Specific reading impairments--are they to be associated with emotional difficulties?

The assumption that specific learning disabilities are not causally related to emotional disturbances is crucial for the cognitive research approaches that endeavor to isolate specific malfunctioning cognitive-computational processes. Because there are conflicting data regarding the emotional status of subjects with learning difficulties, the purpose of this study was the evaluation of the emotional status of subjects with specific learning difficulties in comparison with that of subjects from several relevant control groups. The HSCL-90 profiles of 38 subjects suffering developmental dyslexia, 28 subjects with subjective complaints regarding general concentration and reading comprehension difficulties, and 23 psychiatric patients were compared with those of 44 skilled readers without any known emotional difficulties. All subjects were adolescents and young adults (age range = 15 to 23). A cluster analysis of subjects' HSCL-90 profiles did not reveal any significant differences between subjects with dyslexia and control subjects. Both groups, on the other hand, were easily differentiated from psychiatric patients. Subjects with severe deficits in their ability to remember details of a text, and with subjective complaints regarding concentration difficulties, tended to generate emotional profiles that testify to a high level of anxiety. Although these subjects, as well as individuals with dyslexia, may be regarded at the functional level as inefficient readers, the two groups seem to clearly differ regarding the failure factors that underlie their phenotypic difficulties.


Differences in arachidonic acid metabolism by human myelomonocytic cell lines.

The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines was determined after incubation with interferon-gamma (IFN-gamma, 500 U/ml) or vehicle for 4 days. Cells were prelabeled with tritiated arachidonic acid, [3H]AA, for 4 h, and media supernatants were analyzed by high-performance liquid chromatography. None of the cell lines produced [3H]AA metabolites in large amounts during an unstimulated, basal release period (30 or 60 min). In response to 10 microM calcium ionophore A23187 incubation (30 min), undifferentiated and IFN-gamma-differentiated HL60 cells formed both cyclooxygenase products (thromboxane and prostaglandins) and lipoxygenase products (leukotrienes and hydroxyeicosatetraenoic acids). In contrast to the HL60 cells, IFN-gamma-differentiated U937 cells formed primarily cyclooxygenase products and undifferentiated and IFN-gamma-differentiated ML3 cells did not form any [3H]AA metabolites in response to A23187. These results indicate the need to be careful in selecting a cell line for use in a phagocyte assay system when cyclooxygenase and/or lipoxygenase products could influence the assay results.


In vivo testing confirms a blunting of the human cell-mediated immune mechanism during space flight.

The cell-mediated immune (CMI) mechanism was evaluated in 10 space shuttle astronauts by measuring their delayed-type hypersensitivity response to seven common recall antigens. The Multitest CMI test system was used to administer antigens of tetanus, diphtheria, Streptococcus, Proteus, old tuberculin, Candida, and Trichophyton to the forearm 46 h before nominal mission termination; readings were conducted 2 h after landing. The mean number of reactions was reduced from 4.5 preflight to 3.0 inflight, and the mean reaction score was reduced from 21.4 to 13.7 mm inflight. The data presented suggest that the CMI system is still being degraded by space flight conditions on day 4 and that between day 5 and day 10, the depression maximizes and the system begins to adjust to the new conditions. The relation of these in vivo findings to previously reported in vitro results is discussed.


Steroidal nonspecific esterase metabolism of N-hydroxy-2-acetylaminofluorene: evidence for selective activation by the cellular reductant NADPH.

The significance of the nonspecific esterases of human mononuclear leukocytes (HMLs) in arylamine carcinogenesis is suggested by data showing that the metabolically formed hydroxamic acid derivative of 2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, is a substrate for this class of enzymes. A viable cell assay for the nonspecific esterases using alpha-naphthyl acetate as substrate is described, and data showing this activity to be sensitive to already known substrates for HML esterases as measured by three previously described assays are presented. All four assays of the same esterase activity are shown to be highly sensitive to up- and down-regulation by addition of NADPH or NADP to viable HML cultures. Selective activation of a purified rabbit nonspecific esterase by NADPH, but not by the other cellular reductants, NADH and glutathione, was demonstrated. Cytosols prepared from normal human tissue samples of liver, breast, colon, and brain were also activated by the presence of NADPH. These data do not indicate that steroidal nonspecific esterases are redox-modulated by the presence of mixed disulfides in their structure. Instead, they support the direct and specific influence of NADPH as a widespread activator of esterase activity by a mechanism not yet understood.


Increase in the rat blood leukocyte counts induced by PAF-acether is suppressed by general anesthesia.

Blood leukocyte count alterations induced by PAF-acether in anesthetized and nonanesthetized rats were investigated. Intravenous injection of increasing amounts of PAF-acether (1.5-8 micrograms/kg) in nonanesthetized animals induced dose-dependent hemoconcentration and leukocytosis. The former was apparent within 10 min, peaked from 30 min to 1 h, and diminished thereafter. The leukocytosis was noted within 30 min, was maximal at 1 h, and was over 4 h after injection of PAF-acether (4 micrograms/kg). It was characterized by a marked increase in the blood neutrophil counts under conditions in which the number of lymphocytes, monocytes, and eosinophils remained unchanged. PAF-acether-induced leukocytosis occurred in parallel with a marked decrease in the number of bone marrow nucleated cells, suggesting that the latter phenomenon may determine the former one. Leukocytosis by PAF-acether was inhibited dose-dependently by specific PAF-acether antagonists including BN 52021 (median effective dose ED50 = 4.99 mg/kg), WEB 2086 (ED50 = 4.59 mg/kg), and 48740 RP (ED50 = 9.02 mg/kg). General anesthesia by either pentobarbital, urethane, or ether inhalation, but not by ketamine, also impaired the PAF-acether-induced blood leukocytosis under conditions in which the hemoconcentration was not modified. In addition, pentobarbital-anesthetized rats did not have reduced bone marrow nucleated cell counts after PAF-acether stimulation. These findings are consistent with the assessment that PAF-acether-induced rat leukocytosis is accounted for by a bone marrow neutrophil mobilization process that is clearly suppressed in animals anesthetized by pentobarbital.


Macrophage and lymphocyte functions are down-regulated in newborn rabbits.

We have developed an animal model for congenital syphilis. Treponema pallidum is injected intravenously into pregnant rabbits and fetuses are infected in utero. As a prelude to characterizing the immunologic consequences of fetal infection, it was necessary to expand on the baseline information about newborn rabbit immune capabilities. Studies were undertaken to determine splenic macrophage and T lymphocyte functions with emphasis on newer immunologic parameters. Newborns aged 2 weeks were compared to adults. Macrophage capabilities in newborn rabbits differed from those of their adult counterparts. These cells produced similar basal levels of interleukin 1 (IL-1) but failed to respond to the IL-1 stimulants of lipopolysaccharide (LPS) or T. pallidum. Macrophages also exhibited diminished levels of la expression and increased levels of prostaglandin E2 (PGE2) secretion. T lymphocyte functions were altered in newborn spleen preparations. Following concanavalin A (Con A) stimulation, interferon gamma production was half that of adults; in direct contrast, IL-2 production was twice that of adults. Con A-induced lymphocyte proliferation was markedly decreased in newborn preparations. This diminished response resulted from down-regulation rather than immaturity. When newborn splenic cells were stimulated with Con A in the presence of indomethacin, anti-transforming growth factor (anti-TGF), or exogenous IL-1/IL-2, better proliferation resulted. PGE2, which is well established as a down-regulator of newborn immune functions in human and mouse systems, also appears to play a role in suppressing newborn rabbit functions. TGF is a potent suppressor of a number of adult immunologic reactions. This is the first documentation of the potential role of this factor in down-regulating newborn immune capabilities. These findings provide a framework for future investigations of our congenital syphilis model.


The up- and down-modulation of immunoglobulin G Fc receptors and complement receptors on activated human neutrophils depends on the nature of activator.

Human neutrophils were activated with soluble stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or ionophore A23187, and with opsonized particles, zymosan or Streptococcus pneumoniae bacteria. Monoclonal antibodies and flow cytometry were used to assess the expression of Fc-gamma receptors (FcRI, FcRII, FcRIII) and complement receptors (CR1, CR3). The role of extracellular calcium and magnesium in the modulation of receptor expression was also examined. The low-level expression of FcRI was not affected by any activator tested. fMLP and A23187 did not alter the expression of FcRII, whereas a significant, Ca(2+)- and Mg(2+)-independent down-modulation was observed upon activation with opsonized particles. All activators clearly decreased the surface expression of FcRIII in the presence of Ca2+ and Mg2+, probably as a consequence of shedding of the phosphatidylinositol-glycan-anchored receptor protein. The removal of calcium and magnesium blocked the shedding of FcRIII caused by soluble stimuli, whereas it retarded but did not abolish the fall in FcRIII expression when cells were incubated with opsonized particles. This fall was likely due to internalization of the receptor molecules while the shedding was blocked. A rapid increase in CR1 and CR3 expression was seen upon activation with soluble stimuli. The change in CR1 expression was independent of extracellular Ca2+ and Mg2+. The increase in CR3 number required an influx of divalent cations. No total up-modulation of complement receptors occurred when neutrophils were activated with opsonized particles. However, the kinetic analysis revealed a temporary up-modulation that was followed by a down-modulation. The results indicate that the expression of both Fc-gamma and complement receptors on human neutrophils is changed upon activation and that the up- and down-modulation of these receptors depends on the nature of activator. We also suggest that in neutrophils the FcRIII down-modulation is the result of both receptor shedding and internalization, while FcRII is down-modulated by receptor internalization.


Chronic granulomatous disease with partial deficiency of cytochrome b558 and incomplete respiratory burst: variants of the X-linked, cytochrome b558-negative form of the disease.

Five male patients from four different families presented with a clinical record of chronic granulomatous disease (CGD): recurrent infections of the skin and/or respiratory tract with catalase-positive microorganisms, sometimes in combination with granulomata and/or abscesses in various organs. These patients differed from "classical" forms of the disease in that their neutrophils, although deficient in killing in vitro of Staphylococcus aureus, contained a decreased but measurable amount of cytochrome b558 (10-60% of normal on a heme basis), causing weak staining in the nitroblue tetrazolium dye test and a depressed respiratory burst after contact of the cells with fluid or particulate activators of the NADPH:O2 oxidoreductase. In the cell-free activation system, the defect in the patients' cells was localized in the membrane fraction. In each of the four families, the cellular abnormalities showed an X-linked inheritance. Fusion experiments performed with the monocytes from these patients and those from patients with classical X-linked, cytochrome b558-negative (Xb(0)) or autosomal, cytochrome b558-positive (Ab+) CGD showed complementation of NADPH:O2 oxidoreductase activity in the latter but not in the former combination. Thus, the unusual CGD patients represent variant forms of Xb(0) CGD, with mutations in the gene coding for the beta subunit of cytochrome b558 that do not cause complete loss of this protein.


Granule-associated flavin adenine dinucleotide (FAD) is responsible for eosinophil autofluorescence.

