Showing papers by "Children's Hospital Oakland Research Institute published in 1994"

The animal fatty acid synthase: one gene, one polypeptide, seven enzymes.

Abstract: The animal fatty acid synthase comprises two multifunctional polypeptide chains, each containing seven discrete functional domains, juxtaposed head-to-tail such that two separate centers for fatty acid assembly are formed at the subunit interface. The kinetics and specificities of the component enzymes are well adapted to ensure that, at each of the two centers, the iterative condensation of an acetyl moiety with successive malonyl moieties and complete reduction of the beta-keto intermediates normally results in the formation of palmitic acid as the major product. Nevertheless, utilization of alternative substrates and alternative chain-terminating mechanisms can extend the range of products to include branched-chain, odd carbon-numbered, and shorter chain-length fatty acids. The potential of this multifunctional form of molecular architecture for the elaboration of more complex natural products has been further exploited in microorganisms that, by the use of different fatty acid synthase "modules" that ...

Oxidation of low-density lipoprotein by hypochlorite causes aggregation that is mediated by modification of lysine residues rather than lipid oxidation.

Abstract: Peroxidation of low-density lipoprotein (LDL) lipid is generally thought to represent the initial step in a series of modification reactions that ultimately transform the protein moiety of the lipoprotein into a form recognized by receptors different from those that bind native LDL. Uptake of LDL via these alternative receptors can lead to the formation of lipid-laden cells, which are typical for the early stages of atherogenesis. We have studied the oxidative modification of LDL by hypochlorite (-OCl), a powerful oxidant produced from H2O2 and chloride via the action of myeloperoxidase which is released from activated neutrophils and monocytes. Exposure of LDL to reagent or enzymically generated -OCl at 4 or 37 degrees C resulted in immediate and preferential oxidation of amino acid residues of apolipoprotein B-100, the single protein associated with LDL. Lysine residues quantitatively represented the major target and, like tryptophan, were oxidized to approximately the same extent with reagent or enzymically generated -OCl. In contrast, LDL lipid oxidation was less favoured than protein oxidation, as judged by the amounts of lipid hydroperoxides, chlorohydrins, cholesterol or fatty acid oxidation products formed. Treatment with -OCl caused aggregation of LDL, as shown by an increased turbidity of the oxidized LDL solution and elution from a size-exclusion h.p.l.c. column of high-molecular-mass LDL complexes. Chemical modification of lysine residues before oxidation with -OCl prevented aggregation, while it enhanced the extent of lipid peroxidation. Treatment of LDL with -OCl also caused the formation of carbonyl groups and release of ammonia; both these modifications were inhibited by lysine-residue modification before oxidation. These results demonstrate that aggregation reactions are dependent on initial lysine oxidation by -OCl, followed by deamination and carbonyl formation, but do not involve lipid (per)oxidation. We propose that the observed -OCl-mediated aggregation of LDL is caused, at least in part, by cross-linking of apoproteins by Schiff-base formation independently of lipid peroxidation.

Butyrate derivatives. New agents for stimulating fetal globin production in the beta-globin disorders.

Abstract: PURPOSE Stimulating expression of the normal fetal globin genes is a preferred method of ameliorating sickle cell disease and beta-thalassemia for the majority of patients in North America who do not have appropriate bone marrow donors PATIENTS AND METHODS Due to increased survival of red blood cells that contain both hemoglobin S and hemoglobin F, as little as 4-8% fetal globin synthesis in the bone marrow can produce levels of hemoglobin F of approximately 20% in the peripheral circulation Some success has been achieved in stimulating hemoglobin F using chemotherapeutic agents (such as hydroxyurea and 5-azacytidine) and growth factors (erythropoietin) that alter erythroid growth kinetics However, there is reluctance to treat children with chemotherapeutic agents because of possible undesirable long-term side effects RESULTS Butyric acid and butyrate derivatives are generally safe compounds that stimulate the promoters of individual fetal and embryonic globin genes and thus provide a more specific therapy An initial trial with the parent compound, given as arginine butyrate, has demonstrated rapid stimulation of fetal globin expression to levels that can ameliorate these conditions Phase I trials of an oral butyrate derivative with a long plasma half-life have begun CONCLUSIONS These agents may provide a new and specific approach for ameliorating the clinical manifestations of sickle cell disease and beta-thalassemia

