Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Local host ant specificity of Phengaris (Maculinea) teleius butterfly,an obligatory social parasite of Myrmica ants

1. Phengaris butterflies are obligatory social parasites of Myrmica ants. Early research suggested that there is a different Myrmica host species for each of the five European Phengaris social parasites, but more recent studies have shown that this was an oversimplification. 2. The pattern of host ant specificity within a Phengaris teleius metapopulation from southern Poland is reported. A combination of studying the frequency distribution of Phengaris occurrence and morphometrics on adult butterflies were used to test whether use of different host species is reflected in larval development. 3. Phengaris teleius larvae were found to survive in colonies of four Myrmica species: M. scabrinodis, M. rubra, M. ruginodis, and M. rugulosa. Myrmica scabrinodis was the most abundant species under the host plant but the percentage of infested nests was similar to other host ant species at two sites and lower in comparison to nests of M. rubra and M. ruginodis at the other two sites. Morphometric measurements of adult butterflies reared by wild colonies of M. scabrinodis and M. ruginodis showed that wing size and number of wing spots were slightly greater for adults eclosing from nests of M. ruginodis. 4. Our results suggest that P. teleius in the populations studied is less specialised than previously suggested. The results are consistent with the hypothesis that P. teleius is expected to be the least specific of the European Phengaris species, as it has the largest and best defended fourth‐instar caterpillars and, as a predatory species, it spends less time in the central larval chambers of the host colonies. The fact that individuals reared by M. ruginodis had wider hind wings may suggest that P. teleius had better access to resources in M. ruginodis than in M. scabrinodis colonies.     

Leukotriene C4 production during hypoxic pulmonary vasoconstriction in isolated rat lungs

Leukotriene inhibitors preferentially inhibit hypoxic pulmonary vasoconstriction in isolated rat lungs. If lipoxygenase products are involved in the hypoxic pressor response they might be produced during acute alveolar hypoxia and a leukotriene inhibitor should block both the vasoconstriction and leukotriene production that occurs in response to hypoxia. We investigated in isolated blood perfused rat lungs whether leukotriene C₄ (LTC₄) could be recovered from whole lung lavage fluid during ongoing hypoxic vasoconstriction. Lung lavage from individual rats had slow reacting substance (SRS)-like myotropic activity by guinea pig ileum bioassay. Pooled lavage (10 lungs)_as analyzed by reverse phase high performance liquid chromatography had an ultraviolet absorbing component at the retention time for LTC₄. At this retention time the element had both LTC₄ immunoreactivitiy by radioimmunoassay, and SRS myotropic activity by bioassay. LTC₄ was not found during normoxic ventilation, during normoxic ventilation after a hypoxic pressor response, or during vasoconstriction elicited by KCL. Diethylcarbamazine citrate, a leukotriene synthesis blocker, concomitantly inhibited the hypoxic vasoconstriction and LTC₄ production. Thus 5-lipoxygenase products may play a role in the sequence of events leading to hypoxic pulmonary vasoconstriction.     

Regulation by calmodulin of the calcium affinity of the calcium-transport ATPase in human erythrocytes

The Ca2+ affinity of (Mg2+ + Ca2+)-ATPase in human red blood cells is regulated by a number of intracellular factors, including the association of the enzyme with the cytosolic Ca2+ binding protein, calmodulin. Ghosts prepared by hypotonic lysis in the presence of 0.1 mM CaCl2, or by a gradual stepwise hemolysis procedure, contain an EDTA-extractable protein whose effects are mimicked by calmodulin, whereas ghosts prepared by extensive washes in the absence of added CaCl2 lack calmodulin and contain only a high molecular weight heat stable activator. Purified calmodulin from human red cells or bovine brain shifts the apparent Ca2+ affinity of (Mg2+ + Ca2+)-ATPase activity in extensively washed ghosts to a high Ca2+ affinity state. The shift was most apparent in ghosts in which the Ca2+ affinity was decreased by EDTA treatment. Calmodulin increased the velocity of (Mg2+ + Ca2+)-ATPase in the EDTA-treated ghosts about 36-fold at a low (1.4 microM) Ca2+ concentration, compared with 6-fold before EDTA treatment. The maximum shift in apparent Ca2+ affinity occurred only in the presence of saturating concentrations of calmodulin. It is concluded that red cell calmodulin confers to the Ca2+ transport ATPase the ability to increase its apparent Ca2+ affinity, as well as its maximum velocity, in response to increases in intracellular Ca2+.     