Unstained human eosinophils exhibit marked autofluorescence in comparison to other leukocytes due to a granule-associated fluorescent substance. Fluorescence spectroscopy of granule extracts reveals excitation maxima at approximately 380 and approximately 450 nm with a single emission at approximately 520, characteristic of flavins. The fluorescent material from eosinophil granule extracts was characterized by fluorescence, high-performance liquid chromatographic, and enzymatic analyses. First, acidification to pH 2.6 resulted in increased fluorescence, indicative of flavin adenine dinucleotide (FAD). Second, because flavin mononucleotide (FMN) and riboflavin cannot be distinguished by acidification, high-performance liquid chromatography was performed and revealed a predominance of FAD and smaller amounts (< 15%) of both FMN and riboflavin. Third, the presence of FAD was clearly demonstrated by reconstitution of the activity of D-amino acid oxidase, a FAD-dependent enzyme, when granule extracts were added to the apoenzyme. Thus, we have identified FAD as the predominant fluorophore in eosinophil granules. The small amounts of FMN and riboflavin detected may result from the hydrolysis of FAD under the acidic conditions of granule extraction. Because fluorescent material is deposited onto target cells by eosinophils, it is possible that granule-associated flavoproteins may act as a source of hydrogen peroxide and/or superoxide, which, in conjunction with eosinophil peroxidase, could yield potent cytotoxic agents.


Transcriptional inhibition of endotoxin-induced monokine synthesis following heat shock in murine peritoneal macrophages.

The role of heat shock proteins (HSP) during the inflammatory response has been controversial. The effect of heat shock (HS) on the synthesis of the monokines tumor necrosis factor (TNF) and interleukin 1 (IL-1) by endotoxin-stimulated thioglycollate-elicited peritoneal macrophages was investigated. HS was deemed to have affected macrophages if a 70 kD HSP appeared on SDS gels; identity of this protein as the highly conserved HSP70 was then confirmed by immunoprecipitation. Maximal increases in HSP70 were apparent 2-5 h after HS at 45 degrees C for 12 min. Synthesis of HSP70 was no longer detected 24 h post HS. A reciprocal relationship between HSP70 and TNF was apparent in kinetic studies. TNF was not detected in culture supernatants if macrophages were endotoxin-stimulated 2 to 6 h after HS; however, the same stimulation 24 h later induced significant TNF secretion. RNA analysis of HS and non-HS macrophage cultures demonstrated a 60-fold reduction in TNF message in the HS macrophages 1 h after endotoxin stimulation. TNF mRNA levels remained depressed at 6 h while the HSP70 message had increased 30-fold. The ability of HS macrophages to ingest antibody-coated erythrocytes was not significantly affected following heat treatment. Macrophage response to HS can be said to inhibit transcription of inducible monokines while retaining other macrophage functions.


Ovine mononuclear phagocytes in situ: identification by monoclonal antibodies and involvement in experimental pyogranulomas.

In order to characterize in situ the macrophages present in experimental pyogranulomas induced in lambs with Corynebacterium pseudotuberculosis, a set of monoclonal antibodies (MAbs) was produced following immunization of BALB/c mice with alveolar macrophages from healthy sheep. Three MAbs were retained after two steps of screening using alveolar macrophages, peripheral blood lymphocytes, and polymorphonuclear leukocytes as target cells. Their reactivity was tested not only on macrophages in pyogranulomas but also on sections of various organs in steady-state conditions. One MAb, termed OM1, recognized the monocytes and the majority of cells of the mononuclear phagocyte system in lymphoid and nonlymphoid organs. The two other MAbs, OM2 and OM3, reacted with a subpopulation of alveolar macrophages and with other cell types in tissues, in particular with endothelial cells for the MAb OM2. On sections of experimental pyogranulomas that developed in lymph nodes draining the C. pseudotuberculosis-injected sites, MAb OM1 reacted with all the macrophages distributed in a palisade surrounding the necrotic center of the lesion from day 6 to day 28 postinoculation. The two other MAbs, OM2 and OM3, enabled two types of granulomas to be distinguished: one type was characterized by a large number of epithelioid cells stained by OM2; and the other was characterized by a few OM2-positive macrophages, whereas the OM3-positive cells were more numerous. These results show that macrophages are predominant cells in pyogranulomas and suggest two different histological patterns in the evolution of pyogranulomas induced by C. pseudotuberculosis, according to the immunological status of the host.


Effects of Pseudomonas aeruginosa rhamnolipids on human monocyte-derived macrophages.

Pseudomonas aeruginosa, a major opportunistic gram-negative pathogen, produces and secretes two heat-stable hemolytic glycolipids, a monorhamnolipid and a dirhamnolipid. In this paper a simplified method for the isolation of these rhamnolipids is described. The effect of these two rhamnolipids, both together and individually, on the viability and structural morphology of human monocyte-derived macrophages (MDMs) was examined. These cells were found to be very susceptible to the cytolytic activity of the rhamnolipids, particularly the dirhamnolipid. The monorhamnolipid, although not as cytolytic as the dirhamnolipid, caused extensive blebbing of the MDM plasma membrane. Comparison studies with several detergents confirmed the different yet distinct detergent-like activity of each rhamnolipid form. At sublethal doses, the rhamnolipids produced marked cellular distortions of the MDMs and inhibited the ability of these cells to bind and/or ingest preopsonized bacteria. The potential mechanism of action of these rhamnolipids on the MDM membranes is discussed, as well as the possible significance of these extracellular bacterial glycolipids as a virulence factor in the pathogenesis of P. aeruginosa.


Direct evidence for release of pore-forming protein during NK cellular lysis.

In the present study, we have examined the exocytosis model for cellular lysis. Using monoclonal antibodies reactive with human pore-forming protein (PFP), we examined the localization of PFP at the interaction site of natural killer (NK) cells and the NK tumor targets K562 and Molt-4 as well as during antibody-dependent cellular cytotoxicity. Following the interaction of effector-target cell contact, an increased frequency of PFP was detected on the effector surface, in the micro-environment, and on the target surface of the interaction site. This temporal deposition of PFP was paralleled by loss of target cell integrity and release of chromium. In addition, selective deposition of PFP was seen at the interaction site of the target cell compared to other target cell regions. Collectively, these results are consistent with the exocytosis model and further support the hypothesis that PFP is one of the secreted moieties involved in NK cellular cytotoxicity.


Liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) are excellent adjuvants for induction of an immune response to protein and tumor antigens.

Liposomes containing the synthetic lipophilic analog of muramyl dipeptide, muramyl tripeptide phosphatidylethanolamine (MTP-PE), were used as adjuvants for the induction of humoral and cellular immune responses following immunization with protein or tumor antigens. Cellular immune reactions, including delayed-type hypersensitivity and lymphoproliferation in vitro, were observed following immunization of mice with a mixture of antigen and liposome-MTP-PE. Immunization with murine melanoma K1735 cells, admixed with liposomal MTP-PE, induced a protective immune response as demonstrated by the rejection of transplanted tumor cells. Antibody production was also induced following immunization with protein antigens admixed with liposome-MTP-PE. The efficacy of adjuvant activity following immunization with antigens admixed with liposome-MTP-PE was equal to or better than that of complete Freund's adjuvant (CFA). Moreover, liposome-MTP-PE did not have the toxic side effects associated with CFA. These data suggest that phospholipid liposomes containing MTP-PE are superior adjuvants and should receive consideration for vaccine therapy.


Actin polymerization contributes to neutrophil chemotactic dysfunction following thermal injury.

The agent(s) and mechanism(s) responsible for suppression of neutrophil chemotaxis in association with major thermal injury have not been identified. We have proposed that the reduced random motility characterizing patients' cells may contribute to their generalized chemotactic dysfunction. Here we report that actin polymerization may be responsible for the loss of neutrophil motility associated with major thermal injury. Using a fluorescent ligand specific for polymerized or filamentous actin (NBD-phallacidin) in conjunction with flow cytometry, we have discovered that peripheral blood and exudate neutrophils from patients with major thermal injury contain increased levels of actin in a stably polymerized form. Because cyclic polymerization and depolymerization of actin is essential to cell motility, we suggest that actin polymerization may contribute in a major way to the attenuation of neutrophil random and chemotactic functions induced by major thermal injury.


Infusion of zymosan-activated plasma affects neutrophils in peripheral blood and bone marrow in sheep.

Intravenous infusion of zymosan-activated plasma (ZAP) in sheep results in acute lung injury mediated in part by free radical release from stimulated neutrophils that are retained in increased numbers in the pulmonary microcirculation. ZAP infusion is also associated with long-term reduction in elicited superoxide anion generation from segmented neutrophils in the circulating and bone marrow pools for at least 24 h after the infusion. The purpose of our study was to investigate the effect of ZAP infusion on leukocyte counts and neutrophil granule-associated enzyme activity in the circulating and bone marrow pools. Cytochemical methods were used to characterize enzyme activity in primary granules (acid phosphatase and myeloperoxidase) and secondary granules (alkaline phosphatase). During the infusion period, neutrophil differential counts decreased less in venous blood than in matched arterial blood. Release from bone marrow stores was evident as increased numbers of circulating band neutrophils during and after the infusion. Neutrophils in venous and arterial blood smears were degranulated acutely during and 1-3 h after the infusion was stopped. Band and segmented neutrophils in bone marrow also appeared slightly degranulated 1-2 h after the infusion. In vitro experiments showed that band and segmented neutrophils in bone marrow degranulated in response to ZAP incubation. Immature polymorphonuclear leukocytes did not degranulate. Five to 24 h after ZAP infusion, enzyme activity in circulating and bone marrow neutrophils was at baseline levels, suggesting that new cells were being released into the circulating pool because degranulated neutrophils do not synthesize new granules. Another indication of a long-term effect in bone marrow were slight decreases in the percentage of immature polymorphonuclear leukocytes and band neutrophils and a significant decrease in the percentage of eosinophils, all of which coincided with a significant increase in the percentage of segmented neutrophils. Our results demonstrate that circulating complement anaphylatoxins affect neutrophils in bone marrow as well as the vascular space.


Ontogenic development of macrophage subpopulations and Ia-positive dendritic cells in fetal and neonatal rat spleen.

Development, differentiation, and distribution of macrophage subpopulations and Ia+ dendritic cells in the fetal and neonatal rat spleen were investigated by means of double immunohistochemical staining and immunoelectron microscopy. To characterize these cell populations, a panel of anti-rat macrophage monoclonal antibodies (RM-1, ED2, ED3, TRPM-3, Ki-M2R) and an anti-rat Ia antibody (OX6) were used. In the fetal rat spleen, macrophages were first detected by RM-1 at fetal day 15. ED2+ and/or Ki-M2R+ macrophages appeared at fetal day 16. TRPM-3+ and/or ED3+ macrophages appeared a day later. During the fetal and neonatal development, ED2+ and TRPM-3+ macrophages differentiated independently, maturing into red pulp macrophages and marginal metallophilic and marginal zone macrophages respectively. Intimate topographical relations were observed between ED2+ macrophages and hematopoietic cells and between TRPM-3+ macrophages and marginal zone lymphocytes. Ia+ cells were first observed around arterioles at fetal day 15. In the fetal and neonatal period, the number of Ia+ cells gradually increased, their shape became dendritic, and they matured into interdigitating cells in the inner periarteriolar lymphatic sheath. In ontogeny, Ia+ dendritic cells were not stained with ED2 or TRPM-3. These results suggest that ED2+ macrophages, TRPM-3+ macrophages, and Ia+ dendritic cells are distinct cell lines that pursue independent developmental process in spleen ontogeny.