Shedding of adhesion receptors from the surface of activated platelets

Abstract: When platelets are activated, several receptors are removed from the platelet surface. Cytoskeletal reorganizations can cause receptors to redistribute to intracellular membranes. In addition, receptors can be removed from the platelet surface by shedding of the receptor from the cell. Shedding can occur by at least two mechanisms. First, glycoprotein (GP)Ib alpha and GP V are shed from the membrane as a result of the proteolytic cleavage of the extracellular domain of these receptors from the platelet. The protease responsible for this cleavage appears to be a membrane-bound divalent cation-dependent protease other than calpain. Proteolytic cleavage does not occur until secretion is well under way and occurs whether platelets aggregate or not. Soluble forms of both GP Ib alpha and GP V are present in the plasma, where they may serve as feedback inhibitors limiting the development of thrombi. Future studies will be needed to identify the protease(s) responsible for removing the membrane receptors and to determine whether cleavage of the receptors from activated platelets results from activation of the protease(s), exposure of the protease(s), or an altered exposure of the protease-sensitive sites on the receptors. It will be of particular interest to determine whether the protease(s) that cleaves GP Ib alpha and GP V in platelets is the same as the protease(s) that cleaves receptors from the surface of other activated cells. Receptors also are shed from the surface of activated platelets by the generation of microvesicles from the plasma membrane. These microvesicles appear to contain all of the major membrane glycoproteins but are depleted in those that have been removed from the platelet membrane by proteolytic cleavage. The primary mechanism responsible for the shedding of microvesicles from the surface of platelets stirred with physiological agonists involves activation of calpain, which cleaves components of the membrane skeleton and dissociates it from the plasma membrane GP Ib-IX complex. Microvesicles are present in the circulation and increase under conditions in which platelet activation is known to have occurred. Because they contain functional adhesive receptors and procoagulant activity on their surface, they may function to disseminate procoagulant activity and stabilize the formation of platelet clots.

Universal Screening for Hemoglobinopathies Using High-Performance Liquid Chromatography: Clinical Results of 2.2 Million Screens

Abstract: In screening for hemoglobinopathies, high-performance liquid chromatography (HPLC) achieves excellent sensitivity and specificity, while adding the very important quantitative element to the analysis. Due to the development of a rapid, automated HPLC system, California began screening 600,000 births per year in 1990 using this method. Based on confirmatory testing for 97% of the initial positive results resulting from 2.2 million screens, HPLC has proven to be clinically accurate. In conjunction with the availability of quantitative data, HPLC provides a complete screening system, eliminating the need for a second screening test, and accurately discriminating beta thalassemia compound conditions. The large degree of ethnic diversity and high birth rate in California have produced detailed and reliable birth prevalence rates for most conditions and ethnicities.

Variable region sequences of a protective human monoclonal antibody specific for the Haemophilus influenzae type b capsular polysaccharide.

Abstract: A hybridoma secreting a human immunoglobulin G2 kappa monoclonal antibody (MAb) specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib) was isolated. This MAb, designated CA4, was bactericidal to Hib in vitro and protected infant rats from Hib bacteremia. Nucleotide sequence analysis of CA4 variable (V) region cDNA showed that the heavy (H)-chain V region was of subgroup III and was 96% identical to the VH germ line gene segment DP77 (V3-21). The light (L)-chain V region was of the kappa subgroup III and was 94% identical to the A27 (Humkv325) germ line gene, which is commonly used by rheumatoid factors and other autoantibodies. MAb CA4 did not have rheumatoid factor activity and did not react with histones, DNA, or chromatin. These findings identify an additional VHIII gene segment which can contribute to the anti-Hib capsular polysaccharide repertoire and demonstrate that a VL gene commonly encoding autoantibodies can be utilized for protective immunity.

