Factors affecting quality and safety of fresh-cut produce.

The quality of fresh-cut fruit and vegetable products includes a combination of attributes, such as appearance, texture, and flavor, as well as nutritional and safety aspects that determine their value to the consumer. Nutritionally, fruit and vegetables represent a good source of vitamins, minerals, and dietary fiber, and fresh-cut produce satisfies consumer demand for freshly prepared, convenient, healthy food. However, fresh-cut produce deteriorates faster than corresponding intact produce, as a result of damage caused by minimal processing, which accelerates many physiological changes that lead to a reduction in produce quality and shelf-life. The symptoms of produce deterioration include discoloration, increased oxidative browning at cut surfaces, flaccidity as a result of loss of water, and decreased nutritional value. Damaged plant tissues also represent a better substrate for growth of microorganisms, including spoilage microorganisms and foodborne pathogens. The risk of pathogen contamination and growth is one of the main safety concerns associated with fresh-cut produce, as highlighted by the increasing number of produce-linked foodborne outbreaks in recent years. The pathogens of major concern in fresh-cut produce are Listeria monocytogenes, pathogenic Escherichia coli mainly O157:H7, and Salmonella spp. This article describes the quality of fresh-cut produce, factors affecting quality, and various techniques for evaluating quality. In addition, the microbiological safety of fresh-cut produce and factors affecting pathogen survival and growth on fresh-cut produce are discussed in detail.

Vascular calcifications in homozygote familial hypercholesterolemia.

BACKGROUND: Patients with homozygous familial hypercholesterolemia (hmzFH) attributable to LDL receptor gene mutations have shown a remarkable increase in survival over the last 20 years. Early onset coronary heart disease (CHD) and calcific aortic valve stenosis are the major complications of this disorder. We now report extensive premature calcification of the aorta in patients with hmzFH. METHODS AND RESULTS: We examined 25 hmzFH patients from Canada; mean age was 32 years (range 5 to 54), and mean baseline cholesterol before treatment was 19+/-5 mmol/L (737+/-206 mg/dL). Aortic calcification was quantified using computed tomography (CT). An elevated mean calcium score was found in patients by age 20 and correlated with age (r(2)=0.53, P=0.001). One quarter (24%) of patients underwent aortic valve surgery. CONCLUSIONS: We document premature severe aortic calcifications in all adult hmzFH patients studied. These presented considerable surgical management challenges. Strategies to identify and monitor aortic calcification in hmzFH by noninvasive techniques are required, as are clinical trials to determine whether additional or more intensive therapies will prevent the progression of such calcifications. Whether vascular calcifications in hmzFH subjects are related to sustained increases in LDL-C levels or to other mechanisms, such as abnormal osteoblast activity, remains to be determined.

Bis-pyrene probes of foldamer conformation in solution and in phospholipid bilayers.

Exploring the detailed structural features of synthetic molecules in the membrane phase requires sensitive probes of conformation. Here we describe the design, synthesis and characterization of bis(pyrene) probes that report conformational changes in membrane-active dynamic foldamers. The probes were designed to distinguish between left-handed (M) and right-handed (P) screw-sense conformers of 310-helical α-aminoisobutyric acid (Aib) peptide foldamers, both in solution and in bilayer membranes. Several different bis(pyrene) probes were synthesized and ligated to the C-terminus of Aib tetramers that had different chiral residues at the N-terminus, residues that favored either an M or a P screw-sense in the 310-helix. The readily synthesized and conveniently incorporated N-acetyl-1,2-bis(pyren-1'-yl)ethylenediamine probe proved to have the best properties. In solution, changes in foldamer screw-sense induced substantial changes in the ratio of excimer/monomer fluorescence emission (E/M) for this reporter of conformation, with X-ray crystallography revealing that opposite screw-senses produce very different interpyrene distances in the reporter. In bilayers, this convenient and sensitive fluorescent reporter allowed, for the first time, an investigation of how the chirality of natural phospholipids affects foldamer conformation.

Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier Disease.