Monocyte diapedesis through an in vitro vessel wall construct: inhibition with monoclonal antibodies to thrombospondin.

Human peripheral blood monocytes were examined for migration across an endothelial cell monolayer in an in vitro vessel wall construct. Few monocytes invaded in the absence of a chemotactic gradient, despite significant adhesion to the endothelial monolayer. However, the addition of zymosan-activated human plasma to the lower compartment, to create a chemotactic gradient across the vessel wall, resulted in significantly enhanced monocyte migration. Pretreatment of the monocytes with monoclonal antibodies to thrombospondin (TSP) dramatically inhibited monocyte diapedesis into the vessel wall. The same treatment inhibited monocyte adhesion to endothelial cells in two-dimensional monolayer cultures as well as in vessel wall constructs (no chemotactic gradient). Of interest, however, the monoclonal antibodies had no inhibitory effect on monocyte migration into collagen gels devoid of endothelial cells in response to the same chemotactic gradient, suggesting the importance of TSP in monocyte-endothelial cell interactions. Monoclonal antibodies to fibronectin and normal mouse immunoglobulin G did not inhibit migration in this model of a vessel wall. Furthermore, monoclonal antibodies to TSP showed no inhibition of human peripheral blood neutrophil migration. Previous studies have shown that monocytes synthesize TSP and express this moiety on their surface. The present data suggest that monocytes may utilize TSP to interact with endothelial cells lining the vessel wall during diapedesis.


Activation of human monocyte chemiluminescence response by acylpoly(1,3)galactosides derived from Klebsiella pneumoniae.

The stimulating activity of several preparations isolated from a membrane proteoglycan of a nonencapsulated smooth strain of Klebsiella pneumoniae (Kp-MPG) on the oxidative burst of human blood monocytes was assessed by luminol-enhanced chemiluminescence (CL). Five Kp derivatives were studied: a 34-kd acylpoly(1,3)galactoside (APG), obtained by drastic alkaline hydrolysis and purified by chromatography; an APG preparation subjected to acid hydrolysis that removed the core part and all fatty acids, leaving intact the galactose chain of APG (GC-APG); an APG preparation subjected to mild oxidation (ox APG); a preparation obtained by mild alkaline hydrolysis of Kp-MPG, containing additional ester-linked C14 and C16 fatty acids bound to the APG molecule (EFA-APG); and a polymer of the latter compound, APG pol. EFA-APG directly stimulated monocyte CL, whereas Kp-MPG, APG pol, and the whole bacterial cells had little or no activity. APG itself and ox APG induced a weaker response than EFA-APG. Polymyxin B sulfate completely inhibited the CL response to bacterial lipopolysaccharide (LPS) but not to EFA-APG. The stimulating action of EFA-APG on blood monocytes was dependent on the extracellular levels of both calcium and magnesium. Preincubation of monocytes with monoclonal antibody anti-Mac-1 directed against CD11b, the alpha chain of complement receptor type 3 (CR3; CD11b/CD18), strongly inhibited CL activation by EFA-APG and to a lesser extent CL activation by unopsonized zymosan and rough LPS. Altogether, these findings provide indirect evidence for the contribution of the CD11b/CD18 integrin in the functional interaction of EFA-APG with monocyte membranes. They demonstrate the role of fatty acids in the triggering of monocyte oxidative burst, while the polysaccharide chain itself does not contribute to induction of the CL response in this model. In keeping with the effects of EFA-APG and APG, we show that the monocyte CL response was triggered by bacterial LPS from the rough strain of Salmonella minnesota Re 595 and its lipid A, but not by LPS from smooth strains, again suggesting a critical role for the lipid moiety.


Isolation and characterization of macrophages from rat embryonic muscle culture.

We have previously described the wide distribution of resident macrophages in normal rat skeletal muscle. In this study, we investigated the characteristics of the macrophages that occur in rat embryo muscle cultures. We showed that cells of monocyte-macrophage lineage are present in primary muscle cultures of rat embryos (18 days in gestation) and that these cells form morphologically and phenotypically heterogeneous populations, based on their reaction with monoclonal antibodies ED1, ED2, ED3, and OX43. Constitutively Ia+ cells with dendritic appearance were also observed. Furthermore, we established the procedure for isolation of macrophages from the primary muscle cultures. The isolated cells, mostly ED1+, expressed class I and CD4 antigens and bore complement (C3) receptors on their surfaces. The fact that cell of monocyte-macrophage lineage occur in the embryonic muscle suggests that during embryogenesis these cells may enter the developing muscle and give rise to a population of tissue-associated macrophages.


Augmented expression and release of nonspecific cross-reacting antigens (NCAs), members of the CEA family, by human neutrophils during cell activation.

Nonspecific cross-reacting antigens (NCAs) are a group of human glycoproteins immunologically cross-reactive with carcinoembryonic antigen (CEA). Our previous studies have shown that at least seven NCA glycoproteins different in molecular weight and antigenic reactivity, including a species corresponding to CD67, can be detected in neutrophil granulocytes. In the present paper, it is demonstrated that neutrophil activation induced with soluble stimulators, the calcium ionophore A23187, N-formylmethionyl-leucyl-phenylalanine, and phorbol myristate acetate, results in augmented release and cell surface expression of NCAs. The NCA release was correlated with the discharge of azurophil granules but not with that of specific granules and was attributable to the release of NCA species of 53 and 30 kd. The increased NCA expression on the cell surface was due to increments of the NCAs of 160, 100 (CD67), 95, 90, 30, and 26 kd. These results, together with the previous findings that the CEA family members can mediate intercellular adhesion and bind Escherichia coli in vitro, imply that the neutrophil NCAs participate in the functions of neutrophils such as phagocytosis, chemotaxis, and adherence.


Activation of bovine neutrophils by Pasteurella haemolytica leukotoxin is calcium dependent.

In this study, we used the fluorescent probe Fluo-3 to show that an increase in cytosolic free calcium, [Ca2+]i, occurred when suspensions of bovine neutrophils were incubated with sublethal concentrations of P. haemolytica leukotoxin. This increase in [Ca2+]i was dependent on the concentration of leukotoxin present in the medium and, at a given concentration of leukotoxin, dependent on the external calcium concentration. The calcium channel blocker verapamil and the beta-adrenergic antagonist propranolol inhibited leukotoxin-stimulated Ca2+ gain, as did a neutralizing antileukotoxin monoclonal antibody. As reported previously, incubation of bovine neutrophils with partially purified leukotoxin stimulated a vigorous luminol-dependent chemiluminescence response (LDCL). The present study shows that LDCL stimulation was dependent on the presence of extracellular calcium and was inhibited by the addition of verapamil and propranolol. These data indicate that bovine neutrophils exhibit a considerable increase in cytoplasmic free calcium when they are incubated with P. haemolytica leukotoxin in the presence of external calcium. They also provide evidence that an increased [Ca2+]i is required for functional activation of the bovine neutrophil oxidative burst by P. haemolytica leukotoxin.


Cholesterol esterification rate in plasma depleted of very low and low density lipoproteins is controlled by the proportion of HDL2 and HDL3 subclasses: study in hypertensive and normal middle-aged and septuagenarian men.

The relationship between the fractional rate of cholesterol esterification (FERHDL) in very low density lipoprotein (VLDL)- and low density lipoprotein (LDL)-depleted plasma and the particle size distribution of high density lipoproteins (HDL) were studied in: a) a control group of 9 apparently healthy men (42 +/- 11 years); b) 15 septuagenarians (76 +/- 6 years) who had no clinical signs of coronary artery disease; and c) 32 outpatients with essential hypertension of different stages of severity (51 +/- 10 years). There were small differences between the groups with respect to their plasma total and HDL-cholesterol and plasma triglyceride levels. However, there was a highly significant increase in FERHDL in patients with hypertension compared to control and older men. The HDL of hypertensive patients had a markedly increased relative content of HDL3b, while their HDL2b fraction was reduced by over 50% compared to the other groups. Overall, there was a strong positive correlation between FERHDL and HDL3b (r = 0.89; P less than 0.001) and a negative correlation between FERHDL and HDL2b (r = -0.61; P less than 0.001) and HDL3a (r = -0.77; P less than 0.001). These findings confirm our previous conclusions that FERHDL reflects the relative HDL subclass distribution. In addition, we demonstrate that FERHDL is increased in hypertensive male subjects regardless of the stage of hypertension, i.e., whether or not organic lesions have already become manifest (stage III and stages I plus II, respectively).


Increased serum apoA-IV concentrations in experimental uremic rats.

Normal histochemical analysis localizes apoA-IV within renal proximal tubules, which suggests that the kidney is a major catabolic site. In clinical renal failure and animal models of decreased renal function, low molecular weight proteins cannot be efficiently filtered through the glomerular basement membrane, and therefore they accumulate in plasma. In normal plasma, apoA-IV exists as both lipoprotein associated and lipoprotein-free, low molecular weight forms. To examine this further, uremic serum apolipoprotein and mRNA levels were examined in surgically 5/6 nephrectomized rats. Compared to sham-operated controls, uremic serum apoA-IV was elevated twofold and was distributed to a greater extent in the lipoprotein-free subfraction. Serum triglycerides were unchanged. Despite finding no correlation between serum apoA-IV and triglyceride levels (in either the d less than 1.006 g/ml or 1.006 less than d less than 1.019 g/ml fraction), serum apoA-IV was positively correlated with the renal function parameters of blood urea nitrogen (r = 0.949, P less than 0.001), creatinine (r = 0.952, P less than 0.001), and uric acid (r = 0.903, P less than 0.001). In addition, the concentration of apoA-IV per milligram of renal homogenate protein in uremic rats was significantly higher than that of control rats, whereas there was no difference in the content of apoA-I between the two groups. ApoA-I, apoA-IV, and apoB mRNA levels in hepatic and in intestinal tissue were undistinguishable between the uremic and surgical sham rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Thyroid hormone differentially augments biliary sterol secretion in the rat. I. The isolated-perfused liver model.