Mass Spectrometry in the Characterization of Variant Hemoglobins

Abstract: About 150 million people throughout the world carry abnormal hemoglobins, with consequences ranging from trivial to lethal. More than one in 800 people have hemoglobins that differ from normal by amino acid substitutions, globin chain elongations, contractions, and fusions. In determining this incidence, the most common ones—variants Hb S, Hb E, and Hb C—were not included (1).

Transmembrane Signaling across the Platelet Integrin Glycoprotein IIb‐IIIaa

Abstract: The platelet membrane is lined by a membrane skeleton, which in turn appears to be associated with underlying cytoplasmic actin filaments. Glycoprotein IIb-IIIa appears to associate with the membrane skeleton in unstimulated platelets. Upon platelet activation, unidentified intracellular signals cause GP IIb-IIIa to become competent to bind adhesive ligand. We suggest that the membrane skeleton may play a role in allowing this inside-out signaling. Signaling molecules that appear to associate with the membrane skeleton in unstimulated platelets include pp60c-src, pp62c-yes, and GAP. Preliminary evidence suggests that components of the membrane skeleton may become phosphorylated on tyrosine residues prior to GP IIb-IIIa-ligand interactions. Once GP IIb-IIIa binds adhesive ligand in a platelet aggregate, there is signaling in the opposite direction. One consequence of the outside-in transmembrane signaling is that the membrane skeleton becomes more tightly associated with the underlying actin filaments as focal contact-like structures form. Proteins that accumulate in these focal contact-like structures with a time course identical to that of GP IIb-IIIa and in a GP IIb-IIIa-dependent manner include talin, vinculin, and spectrin. Signaling molecules that accumulate in the focal contact-like structures include pp60c-src, pp62c-yes, phosphoinositide 3-kinase, and protein kinase C. These are potential candidates for the enzymes that mediate the ligand-induced transmembrane signaling. Another enzyme involved in the ligand-induced signaling is calpain. This enzyme is activated as a consequence of ligand-GP IIb-IIIa interactions and cleaves components of the membrane skeleton. Future experiments will be needed to identify other signaling enzymes activated as a consequence of GP IIb-IIIa interactions and to determine which ones are responsible for inducing the cytoskeletal reorganizations that occur in platelets and other cells when integrins bind their adhesive ligands.

Effects of oxidants and antioxidants evaluated using parinaric acid as a sensitive probe for oxidative stress

Abstract: Parinaric acid (PnA) is a fluorescent polyunsaturated fatty acid which can be used as a probe to study lipid peroxidation processes. The basic methodology is simple and sensitive, and offers a direct 'view' of the oxidative decay of a fatty acid and the effects of prooxidant and antioxidant factors. A distinctive feature of the PnA assay is that it does not measure a lipid peroxidation end product, but monitors lipid oxidative stress in its initial stages. This review highlights the methodological characteristics of the PnA assay, and describes the various applications in which PnA and PnA derivatives have yielded useful information. These applications range from oxidant and antioxidant studies in lipid model systems to comparative studies of oxidation processes in normal and pathological red blood cells, and also include studies of lipoprotein oxidation.

Measurement of reaction products from hypochlorous acid and unsaturated lipids

Abstract: Publisher Summary Neutrophils contain the enzyme myeloperoxidase (MPO) that catalyzes the conversion of hydrogen peroxide (H 2 O 2 ) and chloride (Cl - ) to hypochlorous acid (HOCl). HOCl is a highly reactive oxidant that is thought to be important in the antimicrobial action and the cytotoxic effects of activated neutrophils. Although proteins are known to be susceptible to HOCl-mediated oxidative modification, the oxidation by HOCl of unsaturated membrane lipids could be instrumental in destabilizing the membrane lipid matrix, leading to cell disruption. HOCl adds across double bonds to form chlorohydrins. The reaction with unsaturated fatty acids yields a mixture of fatty acid chlorohydrin positional isomers. Monounsaturated fatty acids form monochlorohydrins, whereas in polyunsaturated fatty acids all the double bonds are susceptible to modification. Low HOCl:fatty acid ratios predominantly give monochlorohydrins, and higher ratios increase the proportion of bis- and polychlorohydrin derivatives. This reaction occurs with unsaturated fatty acids in micelles and with sn-2 fatty acyl chains of phospholipids in vesicle membranes. Both reagent hypochlorite and the MPO system can generate these products. Equivalent reactions to form iodohydrins and bromohydrins are shown with various peroxidase systems.