Tangier disease is a rare genetic disorder characterized by extremely low plasma levels of HDL and apo A-I, deposition of cholesteryl esters in tissues, and a high prevalence of cardiovascular disease. We examined the possibility that HDL apolipoprotein-mediated removal of cellular lipids may be defective in Tangier disease. With fibroblasts from normal subjects, purified apo A-I cleared cells of cholesteryl esters, depleted cellular free cholesterol pools available for esterification, and stimulated efflux of radiolabeled cholesterol, phosphatidylcholine, and sphingomyelin. With fibroblasts from two unrelated Tangier patients, however, apo A-I had little or no effect on any of these lipid transport processes. Intact HDL also was unable to clear cholesteryl esters from Tangier cells even though it promoted radiolabeled cholesterol efflux to levels 50-70% normal. Passive desorption of radiolabeled cholesterol or phospholipids into medium containing albumin or trypsinized HDL was normal for Tangier cells. Binding studies showed that the interaction of apo A-I with high-affinity binding sites on Tangier fibroblasts was abnormal. These results indicate that apo A-I has an impaired ability to remove cholesterol and phospholipid from Tangier fibroblasts, possibly because of a defective interaction of apo A-I with cell-surface binding sites. Failure of apo A-I to acquire cellular lipids may account for the rapid catabolism of nascent HDL particles and the low plasma HDL levels in Tangier disease.

Tyrosyl radical generated by myeloperoxidase catalyzes the oxidative cross-linking of proteins.

Phagocytes generate H2O2 for use by a secreted heme enzyme, myeloperoxidase, to kill invading bacteria, viruses, and fungi. We have explored the possibility that myeloperoxidase might also convert L-tyrosine to a radical catalyst that cross-links proteins. Protein-bound tyrosyl residues exposed to myeloperoxidase, H2O2, and L-tyrosine were oxidized to o,o'-dityrosine, a stable product of the tyrosyl radical. The cross-linking reaction required L-tyrosine but was independent of halide and free transition metal ions; the heme poisons azide and aminotriazole were inhibitory. Activated neutrophils likewise converted polypeptide tyrosines to dityrosine. The pathway for oxidation of peptide tyrosyl residues was dependent upon L-tyrosine and was inhibited by heme poisons and catalase. Dityrosine synthesis was little affected by plasma concentrations of Cl- and amino acids, suggesting that the reaction pathway might be physiologically relevant. The requirement for free L-tyrosine and H2O2 for dityrosine formation and the inhibition by heme poisons support the hypothesis that myeloperoxidase catalyzes the cross-linking of proteins by a peroxidative mechanism involving tyrosyl radical. In striking contrast to the pathways generally used to study protein oxidation in vitro, the reaction does not require free metal ions. We speculate that protein dityrosine cross-linking by myeloperoxidase may play a role in bacterial killing or injuring normal tissue. The intense fluorescence and stability of biphenolic compounds may allow dityrosine to act as a marker for proteins oxidatively damaged by myeloperoxidase in phagocyte-rich inflammatory lesions.

High density lipoprotein oxidation: in vitro susceptibility and potential in vivo consequences.

Elevated levels of plasma high density lipoprotein (HDL) are strongly predictive of protection against atherosclerotic vascular disease. HDL particles likely have several beneficial actions in vivo, including the initiation of reverse cholesterol transport. The apparent importance of oxidative modification of low density lipoprotein in atherogenesis raises the question of how oxidative modification of HDL might affect its cardioprotective actions. HDL is readily oxidized using numerous models of lipoprotein oxidation. In vitro evidence suggests oxidation might impair some protective actions, but actually enhance other mechanisms induced by HDL that prevent the accumulation of cholesterol in the artery wall. This article reviews the current literature concerning the relative oxidizability of HDL, the structural changes induced in HDL by oxidation in vitro, and the potential consequences of oxidative modification on the protective actions of HDL in vivo.

Regulation of the uptake of high-density lipoprotein-originated cholesteryl ester by HepG2 cells: role of low-density lipoprotein and plasma lipid transfer protein.