Thyroid hormone lowers serum cholesterol and alters sterol metabolic processes. This laboratory has previously reported increased biliary lipid secretion as an early effect of triiodothyronine (T3) in the rat. To evaluate whether the bile lipid action of T3 is a primary or secondary effect, the isolated-perfused rat liver model was used. Red blood cells in lipid-free buffer were used to perfuse livers of euthyroid and methimazole-hypothyroid rats, as well as hypothyroid rats given T3 at intervals before perfusion. Bile flow was maintained by taurocholate perfusion. Hypothyroid rats had elevated pre-perfusion serum cholesterol compared to euthyroid (107 +/- 4 vs. 65 +/- 2 mg/dl) and decreased biliary cholesterol (0.016 +/- 0.001 vs. 0.031 +/- 0.004 mumol/g liver/h) secretion. Serum cholesterol decreased to euthyroid levels by 18 h after T3, an effect that was prevented by bile duct ligation. Bile cholesterol secretion doubled by 18 h, and reached levels twice euthyroid by 42 h, while phospholipid secretion doubled to levels just above euthyroid. The fourfold increase in biliary cholesterol secretion occurred with lipid-free perfusion and unchanging bile acid uptake or output. It occurred without a fall in hepatic lipoprotein cholesterol secretion. Blockade of cholesterol synthesis with lovastatin failed to alter T3-augmented bile cholesterol secretion. We conclude that T3 induces biliary cholesterol secretion concomitantly with the fall in serum cholesterol. This augmented biliary secretion did not appear to depend upon lipoprotein uptake, increased bile acid transport, or cholesterol synthesis. It did not occur at the expense of hepatic lipoprotein secretion. Facilitated biliary lipid secretion may be a primary effect of T3.


Thyroid hormone differentially augments biliary sterol secretion in the rat. II. The chronic bile fistula model.

To further define thyroid hormone effects on bile acid synthesis and biliary lipid secretion, studies were done in chronic bile fistula rats. Euthyroid and methimazole-hypothyroid rats, with and without triiodothyronine (T3) injection, had total bile diversion for timed bile collections. With interrupted enterohepatic circulation, cholesterol absorption is negligible and bile acid secretion equals bile acid synthesis rate. Hypothyroid rats had diminished levels of bile acid synthesis and biliary secretion of cholesterol and phospholipid. Single dose T3 injection produced a 13-fold increase in bile cholesterol secretion and a 3-fold increase in phospholipid secretion, both initiated 12 h after T3. Bile acid synthesis increased by 50%, but the increase did not begin until 24 h after T3. Neither hypothyroidism nor T3 treatment abolished diurnal rhythms of bile acid synthesis and biliary lipid secretion. Inhibition of cholesterol synthesis with lovastatin resulted in a persistent 33% decrease in bile acid synthesis in euthyroid and hypothyroid rats, while bile cholesterol secretion only transiently decreased. Inhibition of cholesterol synthesis did not alter T3-induced bile cholesterol secretion, with a 10-fold increase seen. However, bile acid synthesis was not stimulated by T3 in the presence of lovastatin. We conclude that facilitated bile acid synthesis and biliary cholesterol secretion are early effects of T3 and may account for the hypocholesterolemia of T3. Cholesterol synthesis does not appear to be required for the T3-induced bile cholesterol secretion.


Role of the intestinal acyl-CoA:cholesterol acyltransferase activity in the hyperresponse of diabetic rats to dietary cholesterol.

Contrary to normal rats, diabetic rats are known to develop marked hypercholesterolemia when fed a cholesterol-enriched diet. The triggering factor involved in this hyperresponse has not been identified. With the aim of clarifying the role of the intestinal acyl-CoA:cholesterol acyltransferase (ACAT), we studied the effects of a high fat diet and the changes of intestinal ACAT activity during the early development of streptozotocin-diabetes in rats. Feeding diabetic rats with a diet enriched in cholesterol and saturated fat produced an increase in plasma and in tissue cholesterol as early as 3 days after streptozotocin injection in the absence of hyperphagia. Under these experimental conditions, treatment with insulin or with the ACAT inhibitor CL-277082 significantly reduced the plasma cholesterol to levels measured in nondiabetic rats fed the same high fat diet. An increase in [14C]cholesterol in plasma very low density lipoprotein was observed after oral administration of labeled cholesterol to 3-day diabetic rats. In parallel experiments, the direct measurement of small intestine microsomal ACAT activity revealed an increase, averaging 288% in diabetic rats 3 days after diabetes induction. This change in ACAT activity occurred simultaneously with an increase in plasma glucagon and was normalized by insulin treatment. The induction of intestinal ACAT activity in diabetic rats, its modulation by insulin, and the hypocholesterolemic effects of insulin or CL-277082 treatment clearly indicate that ACAT activity plays a major role in the initiation of diabetes-associated hypercholesterolemia.


Distribution of phosphatidylcholine molecular species between mixed micelles and phospholipid-cholesterol vesicles in human gallbladder bile: dependence on acyl chain length and unsaturation.

The partitioning of phosphatidylcholine (PC) molecular species between mixed micelles and vesicles was studied in each of seven human gallbladder biles. Biles were fractionated by Sephacryl S-300 SF gel filtration chromatography, and PC species in the micellar and vesicular fractions were quantitated by high performance liquid chromatography. Micelles were enriched in species containing unsaturated acyl groups (e.g., 16:1-18:2, 18:1-18:2, and 18:1-18:3); vesicles were enriched in more highly saturated species (e.g., 16:0-16:1, 16:0-18:1, and 18:0-18:1). Separate multivariate analyses for each bile demonstrated that the distribution of PC species between vesicles and micelles was related to the degree of sn-1 and sn-2 unsaturation, and sn-1, but not sn-2, chain length. In addition, the tendency to partition into the micellar phase was particularly marked when unsaturation was present at both the sn-1 and sn-2 positions. When this interaction was included in the multivariate analyses, the regression models accounted for virtually all of the variation in PC partitioning (for each of the seven patients r2 = 0.92-0.98, P less than 0.03). These results suggest that the partitioning of PC species between micelles and vesicles is strictly determined by sn-1 chain length and the degree of unsaturation at both the sn-1 and sn-2 positions. In light of recent reports that fatty acyl composition influences the cholesterol content of vesicles and micelles in model biles, these results raise the possibility that diet-induced alterations in the phospholipid species and the relative proportions of biliary lipid particles may influence the cholesterol-carrying capacity of bile.


Effect of dietary cis and trans fatty acids on serum lipoprotein[a] levels in humans.

Serum lipoprotein[a] (Lp[a]) is a strong risk factor for coronary heart disease. We therefore examined the effect of dietary fatty acid composition on serum Lp[a] levels in three strictly controlled experiments with healthy normocholesterolemic men and women. In Expt. I, 58 subjects consumed a control diet high in saturated fatty acids for 17 days. For the next 36 days, 6.5% of total energy intake from saturated fatty acids was replaced by monounsaturates plus polyunsaturates (monounsaturated fatty acid diet; n = 29) or by polyunsaturates alone (polyunsaturated fatty acid diet; n = 29). Both diets caused a slight, nonsignificant, increase in median Lp[a] levels, with no difference between diets. In Expt. II, 10% of energy from the cholesterol-raising saturated fatty acids (lauric, myristic, and palmitic acid) was replaced by oleic acid or by trans-monounsaturated fatty acids. Each of the 59 participants received each diet for 3 weeks in random order. The median level of Lp[a] was 26 mg/l on the saturated fatty acid diet; it increased to 32 mg/l (P less than 0.020) on the oleic acid diet and to 45 mg/l (P less than 0.001) on the trans-fatty acid diet. The difference in Lp[a] between the trans-fatty acid and the oleic acid diets was also highly significant (P less than 0.001). Expt. III involved 56 subjects; all received 8% of energy from stearic acid, from linoleic acid, or from trans-monounsaturates, for 3 weeks each. All other nutrients were equal.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibitors of sterol synthesis. Chemical synthesis and spectral properties of 3 beta-hydroxy-5 alpha-cholesta-8(14),24-dien-15-one, 3 beta,25-dihydroxy-5 alpha-cholest-8(14)-en-15-one, and 3 beta,24-dihydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

Side-chain functionalized delta 8(14)-15-ketosterols have been synthesized from 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (VI) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Oxidation of VI to the 24-aldehyde VII, followed by Wittig olefination with isopropyltriphenylphosphonium iodide gave 3 beta-acetoxy-5 alpha-cholesta-8(14),24-dien-15-one (VIII), which was hydrolyzed to the free sterol IX. Oxymercuration of VIII followed by hydrolysis of the 3 beta-acetate gave 3 beta,25-dihydroxy-5 alpha-cholest-8(14)-en-15-one (IV). Hydroboration-oxidation of VIII followed by hydrolysis of the 3 beta-acetate gave 3 beta,24-dihydroxy-5 alpha-cholest-8(14)-en-15-one (V) as a 5:4 mixture of the 24R and 24S epimers. 1H and 13C nuclear magnetic resonance (NMR) assignments and mass spectral fragmentation patterns, supported by high-resolution measurements, are presented for IV and its 3 beta-acetate, V, VII, VIII, and IX. Characterization of IV by NMR and of trimethylsilyl ethers of IV and V by gas chromatography-mass spectrometry was compatible with spectral data for samples of IV and V isolated previously after incubation of I with rat liver mitochondria in the presence of NADPH. Sterols IV, V, and IX were very potent in lowering of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells; their potency was comparable to that of I.


Hepatic apolipoprotein J is secreted as a lipoprotein.

Apolipoprotein J (apoJ) is a unique glycoprotein thought to be involved in a variety of physiological processes, including lipid transport, regulation of complement function, sperm maturation, programmed cell death, and membrane recycling. In the plasma, apoJ is associated with apoA-I in high and very high density lipoproteins. In this report we demonstrate that HepG2 human hepatocellular carcinoma cells secrete apoJ in association with a significant amount of lipid, providing unequivocal evidence that apoJ can transport lipids. The HepG2 cell line has provided important clues about the structural organization of nascent lipoprotein particles. HepG2 cell apoJ-containing lipoproteins are dense and heterogenous in size, ranging from 100 to 910 kDa. Plasma and HepG2 cell apoJ-lipoproteins differ in size distribution. Both have alpha 2 electrophoretic mobility, although their average mobilities differ within the alpha 2 region. In contrast to plasma apoJ-HDL which contain little triglyceride and which can associate with apoA-I, HepG2 cell apoJ-lipoproteins are rich in triglyceride and lack apoA-I. By implication, nascent apoJ-lipoproteins undergo plasma remodeling that results in triglyceride depletion and apoA-I association. We propose that the metabolic consequences of this remodeling play an important role in lipid homeostasis in localized tissue environments, particularly where organs are isolated from the blood by cellular barriers such as in testis and brain. In such tissues, apoJ is expressed constitutively in high level compared to other lipid transport proteins.


Physiologic mechanisms for reduced apolipoprotein A-I concentrations associated with low levels of high density lipoprotein cholesterol in patients with normal plasma lipids.

