finite size (2–29 mm) using blunt probes (total cone angle 60°) driven at 3 mm min⁻¹. Maximum penetrometer pressure increased asymptotically with increase in dimensionless aggregate radius,b/a, wherea andb are the probe and aggregate radii, respectively. A theory was developed for penetration of blunt probes into soil aggregates of finite size. The theory assumed that plastic failure occurs out to a radius,R, and that beyond this only elastic straining occurs. This theory can be applied to estimate the radial and tangential stresses adjacent to a blunt probe. The estimated radial and tangential stresses increased with increase in dimensionless aggregate radius,b/a. The radius of the plastic front,R, around the probe is predicted to increase with increased aggregate size. The results also demonstrate the effect of soil shear cohesion and internal friction angle onR. The results are discussed with reference to root penetration.     

Emperipolesis mediated by CD8+ T cells correlates with biliary epithelia cell injury in primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by chronic destruction of the bile ducts. A major unanswered question regarding the pathogenesis of PBC is the precise mechanisms of small bile duct injury. Emperipolesis is one of cell‐in‐cell structures that is a potential histological hallmark associated with chronic hepatitis B. This study aimed to clarify the pathogenesis and characteristics of emperipolesis in PBC liver injury. Sixty‐six PBC patients, diagnosed by liver biopsy combined with laboratory test, were divided into early‐stage PBC (stages I and II, n = 39) and late‐stage PBC (stages III and IV, n = 27). Emperipolesis was measured in liver sections stained with haematoxylin‐eosin. The expressions of CK19, CD3, CD4, CD8, CD20, Ki67 and apoptosis of BECs were evaluated by immunohistochemistry or immunofluorescence double labelling. Emperipolesis was observed in 62.1% of patients with PBC, and BECs were predominantly host cells. The number of infiltrating CD3⁺ and CD8⁺ T cells correlated with the advancement of emperipolesis (R² = 0.318, P < .001; R² = 0.060, P < .05). The cell numbers of TUNEL‐positive BECs and double staining for CK19 and Ki67 showed a significant positive correlation with emperipolesis degree (R² = 0.236, P < .001; R² = 0.267, P < .001). We conclude that emperipolesis mediated by CD8⁺ T cells appears to be relevant to apoptosis of BEC and thus may aggravate the further injury of interlobular bile ducts.     

Oxidative stress and antioxidant status in patients with autoimmune liver diseases

Abstract

Objective

To estimate oxidative stress and antioxidant components during different stages of autoimmune liver diseases and assess their possible implication on disease progression.

Methods

We determined several markers of oxidative injury (isoprostane, aldehydes, protein carbonyls, 3-nitrotyrosine, and myeloperoxidase) and antioxidant components (glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase) in whole blood, serum, and urine in 49 patients with autoimmune cholestatic liver diseases (AC) and 36 patients with autoimmune hepatitis (AIH) and healthy subjects matched for sex and age.

Results

Both AC and AIH patients had increased levels of all lipid and protein oxidative injury products and significantly decreased whole blood glutathione levels compared to controls. AIH patients had significantly higher levels of aldehydes and glutathione peroxidase activity and significantly lower protein carbonyl levels compared to AC patients. Protein carbonyl and isoprostane levels increased and glutathione levels decreased gradually with progression from mild fibrosis to severe fibrosis and cirrhosis in both AC and AIH patients. In addition, both cirrhotic AC and AIH patients had significantly higher protein carbonyls compared to non-cirrhotics.

Discussion

We provide novel findings in support of a major contribution of oxidant/antioxidant imbalance in the progression of liver injury in AC and AIH.