Cholesteryl ester uptake by the human hepatoma cell line HepG2 was studied in vitro by using radiolabeled cholesteryl ester as a tracer. After the cells were incubated in a lipoprotein deficient condition, the rate of radio labeled cholesteryl ester uptake from low-density lipoprotein (LDL) was estimated to be some 25-times higher than that from high-density lipoprotein (HDL). LDL-cholesteryl ester uptake was suppressed by preincubation of the cells with LDL, but pretreatment of the cells with HDL did not show significant effect. HDL-cholesteryl ester uptake was only slightly suppressed by pretreatment of the cells with LDL, and there was no effect with HDL pretreatment. HDL-cholesteryl ester uptake was not affected either by the presence of LDL or human plasma lipid transfer protein alone in the medium under our experimental conditions. Lipid transfer protein enhanced the uptake of radiolabeled cholesteryl ester originating from HDL by the cells only in the presence of LDL. Thus, lipid transfer protein catalyzes a bypass to LDL for the uptake by HepG2 cells of cholesteryl ester molecules which originate in HDL, and this pathway is much more efficient than direct uptake of cholesteryl ester originating in HDL by these cells.

Functional similarities of human and chicken apolipoprotein A-I: dependence on secondary and tertiary rather than primary structure.

To investigate the sequence requirements for apolipoprotein (apo) AI functions, comparisons of human and chicken apoAI were performed. In lipid binding assays, chicken apoAI was capable of transforming phospholipid vesicles into discoidal bilayer structures, similar in both size and apolipoprotein content to those produced with human apoAI under the same conditions. Human and chicken apoAI were indistinguishable in their relative abilities to prevent phospholipase C-induced aggregation of human low density lipoprotein. This activity, which is dependent upon formation of a stable interaction with the modified lipoprotein, represents a sensitive measure of apolipoprotein association with spherical lipoprotein particles. The ability of chicken versus human apoAI to mobilize the regulatory pool of cholesterol available for esterification by acyl-CoA:cholesterol acyltransferase by human fibroblasts was also assessed. Lipid-free chicken and human apoAI were equivalent in their ability to deplete cholesterol from this pool, as were intact chicken high density lipoprotein (HDL) and human HDL(3). Based on the overall sequence identity of chicken and human apoAI (48%), and comparison of regions thought to be responsible for key apoAI functions, these data indicate that amphipathic alpha-helical structure, rather than specific amino acid sequence, is the major determinant of apoAI lipid binding and ability to mobilize the regulatory pool of cellular cholesterol.

Targeting HDL-mediated cellular cholesterol efflux for the treatment and prevention of atherosclerosis.

The hallmark of the atherosclerotic lesion is the overaccumulation of cholesterol in arterial wall cells. As no pathway exists for the degradation of cholesterol in peripheral cells, a mechanism is necessary to prevent its accumulation to toxic levels in these cells and to allow its delivery to the liver for excretion in bile. Promoting this reverse cholesterol transport pathway is believed to be the main cardioprotective action of high density lipoprotein (HDL). The rate-limiting step in this pathway is likely the initial removal of cholesterol from peripheral cells by HDL. The pathway HDL utilizes for inducing efflux of excess cellular cholesterol represents an important and as-yet untapped mechanism to employ for the treatment and prevention of atherosclerotic vascular disease. This review summarizes the potential cardioprotective actions of HDL, the mechanisms of HDL-mediated cellular cholesterol efflux, and evidence that the specific pathway of cholesterol removal by HDL may be enhanced and used as a novel target in the therapy of atherosclerosis.

The avoidance of surgery in the treatment of subarachnoid cutaneous fistula by the use of an epidural blood patch: technical case report.

In this report, we present two cases of subarachnoid-cutaneous fistula. In both of these cases, in epidural blood patch, a technique frequently used by anesthetists and radiologists, was successfully used to treat the fistulae, thus avoiding the need for open closure.

Human apolipoprotein E N-terminal domain displacement of apolipophorin III from insect low density lipophorin creates a receptor-competent hybrid lipoprotein.