Low plasma concentrations of high density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) are major risk factors for coronary heart disease (CHD). Low HDL levels are common in patients with hypertriglyceridemia, but they also occur in those with normal plasma lipids; the latter include obese patients and cigarette smokers, though other patients with low HDL levels are neither obese nor smokers. The present study was designed to define metabolic causes of low apoA-I levels in normal-weight, normolipidemic patients. ApoA-I tracer studies were carried out in two groups of normolipidemic patients having low HDL levels to determine input rates and residence times for ApoA-I; these patients included 11 nonobese nonsmokers and 11 nonobese cigarette smokers. Their results were compared to those of 20 normal-weight, normolipidemic controls with normal HDL levels and 12 obese nonsmokers also having low HDL. In all three groups manifesting low HDL-cholesterol and low apoA-I levels, residence times for plasma apoA-I were reduced by approximately 30%, compared to control subjects with normal HDL levels. In contrast, average input rates for apoA-I were similar among the three low-HDL patients and control subjects. No differences in apoA-I kinetics were observed among any of the three groups with low apoA-I concentrations. Within each of the four groups of the study, however, input rates for apoA-I were highly correlated with plasma concentrations of apoA-I. Thus, for individuals with normal levels of plasma lipids, both residence times and input rates for apoA-I appeared to be important determinants of apoA-I levels. Residence times for apoA-I were reduced in almost all patients with low apoA-I levels, regardless of concomitant factors, whereas input rates were highly variable among individuals.


Protocol for the study of the metabolism of retinyl esters in plasma lipoproteins during postprandial lipemia.

An efficient protocol is described for the study of the kinetics of retinyl esters in whole plasma and several lipoprotein fractions following the consumption of an oral fat load containing vitamin A (retinol). To allow for a more complete characterization of the kinetics of retinyl esters in different lipoprotein fractions, a simplified two-step ultracentrifugation procedure is reported for the efficient and reproducible isolation of triglyceride-rich chylomicrons from nonfasting subjects, VLDL-sized lipoprotein particles, and the triglyceride-poor lipoprotein fraction. The present method for the determination of retinyl esters is based on the direct application of the lipid fraction onto a normal phase HPLC column without requiring the lipid extract to be desiccated and resolubilized. All of the commonly occurring esters of retinol elute as a single peak with a retention time of 1.6-1.8 min followed by retinyl acetate (serving as the internal standard) and retinol with retention times of 2-2.5 min and 5-5.5 min, respectively. With this system, a new sample can be processed every 10 min and a complete set of 60 samples from a typical oral fat load can analyzed in one working day with minimal technical interaction. By normalizing to the area under the internal standard to correct for variability in the injected volume, the coefficient of variability for the concentration retinyl esters within a single run is less than 5% and less than 10% between runs.


Cryopreservation with sucrose maintains normal physical and biological properties of human plasma low density lipoproteins.

The ability to preserve low density lipoprotein (LDL) preparations frozen for weeks and months without changes in structure or biological properties is of potential use in long-term comparative studies of LDL. We demonstrate that freeze-thawing of LDL causes marked alterations in its structure and biological behavior, and that such changes can be prevented by the addition of sucrose to the LDL solution prior to freezing. Freezing LDL at -70 degrees C in the absence of sucrose resulted in aggregation and fusion of particles as measured by electron microscopy, spectrophotometric absorption, and column gel filtration. This was associated with increased binding affinity of monoclonal antibodies at epitopes distant from the receptor binding region. Functional changes induced by freezing included 3- to 10-fold increases in binding at 4 degrees C and 37 degrees C, and uptake of LDL in fibroblasts, attributable mainly to increases in nonspecific binding processes. Cryopreservation of LDL in 10% sucrose (w/v) completely prevented the structural and functional changes incurred after short-term freezing, and LDL cryopreserved in sucrose for as long as 18 months displayed cell binding, uptake, and degradation very similar to that of freshly obtained LDL.


Apolipoprotein B(Arg3500----Gln) allele specific polymerase chain reaction: large-scale screening of pooled blood samples.

A two-step polymerase chain reaction (PCR) method for the rapid detection of the apolipoprotein B(Arg3500----Gln) mutation in a mixture of pooled blood samples is described. In the first step PCR, a short gene fragment surrounding codon 3500 is amplified. Subsequently the reaction product is subjected to a second amplification in which a mutation-specific primer is used. A PCR product is generated only if the mutant sequence is present in the DNA pool. Individuals carrying the mutation can then be identified by PCR with mutagenic primers and MspI restriction typing, essentially as described by Hansen et al. (J. Lipid Res. 1991. 32: 1229-1233).


Cholesteryl ester storage disease: complex molecular effects of chronic lovastatin therapy.

To better characterize the in vivo effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition on human lipid metabolism, an adolescent male with cholesteryl ester storage disease (CESD) was treated chronically with lovastatin. Therapy was associated with decreased liver-spleen size, improved but not normal serum lipids, a 26% decrease in hepatic cholesteryl ester, a 12% decrease in unesterified hepatic cholesterol, and a fourfold increase in hepatic low density lipoprotein (LDL) receptor protein. Hepatic mRNA levels for the LDL receptor and apolipoprotein (apo) B standardized to levels of hepatic gamma actin mRNA were unchanged with therapy. Kinetic studies revealed no change in the LDL fractional catabolic rate and a decrease in the LDL production rate. Size exclusion chromatography showed striking reductions in plasma very low density lipoprotein (VLDL) cholesterol and intermediate density lipoprotein (LDL) cholesterol but not LDL cholesterol with therapy. Mean LDL particle size and the LDL particle size range were increased by treatment. However, there was no difference in the ability of pretreatment or treatment LDL to bind to the LDL receptor on cultured cells consistent with previous studies in animals, indicating that lovastatin may alter LDL particles to impair interaction with the LDL receptor in vivo but not in vitro. Lovastatin therapy in CESD appears to be clinically beneficial and has complex effects on lipid metabolism that may include a dominant inhibitory effect on hepatic lipoprotein production, posttranscriptionally mediated induction of the LDL receptor, and alterations of LDL particles that interfere with their clearance by the LDL receptor in vivo.


Plasma lipoprotein lipid and Lp[a] changes with substitution of elaidic acid for oleic acid in the diet.

The effect of additional dietary trans fatty acids (7% energy) on plasma lipids was assessed in a double-blind comparison of four separate diets: 1, enriched with butter fat (lauric-myristic-palmitic); 2, oleic acid-rich; 3, elaidic acid-rich; 4, palmitic acid-rich. The total dietary period was 11 weeks and comprised normal foods plus specific fat supplements. In 27 mildly hypercholesterolemic men, total and LDL cholesterol were significantly lower during the 3-week oleic acid-rich diet, and were similar during the other three diets. For the four diets LDL cholesterol levels were in mg/dl: 1, 163; 2, 151; 3, 165; 4, 161. HDL cholesterol was significantly higher with the palmitic acid-rich diet, 42 mg/dl, compared with elaidic acid, 38 mg/dl, which in turn was not lower than with oleic acid, 38 mg/dl. Plasma elaidic acid concentration rose seven-fold with the trans fatty acid diet but did not increase the vulnerability of LDL to oxidative change. The elaidic acid-rich diet led to significant elevations in the level of Lp[a] compared to all the other test diets. The Lp[a] level increased to 296 +/- 220 U/l in the elaidic acid-rich period from 235 +/- 182 (mean +/- SD) in the first ("butter") period (P less than 0.001) compared with 249 +/- 204 in the palmitic acid period (P less than 0.001) and 236 +/- 201 in the oleic acid period (NS).(ABSTRACT TRUNCATED AT 250 WORDS)


ApoB-75, a truncation of apolipoprotein B associated with familial hypobetalipoproteinemia: genetic and kinetic studies.

We have identified a mutation of apolipoprotein B (apoB) in a kindred with hypobetalipoproteinemia. Four affected members had plasma concentrations of total cholesterol of 115 +/- 14, low density lipoprotein (LDL)-C of 48 +/- 11, and apoB of 28 +/- 9 (mg/dl mean +/- SD). The values correspond to approximately 30% the values for unaffected relatives. Triglyceride and high density lipoprotein (HDL)-C concentrations were 92 +/- 50 and 49 +/- 4, respectively, neither significantly different from unaffected relatives. Western blots of plasma apoB of affected subjects showed two major bands: apoB-100 and an apoB-75 (mol wt of approximately 418,000). DNA sequencing of the appropriate polymerase chain reaction (PCR)-amplified genomic DNA segment revealed a deletion of the cytidine at nucleotide position 10366, resulting in a premature stop codon at amino acid residue 3387. In apoB-75/apoB-100 heterozygotes, two LDL populations containing either apoB-75 or apoB-100 could be distinguished from each other by gel permeation chromatography and by immunoblotting of nondenaturing gels using monoclonal antibodies B1B3 (epitope between apoB amino acid residues 3506-3635) and C1.4 (epitope between residues 97-526). ApoB-75 LDL were smaller and more dense than apoB-100 LDL. To determine whether the low concentration of apoB-75 was due to its enhanced LDL-receptor-mediated removal, apoB-75 LDL were isolated from the proband's d 1.063-1.090 g/ml fraction (which contained most of the apoB-75 in his plasma) by chromatography on anti-apoB and anti-apoA-I immunoaffinity columns. The resulting pure apoB-75 LDL fraction interacted with the cells 1.5-fold more effectively than apoB-100 LDL (d 1.019-1.063 g/ml). To determine the physiologic mechanism responsible for the hypobetalipoproteinemia, in vivo kinetic studies were performed in two affected subjects, using endogenous labeling of apoB-75 and apoB-100 with [13C]leucine followed by multicompartmental kinetic analyses. Fractional catabolic rates of apoB-75 VLDL and LDL were 2- and 1.3-fold those of apoB-100 very low density lipoprotein (VLDL) and LDL, respectively. Production rates of apoB-75 were approximately 30% of those for apoB-100. This differs from the behavior of apoB-89, a previously described variant, whose FCRs were also increased approximately 1.5-fold relative to apoB-100, but whose production rates were nearly identical to those of apoB-100. Thus, in contrast to the apoB-89 mutation, the apoB-75 mutation imparts two physiologic defects to apoB-75 lipoproteins that account for the hypobetalipoproteinemia, diminished production and increased catabolism.


Chemiluminescent simultaneous determination of phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide in the liver and brain of the rat.

The quantification of phospholipid hydroperoxides in biological tissues is important in order to know the degree of peroxidative damage of membrane lipids. For this purpose, optimal conditions for the chemiluminescent simultaneous assay of phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) in rat liver and brain were determined. A chemiluminescence detection-high performance liquid chromatography (CL-HPLC) method that incorporates cytochrome c and luminol as a post-column hydroperoxide-specific luminescent reagent was used (Miyazawa et al. 1987. Anal. Lett. 20: 915-925; Miyazawa. 1989. Free Radical Biol. Med. 7: 209-217). An n-propylamine-bound silica column with hexane-2-propanol-methanol-water 5:7:2:1 (v/v/v/v) (flow rate 1.0 ml/min) as eluant was used to determine both PCOOH and PEOOH, which were separated from each other and from other lipids and lipid-soluble antioxidants. High reproducibility and sensitivity as low as 10 pmol hydroperoxide-O2 were observed with a mixture of 10 micrograms/ml cytochrome c and 2 micrograms/ml luminol in 50 mM borate buffer (pH 10.0, flow rate 1.1 ml/min) as luminescent reagent and a post-column mixing joint temperature of 40 degrees C. Using the established analytical conditions, it was confirmed that both PCOOH (1324 +/- 122 pmol/g liver, 114 +/- 18 pmol/g brain, mean +/- SD) and PEOOH (728 +/- 89 pmol/g liver, 349 +/- 60 pmol/g brain, mean +/- SD) are present in the liver and brain of Sprague-Dawley rats bred on a slightly modified AIN-76A semisynthetic diet for 3 months. The phospholipid hydroperoxide content in the rat liver was shown to be affected by dietary oils, but not significantly affected in the brain.


