Kinetic evaluation of lipophilic inhibitors of lipid peroxidation in DLPC liposomes

The authors have developed a kinetic method that allows one to obtain relative reactivity constants for lipophilic antioxidants in free radical systems. Two experimental model systems were developed: (a) a methanolic solution using AMVN as the free radical initiator and linoleic acid as the substrate, and (b) a multilamellar vesicle system composed of dilinoleoylphosphatidylcholine and AAPH as the substrate and the initiator, respectively. The use of these two systems allows researchers not only to determine the intrinsic reactivity of a potential antioxidant, but also to evaluate its potency in a membranous system where the contribution of the physical properties of the antioxidant to the inhibition of lipid peroxidation is important. These results show that all antioxidants tested acted in these systems as free radical scavengers, and they validate the synergism between intrinsic scavenging ability and membrane affinity and/or membrane-modifying physical properties in the inhibition of lipid peroxidation.     

Immobilization of Burkholderia cepacia in polyurethane-based foams: embedding efficiency and effect on bacterial activity

Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam. All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O₂ consumption and CO₂ evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds. Received 23 December 1996/ Accepted in revised form 13 March 1997     

Phosphoinositide 3-kinase regulates excitation-contraction coupling in neonatal cardiomyocytes

The phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 decreased steady-state contraction in neonatal rat ventricular myocytes (NRVM). To determine whether the effect on steady-state contraction could be due to decreased intracellular Ca(2+) content, Ca(2+) content was assessed with fluorescent plate reader analysis by using the caffeine-releasable Ca(2+) stores as an index of sarcoplasmic reticulum (SR) Ca(2+) content. Caffeine-releasable Ca(2+) content was diminished in a dose-dependent manner with LY-294002, suggesting that the decrease in steady-state contraction was due to diminished intracellular Ca(2+) content. Activation of the L-type Ca(2+) channel by BAY K 8644 was attenuated by LY-294002, suggesting the effect of LY-294002 is to reduce Ca(2+) influx at this channel. To investigate whether additional proteins involved in excitation-contraction (EC) coupling are likewise regulated by PI3K activity, the effects of compounds acting at sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), the ryanodine receptor, and the Na/Ca exchanger (NCX) were compared with LY-294002. Inhibition of SERCA2a by thapsigargin increased basal Ca(2+) levels in contrast to LY-294002, indicating that SERCA2a activity is sustained in the presence of LY-294002. Ryanodine decreased SR Ca(2+) content. The additive effect with coadministration of LY-294002 could be attributed to a decrease in Ca(2+) influx at the L-type Ca(2+) channel. The NCX inhibitor Ni(2+) was used to investigate whether the decrease in intracellular Ca(2+) content with LY-294002 could be due to inhibition of the NCX reverse-mode activity. The minimal effect of LY-294002 with Ni(2+) suggests that the primary effect of LY-294002 on EC coupling occurs through inhibition of PI3K-mediated L-type Ca(2+) channel activity.     

Role of Ca2+ and Mg2+ in neutrophil hexose transport

The influence of extracellular Ca²⁺ and Mg²⁺ on the transport of 2-deoxy-[³H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[³H]glucose uptake stimulated by two chemotactic factors (C5a and $\text{N-}\text{formylmethionylleucylphenylalanine}$) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca²⁺ and Mg²⁺ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca²⁺, but not Mg²⁺, supported optimal enhanced hexose transport induced by stimuli.Activation of 2-deoxy-D-[³H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca²⁺ into the cell (as exemplified by the action of the two ionophores), such Ca²⁺ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca²⁺, may be involved in mediating the activation of hexose transport induced by the latter stimuli.     

Evidence for epithelial-mesenchymal transitions in adult liver cells

Both myofibroblastic hepatic stellate cells (HSC) and hepatic epithelial progenitors accumulate in damaged livers. In some injured organs, the ability to distinguish between fibroblastic and epithelial cells is sometimes difficult because cells undergo epithelial-mesenchymal transitions (EMT). During EMT, cells coexpress epithelial and mesenchymal cell markers. To determine whether EMT occurs in adult liver cells, we analyzed the expression profile of primary HSC, two HSC lines, and hepatic epithelial progenitors. As expected, all HSC expressed HSC markers. Surprisingly, these markers were also expressed by epithelial progenitors. In addition, one HSC line expressed typical epithelial progenitor mRNAs, and these epithelial markers were inducible in the second HSC line. In normal and damaged livers, small ductular-type cells stained positive for an HSC marker. In conclusion, HSC and hepatic epithelial progenitors both coexpress epithelial and mesenchymal markers, providing evidence that EMT occurs in adult liver cells.     

Localization and induction of the A2 virulence factor in Leishmania: evidence that A2 is a stress response protein

Leishmaniasis is a vector‐borne infectious disease with a wide range of pathologies depending on the species of Leishmania. Leishmania parasites are transmitted by the sand fly vector as promastigotes; within the mammalian host, Leishmania parasites differentiate into amastigotes and replicate in macrophages. The A2 protein from Leishmania donovani is expressed predominantly in amastigotes and therefore likely plays a role in survival in the mammalian host. In the present study, we have determined that the A2 protein colocalized with the Leishmania endoplasmic reticulum binding protein, BiP, was induced by stress and complexed with BiP following heat shock. The A2 gene in Leishmania major is a non‐expressed pseudogene, and we present evidence that ectopic expression of a transfected A2 gene in L. major enhanced its viability following heat shock. A2 may therefore play a role in protecting L. donovani from stress associated with infection in visceral organs, including the fever typically associated with visceral leishmaniasis. Interestingly, when comparing A2 protein localization, we also observed that the Leishmania secreted acid phosphatase SAcP protein was transported out of the parasite‐containing phagolysosome and was located throughout the macrophage cytoplasm in vesicles, providing the first example of a secreted Leishmania‐derived protein exiting the parasite‐containing phagolysosome.     

Augmentation of ADCC and cytotoxic T-cell activity with ImuVert

We previously reported that natural killer cell activity of peripheral blood mononuclear cells (PBMC) from healthy human subjects was augmented following in vitro incubation in ImuVert, a biologic response modifier derived from the bacterium Serratia marcescens. In the current investigation, we found that exposure of PBMC to ImuVert, 3-40 micrograms/ml, for 18 hr, resulted in significant and dose-dependent augmentation of three other types of cell-mediated cytotoxicity: K cell-mediated antibody-dependent cellular cytotoxicity (ADCC), monocyte-mediated ADCC, and cytotoxic T-cell activity against allogeneic PBMC. These and previous findings suggest that ImuVert may have a broad range of stimulatory effects on immune function.     

Lack of Prion Infectivity in Fixed Heart Tissue from Patients with Creutzfeldt-Jakob Disease or Amyloid Heart Disease

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP^Sc) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP^Sc in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP^Sc. Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.