Cytosine arabinoside and mitoxantrone treatment of relapsed or refractory childhood leukemia: Initial response and relationship to multidrug resistance gene 1

Abstract: The objective of this study was to determine the response rate and toxicity of high-dose cytosine arabinoside (AC) and mitoxantrone (M) in relapsed or refractory childhood acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) and to correlate response with the expression of the multidrug resistance gene 1 (mdr1). Twenty-nine patients were treated with AC 1.0 g/m2 infused over 2 h every 12 h for eight doses (days 1-4) and M 12 mg/m2 infused over 1 h (days 3-6). Mdr1 expression was determined by a polymerase chain reaction (pcr) assay. Ten of 15 patients (67%) with AML obtained a complete remission (CR) of 3 to 30+ months duration. Eight of 14 (57%) ALL patients obtained a CR of 1 to 23+ months duration. The major toxicities were hematopoietic and infectious. Seventy-nine per cent of patients developed a documented infection during induction. Mdr1 did not correlate with a lower induction rate. This AC/M regimen is active in childhood AML and ALL.

Cytosine arabinoside and mitoxantrone induction chemotherapy followed by bone marrow transplantation or chemotherapy for relapsed or refractory pediatric acute myeloid leukemia.

Abstract: The purpose of this study was to determine the induction rate, duration of response and toxicity of cytosine arabinoside (1.0 gm/m2 i.v. over 2 h q 12 h x 8 doses days 1 through 4) and mitoxantrone (12 mg/m2 over 1 h daily x 4 doses days 3 through 6) in pediatric patients with acute myeloid leukemia (AML). Patients achieving a complete remission received either bone marrow transplantation or further chemotherapy. Twenty-seven of 37 evaluable patients (73% (95% confidence interval 59-87%)) achieved a complete remission. For all responding patients, the projected median time to relapse is 12 months. The projected 1 and 2 year disease-free survival is 47% (28-66) and 41% (21-61) with a range of follow-up of 0 to 48+ months. The major toxicity was bone marrow suppression and infection. This therapy is very active in pediatric AML and has acceptable toxicity. Some patients treated achieve prolonged survival.

Induction of a protective human polysaccharide-specific antibody response in hu-PBL SCID mice by idiotypic vaccination.

Abstract: The human Ab repertoire to the Haemophilus influenzae type b (Hib) polysaccharide (PS) is dominated by Abs that use the kappa II-A2 VL region and that express an idiotype (Id) designated Hibld-1. In this study we determined whether a human Hib PS-specific Ab response could be induced by idiotypic manipulation. We prepared a bispecific vaccine consisting of the F(ab')2 fragment of a mAb specific for Hibld-1, coupled to the F(ab')2 fragment of a mAb specific for CD3, a component of the TCR complex. This bispecific idiotypic vaccine stimulated production of human Abs to Hib PS in severe combined immunodeficient mice engrafted with normal human adult PBLs. The induced Abs uniformly expressed Hibld-1 and protected neonatal rats from Hib bacteremia. Experiments using additional conjugates demonstrated that covalent coupling of the CD3-specific moiety to the anti-Id was required for immunogenicity in this model, a result suggesting that engagement of B cell Id and proximate delivery of T cell signals are both necessary for B cell activation and differentiation. These findings demonstrate that human Ids can serve as targets for induction of a protective anti-PS Ab response.