Improved preparation method for lysogangliosides.

Lyso-GM3 and -GM1 gangliosides were prepared from the corresponding N,N'-dideacylated gangliosides using N-trifluoroacetylation at the sphingosine moiety, followed by N-acetylation and mild saponification. The blocking reaction was performed using a water-ether bilayer system at alkaline medium, in which the N-trifluoroacetylation occurred predominantly at the lipid moiety. Through the procedure, lysoGM3 and lysoGM1 were obtained with higher yields from the corresponding dideacylated gangliosides than through the previous method using 9-fluorenylmethoxycarbonyl chloride as a blocking group or of direct N-acetylation of it on liposomes containing starting ganglioside and other lipid. Chemical structures of the lysogangliosides and the synthetic intermediates were confirmed by the proton nuclear magnetic resonance spectrometry and negative fast atom bombardment-mass spectrometry.


A simple and rapid method for the preparation of apolipoproteins for electrophoresis.

A new method is described for the rapid preparation of apolipoproteins for polyacrylamide gel electrophoresis. It is suitable for all serum lipoproteins including chylomicrons. The procedure involves extraction with diethyl ether in the presence of trichloroacetic acid and sodium deoxycholate. The method gives an improved protein recovery, in particular with chylomicrons. In addition, samples do not require dialysis to remove salts (e.g., KBr) prior to processing; as a consequence, the procedure requires only 1 h. Due to this rapidity and the high yields, the procedure is superior to present methods utilizing ethanol-diethyl ether extraction.


Sitosterolemia.

Sitosterolemia is a rare inherited lipid storage disease characterized chemically by the accumulation of plant sterols and 5 alpha-saturated stanols in plasma and tissues. Very low cholesterol synthesis due to a deficiency of HMG-CoA reductase associated with increased intestinal plant sterol absorption and slow hepatic sterol removal are major biochemical features. Because cholesterol synthesis cannot up-regulate, bile acid malabsorption mobilizes body sterols for bile acid synthesis and dramatically lowers plasma and monocyte sterol concentrations and may halt the progression of the atherosclerotic process.


Computational sequence analysis revisited: new databases, software tools, and the research opportunities they engender.

The increasing quantity and complexity of sequences and structural data for proteins and nucleic acids create both problems and opportunities for biomedical researchers. Fortunately, a new generation of practical computer tools for data analysis and integrated information retrieval is emerging. Recent developments in fast database searching, multiple sequence alignment, and molecular modeling are discussed and windows-based, mouse-driven software for CD-ROM and network information retrieval are described. Each method is illustrated with a practical example pertinent to lipid research. In particular, the connection among cholesteryl ester transfer protein, bactericidal permeability-increasing protein, and lipopolysaccharide-binding proteins is determined; novel repetitive sequence motifs in mammalian farnesyltransferase subunits and related yeast prenyltransferases are derived; biochemical insights from a three-dimensional model of human apolipoprotein D based on two insect lipocalins are discussed; the relationship between apolipoprotein D and gross cystic disease fluid protein from human breast is reviewed; and prospects for modeling apolipoprotein E-related proteins are described. In addition, information on a number of general and special-purpose sequence, motif, and structural databases is included.


Lipoprotein lipase in plasma after an oral fat load: relation to free fatty acids.

Lipoprotein lipase (LPL) releases fatty acids from triglyceride-rich lipoproteins for use in cellular metabolic reactions. How this hydrolysis, which occurs at the vascular endothelium, is regulated is poorly understood. A fatty acid feedback system has been proposed by which accumulation of fatty acids impedes LPL-catalyzed hydrolysis and dissociates the enzyme from its endothelial binding sites. We examined this hypothesis in humans who were subjected to an oral fat tolerance test of a mixed-meal type. Plasma triglycerides, free fatty acids, and LPL activity were measured before and repeatedly during a 12-h period after intake of the fat load. Since soybean oil with a high content of linoleic fatty acid was the source of triglycerides, a distinction could be made between endogenous free fatty acids (FFA) and FFA derived directly from lipolysis of postprandial triglyceride-rich lipoproteins. Mean LPL activity was almost doubled (P less than 0.01) 6 h after intake of the oral fat load. The rise in LPL activity was accompanied by an increase of plasma triglycerides and linoleic free fatty acids (18:2 FFA), but not of total plasma FFA, which instead displayed a heterogeneous pattern with essentially unchanged mean levels. The postprandial response of LPL activity largely paralleled the postprandial responses of 18:2 FFA and triglycerides. The highest degree of parallelism was seen between postprandial 18:2 FFA and LPL activity levels. Furthermore, the integrated response (area under the curve, AUC) for plasma measurements of LPL correlated with the AUC for 18:2 FFA (r = 0.40, P less than 0.05), but not with the AUC for plasma triglycerides (r = 0.21, ns). The high degree of parallelism and significant correlation between postprandial plasma LPL activity and 18:2 FFA support the hypothesis of fatty acid control of endothelial LPL during physiological conditions in humans.


Calcium binding to phospholipid: structural study of calcium glycerophosphate.

To consider possible interaction of the phospholipid membrane with calcium ions, crystal structures of calcium dl-alpha- and beta-glycerophosphates (alpha- and beta-CaGs, respectively) were investigated by X-ray diffraction methods. After many attempts, relatively large single crystals of beta-CaG were prepared from the aqueous solution containing HCl, while crystals of CaHPO4.2H2O were obtained from alpha-CaG solution under the same crystallization conditions. The crystal structure of beta-CaG is orthorhombic with space group Pna2(1) and cell dimensions of a = 8.251(1), b = 13.038(3), c = 25.483 (10) A, V = 2741.5 (13) A3 and Z = 16 [four molecules (A to D) in an asymmetric unit]. Molecules of A to D took, as a whole, similar extended conformations, although A and B were different from C and D in the orientation about a glycerol C-C bond. Four independent beta-glycerophosphates commonly act as two types of bidentate ligands, where one is the coordination to the calcium ion by the glycerol O(1) and phosphate O(22) atoms, and the other by the phosphate O(22) and O(23) atoms, thus forming the calcium coordination of a distorted square plane, respectively. Each of four independent calcium ions forms the same coordination geometry of a distorted pentagonal bipyramid. Infinite double layers consisting of alternate A/B molecules and of alternative C/D ones and sandwiching calcium ions were arranged face-to-face along the b-direction and were piled up in the a-direction, thus forming the stacked bilayer unit with the thickness of d002 = 12.75 A. The elaborate networks of calcium coordinations and hydrogen bondings were formed among the layers and stabilized the crystal structure. Based on the structural parameters of the present beta-CaG crystal, a possible interaction model of phospholipid with calcium ions was proposed.


Inhibition of lipoprotein lipase activity by synthetic peptides of apolipoprotein C-III.

In this study we have examined effects of synthetic polypeptide fragments of apoC-III on the kinetic properties of lipoprotein lipase (LPL) activity. Based on the loss of 79% of LPL-inhibitory activity after CNBr cleavage at the N-terminal portion of apoC-III and a systematic search for synthetic peptides with LPL-inhibitory activity spanning the apoC-III sequence, we concluded that the N-terminal domain is the most important in the modulation of LPL activity. In addition, there are multiple attachment sites in apoC-III for its interaction with LPL and these sites reside in the hydrophilic sequences of apoC-III. Probably for this reason the intact apo-CIII exhibited higher inhibitory potential than its peptide components. Based on the deduced inhibition constants derived for the synthetic apoC-III1-79 we concluded that apoC-III is likely to exhibit a physiological role in regulating LPL activity since the derived dissociation constants for the LPL-apoC-III interaction are within the physiological concentration range of plasma apoC-III. In addition, as the synthetic apoC-III1-79 lacks the carbohydrate moiety, we also concluded that the presence of the oligosaccharide in native apoC-III is not essential for its inhibitory activity on LPL. The fact that the I50 (concentration for inhibition of LPL at 50% activity) decreases for apoC-III-1 when assayed in the presence of apoC-II indicated that the activator actually caused an increased affinity between LPL and apoC-III and demonstrated that apoC-III does not compete for the activator site of apoC-II.


Apolipoproteins, membrane cholesterol domains, and the regulation of cholesterol efflux.

Published data related to both cell membrane biology and apolipoprotein structure are reviewed and used to formulate a new model describing the mechanisms of cholesterol efflux from cell plasma membrane to high density lipoprotein (HDL) particles. The central premise of this model is the existence of heterogenous domains of cholesterol within plasma membranes. We propose that cholesterol efflux from cell membranes is influenced by three factors: 1) the distribution of cholesterol between cholesterol-rich and cholesterol-poor membrane domains, 2) the diffusion of cholesterol molecules through the extracellular unstirred water layer, and 3) the transient interaction of segments of the amphipathic helix of the HDL apolipoprotein with cholesterol-poor membrane domains resulting in enhanced cholesterol efflux.


Molecular basis of mitochondrial fatty acid oxidation defects.

A dozen separate inherited disorders of mitochondrial fatty acid beta-oxidation have been described in humans. This represents about half of the potential sites for genetic error that can affect this important pathway of energy metabolism. As the characterization of these disorders at the clinical and biochemical levels has progressed rapidly, so has the delineation of the molecular defects that underlie them. The most commonly recognized disorder of beta-oxidation is medium-chain acyl-CoA dehydrogenase deficiency; a striking feature of this disorder is that there is a single point mutation that accounts for 90% of the variant alleles among patients with medium-chain acyl-CoA dehydrogenase deficiency. Molecular defects of other enzymes in the pathway have been identified, and it seems likely that a complete description of these defects at the molecular level is a realistic goal. In basic biological terms, such studies will lead to a better understanding of the genetic control exerted on this pathway. In clinical terms, they will lead to improved understanding of the molecular pathophysiology of these diseases and may well provide the necessary techniques to proceed with the screening of these disorders.


Ionization and self-association of unconjugated bilirubin, determined by rapid solvent partition from chloroform, with further studies of bilirubin solubility.

