Plaque stabilizing effects of apolipoprotein A-IV.

BACKGROUND AND AIMS: Apolipoprotein (apo) A-IV, the third most abundant HDL-associated protein, is atheroprotective and shares similar properties as apoA-I. We have reported previously that apoA-I, the most abundant apolipoprotein in HDL, inhibits plaque disruption in a mouse model. We aimed at examining the effects of apoA-IV on markers of plaque stability in vivo. METHODS: Plaques within brachiocephalic arteries of 16-week old apoE-knockout C57BL/6 mice were examined for changes in composition after 10 weeks on a high-fat diet (HFD). The animals received twice-weekly injections of human lipid-free apoA-IV (1 mg/kg, n = 31) or PBS (n = 32) during the 9th and 10th weeks of the HFD. RESULTS: In the apoA-IV treated mice, there were significantly fewer hemorrhagic plaque disruptions (9/31 vs. 18/32, p < 0.05), thicker fibrous caps, smaller lipid cores, a lower macrophage:SMC ratio, less MMP-9 protein, more collagen, and fewer proliferating cells. In the plaques of mice given apoA-IV, MCP-1, VCAM-1, and inducible NOS were also significantly lower. Based on the percentage of cleaved PARP-positive and TUNEL-positive plaque nuclei, apoA-IV reduced apoptosis. in HMDMs, apoA-IV reduced MMP-9 mRNA expression by half, doubled mRNA levels of TIMP1 and decreased MMP-9 activity. CONCLUSIONS: ApoA-IV treatment is associated with a more stable plaque phenotype and a reduced incidence of acute disruptions in this mouse model.

Kavalactones Yangonin and Methysticin induce apoptosis in human hepatocytes (HepG2) in vitro.

While cases of severe kava hepatotoxicity have been reported, studies examining the toxicity of individual kavalactones are limited. The present study examined the in vitro hepatotoxicity of kavain, methysticin and yangonin on human hepatocytes (HepG2) and the possible mechanism(s) involved. Cytotoxicity was assessed using lactate dehydrogenase (LDH) and ethidium bromide (EB) assays. The mode of cell death was analysed with acridine orange/ethidium bromide dual staining with fluorescence microscopy. Glutathione oxidation was measured using the ortho-phthalaldehyde (OPT) fluorescence assay. Kavain had minimal cytotoxicity, methysticin showed moderate concentration-dependent toxicity and yangonin displayed marked toxicity with ~ 40% reduction in viability in the EB assay. Acridine orange/ethidium bromide staining showed the predominant mode of cell death was apoptosis rather than necrosis. No significant changes were observed in glutathione levels, excluding this as the primary mechanism of cell death in this model. Further studies may elucidate the precise apoptotic pathways responsible and whether toxic kavalactone metabolites are involved.

Metabolism of protein-bound DOPA in mammals.

Protein-bound 3,4-dihydroxyphenylalanine (DOPA) can be generated in mammalian cells by both controlled enzymatic pathways, and by uncontrolled radical reactions. Protein-bound DOPA (PB-DOPA) has reducing activity and the capacity to inflict secondary damage on other important biomolecules such as DNA. This may be mediated through replenishment of transition metals or from catechol-quinone-catechol redox cycles in the presence of cellular components such as ascorbate or cysteine, resulting in amplification of radical damaging events. The generation of PB-DOPA confers on protein the ability to chelate transition metals generating protein 'oxychelates'; this may be amongst the factors, which localise such damage. Tissue levels of PB-DOPA are increased in a number of age-related pathologies such as atherosclerosis and cataract formation. We discuss the detoxification, and the subsequent proteolysis and excretion of components of PB-DOPA. We contrast the fact that in marine organisms, and particularly in extracellular proteins, PB-DOPA and other DOPA-polymers can play important functional roles in adhesion and the provision of tensile properties.

Induction of peptidylglycine alpha-amidating monooxygenase in N(18)TG(2) cells: a model for studying oleamide biosynthesis.