The surface of Manduca sexta low density lipophorin (LDLp) particles was employed as a template to examine the relative lipid binding affinity of the 22 kDa receptor binding domain (residues 1-183) of human apolipoprotein E3 (apo E3). Isolated LDLp was incubated with exogenous apolipoprotein and, following re-isolation by density gradient ultracentrifugation, particle apolipoprotein content was determined. Incubation of recombinant human apo E3(1-183) with LDLp resulted in a saturable displacement of apolipophorin III (apo Lp-III) from the particle surface, creating a hybrid apo E3(1-183)-LDLp. Although subsequent incubation with excess exogenous apo Lp-III failed to reverse the process, human apolipoprotein A-I (apo A-I) effectively displaced apo E3(1-183) from the particle surface. We conclude that human apo E N-terminal domain possesses a higher intrinsic lipid binding affinity than apo Lp-III but has a lower affinity than human apo A-I. The apo E3(1-183)-LDLp hybrid was competent to bind to the low density lipoprotein receptor on cultured fibroblasts. The system described is useful for characterizing the relative lipid binding affinities of wild type and mutant exchangeable apolipoproteins and evaluation of their biological properties when associated with the surface of a spherical lipoprotein.

The glutamate decarboxylase acid resistance mechanism affects survival of Listeria monocytogenes LO28 in modified atmosphere-packaged foods.

AIMS: The contribution of the glutamate decarboxylase (GAD) acid resistance system to survival and growth of Listeria monocytogenes LO28 in modified atmosphere-packaged foods was examined. METHODS AND RESULTS: The survival and growth of the wild-type LO28 and four GAD deletion mutants (DeltagadA, DeltagadB, DeltagadC, DeltagadAB) in packaged foods (minced beef, lettuce, dry coleslaw mix) during storage at 4, 8 and 15 degrees C were studied. Survival and growth patterns varied with strain, product type, gas atmosphere and storage temperature. In minced beef, the wild-type LO28 survived better (P < 0.05) than the GAD mutant strains at 8 and 15 degrees C. In both packaged vegetables at all storage temperatures, the wild-type strain survived better (P < 0.05) than the double mutant DeltagadAB. The requirement for the individual gad genes varied depending on the packaged food. In the case of lettuce, gadA played the most important role, while the gadB and gadC genes played the greatest role in packaged coleslaw (at 15 degrees C). CONCLUSIONS: This work demonstrates that elements of the GAD system play significant roles in survival of L. monocytogenes LO28 during storage in modified atmosphere-packaged foods. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of how L. monocytogenes behaves in modified atmosphere-packaged foods, and how it responds to elevated carbon dioxide atmospheres.

Effects of vegetable type, package atmosphere and storage temperature on growth and survival of Escherichia coli O157:H7 and Listeria monocytogenes.

The survival and growth of Escherichia coli O157:H7 (ATCC 43888 and NCTC 12900) and Listeria monocytogenes (ATCC 19114 and NCTC 11994) during storage (4 and 8 degrees C) on ready-to-use (RTU) packaged vegetables (lettuce, swedes (rutabaga), dry coleslaw mix, soybean sprouts) were studied. The vegetables were sealed within oriented polypropylene packaging film, and modified atmospheres developed in packs during storage due to produce respiration. Survival and growth patterns were dependent on vegetable type, package atmosphere, storage temperature and bacterial strain. Populations of L. monocytogenes and E. coli O157:H7 increased (P<0.05, by 1.5 to 2.5 log cycles, depending on strain) during a 12-day storage period on shredded lettuce (8 degrees C). L. monocytogenes populations also increased (by approximately 1 log cycle) on packaged swedes, did not change significantly (P>0.05) in packages of soybean sprouts and decreased by approximately 1.5 log cycles (P<0.05) on coleslaw mix (8 degrees C). E. coli O157:H7 populations on packaged coleslaw and soybean sprouts increased (by 1.5 to 2.5 log cycles) up to day 5, but declined during subsequent storage (8 degrees C). On packaged swedes (8 degrees C), populations of E. coli O157:H7 strain ATCC 43888 increased (by approximately 1 log cycle) during storage, whereas populations of strain 12900 increased between days 2 and 5, and declined during subsequent storage. Reducing the storage temperature from 8 to 4 degrees C reduced the growth of L. monocytogenes and E. coli O157:H7 on packaged RTU vegetables. However, viable populations remained at the end of the storage period at 4 degrees C.

Bradycardia associated with insertion and inflation of tissue expanders under the scalp.