Our studies of equilibrium solubilization of crystals of unconjugated bilirubin (UCB) in buffered aqueous NaCl (1988. J. Lipid Res. 29: 335-348) suggested that the two carboxylic pKa values were 6.8 and 9.3 and the solubility of UCB diacid was 0.1 microM. These data, however, were not ideal, due to possible effects of crystal size, metastability, 96-h incubation times with formation of polar derivatives, impurities in the bilirubin, and imprecision of analyses at low concentrations of UCB ([UCB]). In the present study, designed to determine the pKa values and self-association of UCB, these problems were minimized by solvent partition of UCB from solution in CHCl3 into buffered aqueous NaCl. There was no crystal phase. Equilibrium was attained rapidly (10 min); UCB and CHCl3 were highly purified; and accurate diazo assay of low [UCB] in the aqueous phase, [Bw], was achieved by concentrating the UCB through back-extraction into a small volume of CHCl3. By determining effects on partition rations of varying the [UCB] in the CHCl3 phase, [Bc], we could assess also the self-association of UCB species in the aqueous phase. Partition ratios (P = Bw/Bc) did not differ between initial and repeat extractions, indicating insignificant concentrations of polar UCB derivatives. Similar P ratios were obtained when equilibrium was approached from a supersaturated aqueous phase. At 21-25 degrees C, mu = 0.15, the data (n = 76) fit the equation: log P = log Po + log[1 + 10(pH-A) + 10(2pH-B) + Bc.10(4pH-D)]; the bracketed terms reflect P for H2Bo (diacid), HB- (monoanion), B= (dianion), and (B=)2 dimer, respectively. Computer-fitted values for constants (+/- SD) were: Po = P for H2Bo = 5.79 x 10(-5); A = pK1 = 8.12 +/- 0.23; B = pK1 + pK2 = 16.56 +/- 0.10; pK2 = 8.44 +/- 0.33; D = pk22 + 2(pK1 + pK2) -log(2Po) = 37.64 +/- 0.07, and k22 = 0.26 microM-1 [formation constant of (B=)2 dimer]. In ancillary studies, multiple cycles of direct dissolution of UCB crystals revealed a progressive decrease in aqueous solubility of UCB as fine crystals were removed; this effect was minimal in CHCl3. Unlike in water, moreover, varied UCB crystal forms had similar solubilities in CHCl3, with [Bc] = 1.14 mM at saturation. As determined from [Bc]sat.Po, the aqueous solubility of H2Bo was 66 nM.(ABSTRACT TRUNCATED AT 400 WORDS)


HepG2 cell LDL receptor activity and the accumulation of apolipoprotein B and E in response to docosahexaenoic acid and cholesterol.

In the present study, the accumulation of apolipoproteins (apo) A-I, B, and E in culture medium was measured after 0, 3, 6, 12, and 24 h of incubation with 150 microM docosahexaenoic acid complexed to 75 microM bovine serum albumin (BSA-22:6), either in the presence or absence of 50 micrograms/ml cholesterol and 4 micrograms/ml 25-hydroxycholesterol (C/25-OH). HepG2 cells incubated with BSA + C/25-OH for 24 h accumulated approximately 2.0-fold greater apoE and B as compared to BSA-treated cells. Moreover, HepG2 cell apoB accumulation after 24 h of BSA-22:6 treatment was approximately 2.0-fold greater than apoB accumulation from cells treated with BSA alone. When BSA-22:6 and C/25-OH were both included in the incubation, apoB accumulation was approximately 5.0-fold greater than BSA-treated cells. Comparative studies using BSA-18:1 were carried out for 24 h and showed similar levels of apoA-I, B, and E accumulation in culture medium as compared to BSA-22:6-treated cells. In addition, apoA-I, B, and E mRNA abundance were found to be unaffected by type of fatty acid treatment or length of incubation, averaging 48.2 +/- 7.5, 222 +/- 33.6, and 17.1 +/- 0.7 pg mRNA/micrograms RNA (mean +/- SEM), respectively. As the accumulation of apoB and apoE in culture medium may be modified by HepG2 cell LDL receptor expression, LDL receptor mRNA abundance and LDL receptor activity were quantified at various times over the course of the study. By 6 h of BSA + C/25-OH treatment, LDL receptor mRNA was reduced approximately 2.3-fold, while receptor activity was reduced approximately 1.5-fold, as compared to BSA controls. In an experiment designed to determine uptake of HepG2 cell lipoproteins, 3H-labeled apoB-containing lipoproteins derived from HepG2 cells were prepared. The 3H-labeled lipoproteins were 1.25-fold more likely to be removed from the media of HepG2 cells treated with BSA than from cells treated with BSA + C/25-OH. From these results, we postulate that HepG2 cell LDL receptor activity mediates the removal of apoB, E-containing lipoproteins from culture medium and contributes to the lower accumulation of apoB and E observed in culture medium from cells treated with BSA as compared to cells treated with C/25-OH.


Discrimination between forms of vitamin E by humans with and without genetic abnormalities of lipoprotein metabolism.

To study the mechanisms of discrimination between various forms of vitamin E, four normal subjects, one patient with lipoprotein lipase deficiency, and three patients with abnormal apolipoprotein B-100 production were given an oral dose containing three tocopherols labeled with differing amounts of deuterium (2R,4'R,8'R-alpha-(5,7-(C2H3)2)tocopheryl acetate (d6-RRR-alpha-tocopheryl acetate), 2S,4'R,8'R-alpha-5-(C2H3)tocopheryl acetate (d3-SRR-alpha-tocopheryl acetate), and 2R,4'R,8'R-gamma-(3,4-2H)tocopherol (d2-RRR-gamma-tocopherol). The tocopherol contents of plasma, red cells, and lipoproteins were measured up to 76 h after the dose. In normal subjects all three tocopherols were absorbed and secreted in chylomicrons with equal efficiencies. Both d2-gamma- and d3-SRR-alpha-tocopherols peaked at similar concentrations in the other lipoprotein fractions, then decreased similarly, but 2-4 times more rapidly than did d6-RRR-alpha-tocopherol. A lipoprotein lipase-deficient patient and a patient with prolonged production of chylomicrons with absent apolipoprotein B-100 also demonstrated the lack of discrimination between tocopherols during absorption. Despite abnormal apolipoprotein B-100 production in two patients, the "VLDL" was preferentially enriched in d6-RRR-alpha-tocopherol. Our results show that there is no discrimination between the three tocopherols during absorption and secretion in chylomicrons, but subsequently there is a preferential enrichment of very low density lipoprotein (VLDL) with RRR-alpha-tocopherol. Catabolism of this VLDL results in the maintenance of plasma RRR-alpha-tocopherol concentrations.


Effects of psyllium hydrophilic mucilloid on LDL-cholesterol and bile acid synthesis in hypercholesterolemic men.

The goal of the current study was to determine the mechanism of the hypocholesterolemic effect of psyllium using a randomized, double-blind, crossover design. Twenty males (age 44 +/- 4 yr, weight 79 +/- 10 kg) with moderate hypercholesterolemia (total 265 +/- 17 mg/dl, low density lipoprotein (LDL) 184 +/- 15 mg/dl) were studied at baseline (B) and after randomization to receive a 40-day course of 15 g/day of either psyllium (Ps) or placebo (Pl) (cellulose). After a washout period (11 +/- 2 days), subjects were crossed over to the other fiber treatment for an additional 40 days and restudied. Intestinal cholesterol absorption, cholesterol synthesis in isolated peripheral blood mononuclear cells, bile acid kinetics, gallbladder motility, and intestinal transit were measured at each study period. Psyllium lowered LDL cholesterol (x:184 (B), 169 (Ps), and 179 (Pl) mg/dl; Ps vs. B,Pl: P less than 0.004, P less than 0.02), decreased relative cholesterol absorption (x:51 (B), 45 (Ps), and 49 (Pl) %; Ps vs. B,Pl: P less than 0.03, P less than 0.03), did not alter absolute cholesterol absorption, and increased the fractional turnover of both chenodeoxycholic acid (x:0.176 (B), 0.203 (Ps), and 0.170 (Pl) day-1; Ps vs. B,Pl: P less than 0.0001, P less than 0.01) and cholic acid (x:0.303 (B), 0.411 (Ps), and 0.301 (Pl) d-1; Ps vs. B, Pl: P less than 0.006, P less than 0.002). Bile acid synthesis increased in subjects whose LDL cholesterol was lowered by more than 10% (Ps vs. B: 1304 +/- 489 vs 992 +/- 307 mumol/day, P less than 0.006; Ps vs. PI: 1304 +/- 489 vs. 914 +/- 321 mumol/day, P less than 0.0002). We conclude that psyllium lowers LDL cholesterol primarily via stimulation of bile acid synthesis.


Differing effects of cholesterol and taurocholate on steady state hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase activities and mRNA levels in the rat.

We investigated the effects of cholesterol, cholestyramine, and taurocholate feeding on steady state specific activities and mRNA levels of hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and cholesterol 7 alpha-hydroxylase in the rat. Interruption of the enterohepatic circulation of bile acids (cholestyramine feeding) increased total HMG-CoA reductase activity 5-fold. Cholesterol and taurocholate administration suppressed total microsomal HMG-CoA reductase activities 87% and 65%, respectively. HMG-CoA reductase mRNA levels increased 3-fold with cholestyramine, did not decrease significantly with cholesterol feeding, but were markedly decreased after taurocholate treatment. Cholesterol 7 alpha-hydroxylase activity increased 4-fold with cholestyramine and 29% during cholesterol feeding, but decreased 64% with taurocholate. Cholesterol 7 alpha-hydroxylase mRNA levels rose 150% and 50% with cholestyramine and cholesterol feeding, respectively, but decreased 73% with taurocholate. The administration of cholesterol together with taurocholate prevented the decline in cholesterol 7 alpha-hydroxylase mRNA levels, but inhibition of enzyme activity persisted (-76%). Hepatic microsomal cholesterol concentrations increased 2-fold with cholesterol feeding but did not change with taurocholate or cholestyramine treatment. These results demonstrate that mRNA levels of HMG-CoA reductase are controlled by the hepatic taurocholate flux, whereas mRNA levels of cholesterol 7 alpha-hydroxylase are controlled by the cholesterol substrate supply. These end products, cholesterol and bile acids, exert post-transcriptional regulation on HMG-CoA reductase and cholesterol 7 alpha-hydroxylase, respectively.


beta-Glucocerebrosidase activity in murine epidermis: characterization and localization in relation to differentiation.

The intercellular lipids of the stratum corneum, which are highly enriched in ceramides, are critical for the mammalian epidermal permeability barrier. During the terminal stages of epidermal differentiation, the glucosylceramide content is dramatically reduced, while the content of free ceramides increases. To investigate whether beta-glucocerebrosidase (beta-GlcCer'ase) could be responsible for this change in lipid content, we characterized its activity in murine epidermis, compared enzyme activity to other murine tissues, and localized beta-GlcCer'ase activity within the epidermis. Epidermal extracts demonstrated linear 4-methylumbelliferyl-beta-D-glucose hydrolysis (to 3 h) with protein concentrations between 1 and 250 micrograms/ml. Whole epidermis contained comparable beta-glucosidase activity (9.1 +/- 0.4 nmol/min per mg DNA) to murine brain and liver, and 5-fold higher activity than spleen. Epidermal beta-glucosidase activity was stimulated greater than 15-fold by sodium taurocholate at pH 5.6, and inhibited at acidic pH (3.5-4.0). Bromoconduritol B epoxide (greater than or equal to 1.0 microM), inhibited epidermal enzyme activity by greater than 75%, while activity in brain, liver, and spleen was only inhibited by 6, 17, and 14%, respectively. Moreover, beta-GlcCer'ase mRNA expression in murine epidermis exceeded levels in liver, brain, and spleen. Finally, beta-GlcCer'ase activity was highest in the outer, more differentiated epidermal cell layers including the stratum corneum. In summary, mammalian epidermis contains an usually high percentage (approximately 75%) of beta-glucocerebrosidase activity, and the concentration of activity in the more differentiated cell layers may account for the replacement of glucosylceramide by ceramides in the outer epidermis.