The fatty-acid primary amide, oleamide, is a novel signaling molecule whose mechanism of biosynthesis is unknown. Recently, the N(18)TG(2) cell line was shown to synthesize oleamide from oleic acid, thereby demonstrating that these cells contain the necessary catalytic activities for generating the fatty-acid primary amide. The ability of peptide alpha-amidating enzyme, peptidylglycine-alpha-amidating monooxygenase (PAM; EC 1.14.17.3), to catalyze the formation of oleamide from oleoylglycine in vitro suggests this as a function for the enzyme in vivo. This investigation shows that N(18)TG(2) cells, in fact, express PAM and that cellular differentiation dramatically increases this expression. PAM expression was confirmed by the detection of PAM mRNA, PAM protein, and enzymatic activity that exhibits the functional characteristics of PAM isolated from mammalian neuroendocrine tissues. The regulated expression of PAM in N(18)TG(2) cells is consistent with the proposed role of PAM in the biosynthesis of fatty-acid primary amides and further establishes this cell line as a model for studying the pathway.

N-acylglycine amidation: implications for the biosynthesis of fatty acid primary amides.

Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the O2-dependent conversion of C-terminal glycine-extended prohormones to the active, C-terminal alpha-amidated peptide and glyoxylate. We show that alpha-AE will also catalyze the oxidative cleavage of N-acylglycines, from N-formylglycine to N-arachidonoylglycine. N-Formylglycine is the smallest amide substrate yet reported for alpha-AE. The (V/K)app for N-acylglycine amidation varies approximately 1000-fold, with the (V/K)app increasing as the acyl chain length increases. This effect is largely an effect on the KM,app; the KM,app for N-formylglycine is 23 +/- 0.88 mM, while the KM,app for N-lauroylglycine and longer chain N-acylglycines is in the range of 60-90 microM. For the amidation of N-acetylglycine, N-(tert-butoxycarbonyl)glycine, N-hexanoylglycine, and N-oleoylglycine, the rate of O2 consumption is faster than the rate of glyoxylate production. These results indicate that there must be the initial formation of an oxidized intermediate from the N-acylglycine before glyoxylate is produced. The intermediate is shown to be N-acyl-alpha-hydroxyglycine by two-dimensional 1H-13C heteronuclear multiple quantum coherence (HMQC) NMR.

Purification and characterisation of 6 and 58 kDa forms of the endogenous serine proteinase inhibitory proteins of ovine articular cartilage.

The major ovine articular cartilage (AC) serine proteinase inhibitory protein (SPI), a 58 kDa glycoprotein (SPI-58), was purified to homogeneity by sequential Sephacryl S-300 gel permeation, concanavalin A affinity, Mono Q anion exchange and Superose 12 FPLC. If precautions to prevent degradation of the native 58 kDa SPI were not undertaken during the early stages of its purification a SPI of approximately 6 kDa (SPI-6) was generated. SPI-6 could also be generated from SPI-58 by chymotrypsin affinity chromatography, suggesting that SPI-6 could be produced from SPI-58 in vivo by proteolytic processing within the tissue. SPI-6 was indistinguishable from the Kunitz inhibitor, bovine pancreatic trypsin inhibitor (BPTI) by SDS-PAGE under both reducing and non reducing conditions and showed a strong homology to BPTI in N-terminal sequence. These data suggest that the BPTI-like 6 kDa SPI constituted the inhibitory domain of the native 58 kDa SPI of ovine AC. Detection of [14C]-lysine-SPI-6 and SPI-58 in the serum free culture medium from ovine chondrocytes cultured in alginate beads in the presence of [14C]-lysine indicated that these SPIs were chondrocyte biosynthetic products. The inhibitory profiles of SPI-58 and SPI-6 differed somewhat suggesting that each may have an independent role in vivo.

Biotinylated trypsin and its application as a sensitive, versatile probe for the detection and characterisation of an ovine chondrocyte serine proteinase inhibitor using Western blotting.