A protein activator of Mg2+-dependent, Ca2+-stimulated ATPase in human erythrocyte membranes distinct from calmodulin

The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in chicken kidney was investigated. By separation on density gradients of the silica sol Ludox AM, the enzyme was localized in the mitochondrial fraction. This mitochondrial fraction was shown to be substantially free of lysosomal contamination. Subfractionation of the purified mitochondria indicates that the enzyme is located on the outer membrane, can be solubilized, and may be a suitable marker enzyme for kidney mitochondria.     

Evaluation of energetic and dynamic coupling networks in a PDZ domain protein

A number of computational and experimental studies have identified intramolecular communication "pathways" or "networks" important for transmitting allostery. Here, we have used mutagenesis and NMR relaxation methods to investigate the scope and nature of the communication networks found in the second post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain of the human protein tyrosine phosphatase 1E protein (hPTP1E) (PDZ2). It was found that most mutations do not have a significant energetic contribution to peptide ligand binding. Three mutants that showed significant changes in binding also displayed context-dependent dynamic effects. Both a mutation at a partially exposed site (H71Y) and a buried core position (I35V) had a limited response in side-chain (2)H-based dynamics when compared to wild-type PDZ2. In contrast, a change at a second core position (I20F) that had previously been shown to be part of an energetic and dynamic network, resulted in extensive changes in side-chain dynamics. This response is reminiscent to that seen previously upon peptide ligand binding. These results shed light on the nature of the PDZ2 dynamic network and suggest that position 20 in PDZ2 acts as a "hub" that is energetically and dynamically critical for transmitting changes in dynamics throughout the PDZ domain.     

Unique features of the skin barrier in naked mole rats reflect adaptations to their fossorial habitat

The stratum corneum (SC), the top layer of the epidermis, is the functional site of the skin barrier and serves to maintain hydration of the body by preventing water loss and thwarting the entrance of pathogens. The naked mole rat (NMR) (Heterocephalus glaber) is a rodent that resides in hypoxic underground tunnels in arid Africa. NMRs are not only hairless; their skin is devoid of glands and pain sensation. To understand how the skin barrier of the NMR is uniquely adapted to this environment, skin samples from the dorsum and ventral abdomen in one adult and one neonate were examined by transmission electron microscopy using both reduced osmium tetroxide to assess overall structure and ruthenium tetroxide post-fixation to assess lipid organization. These findings were compared with that of hairless mice—a well-defined model for skin barrier studies. The plasticity of the skin was evaluated on 10 NMRs from a colony at the Philadelphia Zoo in humid and dry conditions by measuring cutaneous hydration, transepidermal water loss (TEWL), and pH. The epidermal ultrastructure of the NMR differed from hairless mice by having the following features: decreased content of lamellar bodies (LBs), higher LB pleomorphism, periodic presence of abnormal lipid bilayers, and an unusually thick SC. The NMRs developed significant TEWL and a trend toward decreased hydration when subjected to dry conditions. While these features illustrate an imperfect skin barrier in terrestrial mammals, they likely represent adaptations of the poikilothermic NMRs to their unique natural fossorial climate. Prolonged exposure to decreased humidity could possibly lead to adverse health effects in this species.     

Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-gamma expression and decrease of PKA activity

To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca(2+)-dependent PKC isoforms and 2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca(2+)-dependent PKC-gamma but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.     

Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis

Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7–8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (10⁴–10⁵ M⁻¹) and number of binding sites (10⁶–10⁷sites/erythrocyte) for CRP on patients’ erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10 μg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two diseases.