Preparation of lyso-GM1 (II3Neu5AcGgOse4-long chain bases) by a one-pot reaction.

A simple procedure is described for preparing lyso-GM1, a GM1 derivative that lacks the fatty acid moiety, starting from GM1 ganglioside using a one-pot reaction. Ganglioside deacylation was carried out in KOH/propan-1-ol in the absence of oxygen. The yield of lyso-GM1 under optimal conditions (6 h, 90 degrees C, 0.2 N KOH, 1 mM GM1) was 54%. The chemical structure of lyso-GM1 was determined by 1H-NMR and FAB-MS analyses, thus proving that the acetamide groups of galactosamine and sialic acid units were not affected during the deacylation reaction.


Galactosylceramide containing omega-amino-fatty acids: preparation, characterization, and sulfotransferase acceptor.

For preparation of an affinity ligand, an N-fatty acyl moiety of galactosylceramide (GalCer) was chemically replaced with omega-amino-fatty acid including amino-n-hexanoic acid or amino-n-dodecanoic acid to obtain omega-aminoGalCer. For the synthesis of the compound, galactosylsphingosine (GalSph) was coupled with N-trifluoroacetyl omega-amino-fatty acid which was prepared by a reaction with S-ethyltrifluorothioacetate. After removal of the N-trifluoroacetyl group in a mild alkaline solution, in which an N-fatty acyl group was retained, aminoGalCer composed of an N-hexanoyl or an N-dodecanoyl group was obtained with an overall yield of 90%. Their chemical structures were confirmed by proton nuclear magnetic resonance and fast atom bombardment-mass spectrometries. These aminoGalCers and GalSph as well as immobilized aminoGalCer were sulfated by a glycolipid sulfotransferase from rat kidney. Furthermore, immobilized aminoGalCer on gel matrix was used for affinity chromatography of the sulfotransferase, resulting in an excellent increase in the purification (14,000-fold) with a recovery rate of 40%.


Improved procedures for the determination of lipid phosphorus by malachite green.

We have developed two procedures for the measurement of lipid phosphorus based on interaction between phosphomolybdenum and malachite green. One method, the "micro" assay uses 50-200 microliters of HClO4 and has a sensitivity range of 0.01-1.5 micrograms phosphorus. The second method, the "macro" assay, has a sensitivity range of 0.03-5.0 micrograms phosphorus with 100-500 microliters HClO4. Both assays are very reproducible with day to day standard deviations of less than 6% between triplicates irrespective of the HClO4 content used. At different concentrations of HClO4, each method was successfully used to determine the phosphorus content in phosphatidylethanolamine and sphingomyelin standards that covered the proposed sensitivity ranges. The increased range, sensitivity and greater volumes of HClO4 permitted in the procedures represent significant improvements over existing methods.


[Circulating lupus coagulants. A paradox in vascular pathology].

Lupus anticoagulants (LA) are acquired inhibitors of coagulation related to the antiphospholipid antibodies. Paradoxically, these anticoagulants do not expose patients to the risk of hemorrhage but, on the contrary, to a thrombotic risk. The association in a patient of an antiphospholipid antibody and a clinical manifestation (thrombosis or equivalent) defines the antiphospholipid syndrome. This syndrome is termed primary or secondary according to whether it appears as an isolated disorder or is associated with an identified disease, frequently autoimmune (systemic lupus erythematous or lupus related syndrome). Clinical complications of LA are arterial or venous thrombosis at various sites. They are frequently recurrent, and deep venous thrombosis of leg, oculo-cerebral ischemic lesions and heart valve complications have all been well documented. Thrombosis of the microcirculation can cause tissue or organ disfunction, the most characteristic effect being repeated abortions. Laboratory demonstration of LA is difficult when the inhibitor is weak, and this should be completed by tests for other antiphospholipid antibodies. Clinical studies are necessary to assess the thrombotic risk of the LA in different clinical conditions and to evaluate the need and type of antithrombotic treatment. The LA are heterogeneous and only a small proportion of patients with LA will develop thrombosis. New tests capable of predicting the thrombotic risk, bases on the physiopathological mechanisms with which LA interfere in vivo, are currently being investigated.


[Surgery of aneurysms of the abdominal aorta in 1992. Lessons of a decade].

Perioperative mortality of abdominal aortic aneurysm (AAA) remains at a mean of 3% in asymptomatic cases, 6% when symptomatic and 50% when ruptured while many patients with ruptured AAA die prior to surgery. This has changed little over the last decade. The causes of this stagnation are analyzed, strategic choices as a function of clinical situations, size of aneurysm, and associated lesions exposed, and means explored to improve the prognosis. Since the perioperative mortality rate appears incompressible, an improvement in the prognosis of AAA can be obtained only by a combined medical effort. This requires the more rapid recognition of prodromal symptoms of rupture, the detection of a larger number of asymptomatic AAA and improved postoperative life expectancy, already reasonable, by more rigorous vascular and cardiologic surveillance.


[Duplex ultrasonography in the diagnosis of deep vein thrombosis of the legs. Agreement between two operators].

The operator-dependent nature of venous Duplex-ultrasonography diagnosis of deep venous thrombosis (DVT) of the leg is the principal reproach applied to it and a reason for delaying use. The aim of the present study was to evaluate concordance between two operators, with rigorous application of the same methodology of exploration of the venous axes. The study included 82 patients under hospital care, randomly selected from among those referred for the first Duplex-ultrasonography examination for DVT, and having accepted to undergo 2 consecutive explorations, one by each of the two operators. The two operators evaluated independently, for the 19 venous axes of the legs: 1: the interpretability of the examination, 2: the differential diagnosis between: absence of thrombus, presence of thrombus, nonconclusive examination, and 3: the score for the DVT extension. Evaluation of concordance of diagnosis was by Cohen's kappa coefficient, calculated on the positive diagnosis of DVT and the site of the most proximal thrombus. Extension scores were compared by calculation of the coefficient of correlation "r". Interpretability rates were 92% and 91% respectively for the two operators, 35 DVT being diagnosed in 27 patients by operator A and 36 DVT in 27 patients by operator B. The kappa coefficient for diagnosis of DVT with localization in the affected leg was 0.90 [0.81-0.98]. It was 0.89 [0.73-1] for proximal lesions, 0.86 [0.74-0.97] for the lower leg level, and 0.79 for localization of the proximal extremity of the thrombus. Extension scores evaluated by the two operators were 2.88 and 3.14 respectively, with a coefficient of correlation between the extension scores of 0.96. The 5 diagnostic divergences concerned the lower leg level; the 2 localization discordances concerned: the frontier zones leg-lower popliteal, lower femoral-upper popliteal. Good concordance between results of the two operators using Duplex-ultrasonography exploration was obtained even though the majority of the DVT were in the calf, a region known to be explored with difficulty. Overcoming the operator-dependent character of Duplex-ultrasonography by a rigorous exploratory methodology could make it the future reference examination for the diagnosis of DVT.


[Use of an active clinical maneuver for diagnosis of popliteal artery entrapment].

Overall incidence rate of popliteal artery entrapment syndrome is certainly underestimated. The aim of this work was to test and to evaluate the interest of a clinical maneuver of active and repetitive ankle extensions in upright position, for the screening, the diagnosis and the follow-up of patients with popliteal artery entrapment. During a 3 year period, this maneuver was used in 10 patients (16 popliteal artery entrapments) and in 50 age-matched controls. The results of the maneuver were compared to the results of functional vascular investigations. For all the patients the maneuver had to be stopped before 20 movements whereas it could be continued up to 50 movements for all the controls. The maneuver was easy to perform, reliable and constant in its results. The earlier the maneuver was stopped the more critical was the trouble at functional vascular investigations. We believe that this maneuver is of great help for early diagnosis of popliteal artery entrapment, assessment of its severity and follow-up treated patients. It could also help to detect athletes with asymptomatic popliteal entrapment.


[Electric stimulation of the spinal cord in arterial diseases of the legs. A multicenter study of 244 patients].

From January 1985 through January 1990, 244 patients (168 males, 76 females, mean age: 69 +/- 14 years) received epidural spinal cord stimulation for the treatment of advanced, nonreconstructable, peripheral vascular disease of the lower limbs due to atherosclerosis in 180 patients, atherosclerosis and/or diabetes in 49, and thromboangiitis obliterans in 15 patients: previous surgery included 101 bypass-grafts in 70 patients, 51% of which below the knee, and 117 sympathectomies in 113 patients as the last resource in face of distal peripheral vascular disease of the lower limbs. Mean ankle-to brachial systolic pressure ratio was .31 +/- .34 on symptomatic limbs; due to pain and advanced disease, walking capacity was assessed in only 151 patients, either on treadmill in 25, or in a metered corridor in 126; angiogram of the lower limbs was performed in every patient unless one not older than three months was readily available; pain at rest was assessed after an analogical scale; partial transcutaneous oxygen tension was measured on the dorsum of the fore-foot of 77 symptomatic limbs (mean: 13.35 +/- 14 mmHg). According to clinical and functional evaluation, 18 patients had exertional ischemia (group I), 87 had permanent ischemia with pain at rest and no tissue loss (group II), and 139 had chronic tissue loss (group III), including 93 ischemic ulcers (mean surface: 3.7 cm2, mean duration: 3.5 months) in 88 patients, 27 limited gangrene, and 24 previous limited non-healing distal amputation. After temporary spinal cord stimulation at T12-L1 level (mean duration: 9 +/- 4 days) with a percutaneous quadripolar electrode lead had allowed for selection of responders, 212 patients received an implantable neurostimulator.(ABSTRACT TRUNCATED AT 250 WORDS)


Oscillometry of radial artery in acrocyanosis and cold sensitivity.

Acrocyanosis is reassessed on the basis of 34 new patients with diagnostic clinical features. The most useful tests are the skin temperature differences (STD) between wrist and end of fingers (or ankle and end of toes) and oscillometry over the radial and dorsalis pedis and posterior tibial arteries. Capillaroscopy of the conjunctival and nailfold vessels is useful in half the patients. Capillary resistance was not helpful. STD of 1.5 degrees C or more between skin of wrist or ankle and fingertip or toetip, is usually associated with more severe disease and capillary abnormalities on microscopy, and with reduced oscillometry (to 1 or less) over the radial arteries and dorsalis pedis and posterior tibial arteries. Reduced oscillometry is more associated with abnormalities than the other tests, and more than STD. The work of Mulvany and his colleagues suggests an explanation for the low oscillometry. Larger vessels than the arterioles and digital vessels can become resistance vessels in various circumstances. It is likely that the radial artery and vessels of similar diameter can temporarily act as resistance vessels in patients with acrocyanosis in a low temperature environment.