A case of bradycardia during the insertion and inflation of tissue expanders under the scalp on two separate occasions is described. The possible mechanisms of this complication are discussed.

Localization of photosynthetic membrane components in Rhodopseudomonas sphaeroides by a radioactive labeling procedure.

Reduction with [3H]KBH4 of Schiff's bases generated by reaction with pyridoxal 5'-phosphate (which cannot penetrate the intact cytoplasmic membrane) yields tritium-labeled derivatives of both proteins and lipids accessible on the periplasmic side of the cytoplasmic membrane. Application of this technique to phototrophically grown Rhodopseudomonas sphaeroides labeled both the cell envelope and chromatophore fractions. The technique was also applied to R. sphaeroides harvested at various times during an adaptation from heterotrophic to phototrophic growth conditions. The specific activity of the chromatophore fraction after 20 h of adaptation was 76% of that found at the beginning, indicating that the intracytoplasmic membranes and cytoplasmic membrane form a continuous membrane system, with the majority of the intracytoplasmic membranes accessible to the external medium throughout the adaptation. The identity of the proteins labeled by this technique was investigated in two fractions labeled after cell disruption: normal "inside-out" chromatophores and "right-side-out" membrane vesicles isolated by lysozyme--osmotic shock treatment of cells grown in high light intensity (15000 lx). The results after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and fluorography indicated that the 28000-dalton subunit (and to a lesser extent the 21000-dalton subunit) of the reaction center complex and two polypeptides in the light-harvesting region of the gel were heavily labeled in the chromatophores and were thus accessible on the cytoplasmic side of the membrane. At least one of the latter two polypeptides was also labeled in the membrane vesicles and was thus also accessible on the periplasmic side of the membrane. None of the reaction center subunits was significantly labeled in a reaction center complex prepared from the membrane vesicle sample.

A serious complication of minitracheotomy.

A previously unreported complication of minitracheotomy is described. The minitracheotomy introducer was lost into the pleural cavity and a thoracotomy was required to remove it.

Cholesterol mobilization by free and lipid-bound apoAI(Milano) and apoAI(Milano)-apoAII heterodimers.

Despite very low plasma levels of HDL, carriers of the apolipoprotein AI Arg173 --> Cys mutation apoAI(Milano) (AIM) have no apparent increase in risk for atherosclerotic vascular disease. HDL apolipoprotein species in AIM carriers include apoAI-AII heterodimers, previously found to confer the enhanced ability of tyrosyl radical-oxidized HDL to mobilize cholesterol for removal from cultured cells. To determine whether enhanced mobilization of cholesterol by apoprotein species in AIM explains a cardioprotective action of this mutation, we examined the ability of lipid-free and lipid-bound AIM and AIM-AII heterodimers to deplete cholesterol from cultured cells. Free AIM and AIM-AII heterodimers showed a decreased capacity to act as acceptors of cholesterol from cholesterol-loaded human fibroblasts compared with native apoAI but similar capacities to deplete fibroblasts of the pool of cholesterol available for esterification by acyl-CoA:cholesterol acyltransferase (ACAT). Discoidal reconstituted HDL (rHDL) containing apoAI depleted both of these cholesterol pools more readily than AIM-containing rHDL when compared at equivalent rHDL protein levels, but similar abilities of these rHDL to deplete cell cholesterol were seen when compared at equivalent phospholipid levels. Spherical rHDL generated using the whole lipid fraction of HDL and apoAI or AIM showed similar capacities to deplete total and ACAT-accessible cell cholesterol when compared at similar protein levels, but an increased capacity of AIM-containing particles was seen when compared at equivalent phospholipid levels. Unlike the apoAI-AII heterodimer in tyrosylated HDL, AIM-AII heterodimer-containing spherical rHDL showed no increased capacity to deplete either of these pools of cholesterol. These results suggest a similar or better capacity of native apoAI in lipid-free or lipid-bound form in discoidal rHDL to enhance the mobilization of cellular cholesterol when compared to AIM in its free or lipid-bound forms. Any increase in depletion of cellular cholesterol by lipid-bound AIM in spherical rHDL appears related to altered phospholipid-binding rather than intrinsic cholesterol-mobilizing characteristics of this protein compared to native apoAI. The lack of major differences in these studies in cholesterol mobilization by native apoAI and AIM, or by apoAIM-AII heterodimers, suggests that any protection against atherosclerosis conferred by this mutation is likely related to other beneficial vascular effects of AIM.