Biotinylated trypsin (bT) was used as a probe on Western blots of 10-20% polyacrylamide gradient Tris-Tricine gels for the detection of serine proteinase inhibitors (SPIs) isolated from extracts of ovine articular cartilage and from chondrocyte conditioned culture medium. The major cartilage SPI, a 58 kDa glycoprotein, was purified by sequential Sephacryl S--300 gel permeation, concanavalin A affinity, anion exchange and Superose 12 fast protein liquid chromatography (FPLC). A low molecular weight SPI of approximately 6 kDa was also detectable in cartilage extracts after prolonged storage at 4 degrees C and following affinity chromatography on immobilised chymotrypsin, suggesting that the 6 kDa inhibitor may have arisen from proteolytic modification of the 58 kDa SPI. Analysis of chondrocyte conditioned culture medium using the bT detection system revealed that the major SPI present was the 6 kDa species although a small amount of the 58 kDa SPI was also detectable. The large molecular weight 'native' cartilage SPI was distinct from ovine alpha1-proteinase inhibitor (alpha1-PI) when examined by native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). The 6 kDa cartilage SPI was indistinguishable from basic pancreatic trypsin inhibitor (BPTI, aprotinin) following sodium dodecyl sulfate (SDS)-PAGE under reduced and nonreduced conditions. Amino terminal sequencing of the 6 kDa cartilage SPI revealed a strong (90%) homology with BPTL.

Biotin-labeled potato chymotrypsin inhibitor-1: a useful probe for the detection and quantitation of chymotrypsin-like serine proteinases on western blots and its application in the detection of a serine proteinase synthesised by articular chondrocytes.

Potato chymotrypsin inhibitor-1 (pCTI-1) was biotinylated by reaction with sulfosuccinimidyl-6-(biotinamido)hexanoate. This derivative was used as a probe on Western blots for the detection and quantitation of chymotrypsin and the detection of a chymotrypsin-like serine proteinase synthesized by ovine chondrocytes in alginate bead culture. Densitometric analysis demonstrated that there was a linear relationship between the amount of chymotrypsin electrophoresed, over the range 0.1 to 10 ng, and the intensity of the band detected on Western blots using biotinylated pCTI-1 as probe, indicating that the technique could be used for the quantification of active proteinases. The biotinylated pCTI-1 detection technique was convenient to use, reproducible, and more sensitive than zymography.

Modification of Bick's procedure for treatment of eyelid laxity.

A modification of Bick's procedure is presented that offers a simple, effective treatment of ectropion and entropion secondary to eyelid laxity. A full-thickness lid-shortening procedure performed at the lateral canthus avoids lid notching, with good cosmetic results. The success of the procedure is determined by suture of the tarsus directly to the orbital periosteum. The modification of Bick's procedure improves the outcome of the operation and simplifies its performance.

Corneal wedge resection in rabbits.

Corneal wedge resection was performed on 30 albino rabbits. There was a significant increase in astigmatism produced when the width or length of the wedge was increased.

Prevention and management of postoperative lateral upper-lid retraction in Graves' disease.

Patients with Graves' disease often have eyelid retraction, the upper lid being more commonly affected, and its lateral aspect being more retracted than its medial aspect. This paper describes the surgical treatment of upper-lid retraction, including methods of preventing residual postoperative lateral retraction and medial ptosis. Techniques of dealing with these postoperative problems are also described.

Management of acquired dacryocystitis.

Acquired dacryocystitis that has its onset after the first year of life is due to fibrotic obstruction within the lacrimal drainage pathways that is usually secondary to trauma or infection. Staphylococcus aureus is the most common pathogen, but attempts should be made to express or aspirate material from the lacrimal sac for culture and antibiotic sensitivity testing. When the strain is resistant to penicillin or when a culture specimen is not obtainable, cloxacillin is the antibiotic of choice. The timing of application of hot compresses is important so that the swelling can be localized to the sac but perforation will not be induced. Once the inflammation has resolved, dacryocystorhinostomy may be needed to drain residual fluid. Dacryocystography is useful to indicate whether there is persistent obstruction or stenosis; if the results are normal but symptoms persist, scintiscanning may demonstrate a delay of tear flow out of the sac or may reveal a stone.