Apolipoprotein AI efficiently binds to and mediates cholesterol and phospholipid efflux from human but not rat aortic smooth muscle cells.

Aortic smooth muscle cells (SMC) from several animal species have been reported to resist depletion of cellular cholesterol by the major apolipoprotein of HDL, apoAI. Resistance of SMC to this protective action of apoAI, if present in humans, could contribute to the overaccumulation of arterial wall cholesterol seen in atherosclerosis. We investigated the ability of human aortic medial SMC to bind and be depleted of cholesterol and phospholipids by apoAI. In contrast to rat aortic SMC, but similar to human fibroblasts, human SMC were readily depleted of cholesterol by apoAI, measured by a marked depletion of intracellular cholesterol available for esterification, and an increase in cholesterol efflux to the medium. Human SMC were also actively depleted of the phospholipids phosphatidylcholine and sphingomyelin by apoAI. In contrast, rat SMC released only a small fraction of these cellular phospholipids to apoAI-containing medium. (125)I-labeled apoAI bound with high affinity and specificity to human SMC, but failed to bind to rat SMC. Similar levels of expression of class B, type I scavenger receptor (SR-BI) and caveolin in human and rat SMC suggested these proteins do not account for the differences in apoAI binding or lipid efflux seen in these cells. An enhancer of apolipoprotein-mediated cholesterol efflux, tyrosyl radical-oxidized HDL, markedly amplified the depletion of cholesterol available for esterification in human SMC compared to HDL, but had no enhanced effect in rat SMC. These results show that human SMC bind and are readily depleted of cellular lipids by apoAI, and suggest that apoAI-mediated cholesterol efflux from arterial SMC may contribute significantly to the circulating pool of HDL cholesterol in vivo. The marked difference in apoAI binding to human and rat arterial SMC provides an excellent model to study the nature of the apoAI-cell binding interaction.

Oxidative tyrosylation of HDL enhances the depletion of cellular cholesteryl esters by a mechanism independent of passive sterol desorption.

It is believed that HDL protects against atherosclerosis by removing excess cholesteryl esters from cells of the artery wall. Previous studies have suggested that HDL depletes cells of cholesteryl esters both by stimulating cholesterol efflux from the plasma membrane and by activating transport processes that divert cholesterol from the cholesteryl ester cycle, but it is unknown if these are independent processes. We previously found that HDL oxidized by tyrosyl radical has a markedly enhanced ability to promote the removal of cholesterol from cultured cells [Francis, G. A., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6631-6635]. Here we show that incubation of cholesterol-loaded human fibroblasts with low concentrations of tyrosylated HDL depleted cells of cholesteryl esters and increased cellular free cholesterol without increasing efflux of cholesterol into the medium as compared to incubation with untreated HDL. Cells preincubated with tyrosylated HDL and then exposed to a variety of cholesterol acceptors exhibited significantly higher rates of free cholesterol efflux than did cells preincubated with HDL. This effect was observed in the presence or absence of an inhibitor of acyl CoA:cholesterol acyltransferase (ACAT) and was independent of cholesteryl ester hydrolysis, suggesting that alterations in cholesteryl ester cycle enzymes were not responsible for the loss of cholesteryl esters. In contrast to the reduction of cholesteryl esters, the rates of cholesterol and phospholipid efflux from the plasma membranes of cells exposed to tyrosylated HDL and HDL were identical. These results suggest for the first time that a mechanism exists to deplete cellular cholesteryl esters and the cholesterol substrate pool for esterification by ACAT prior to the removal of cholesterol from the plasma membrane. Identification of products in tyrosylated HDL responsible for this redistribution of cellular cholesterol may provide important insights into mechanisms of intracellular cholesterol trafficking and the ability of modified forms of HDL to protect the artery against wall pathological cholesterol accumulation.