Showing papers by "Children's Hospital Oakland Research Institute published in 1993"
TL;DR: In patients with beta-hemoglobinopathies butyrate, a natural fatty acid, can significantly and rapidly increase fetal-globin production to levels that can ameliorate beta- globin disorders.
Abstract: Background Fetal-globin (γ-globin) chains inhibit the polymerization of hemoglobin S (sickle hemoglobin) and can functionally substitute for the β-globin chains that are defective or absent in patients with the β-thalassemias. Identifying safe mechanisms to stimulate fetal-hemoglobin production is therefore of great interest. Previous studies have shown that administering butyrate selectively stimulates the promoter of the human fetal-globin gene and leads to increases in γ-globin-gene expression in the developing fetus, cultured cells, and animal models. Methods To determine whether butyrate can stimulate fetal-globin production in humans, we treated three patients (3 to 13 years old) with sickle cell anemia and three patients (7 to 27 years old) with β-thalassemia syndromes with a short course of intravenous infusions of arginine butyrate. The drug was infused continuously for either two or three weeks; the initial dose was 500 mg per kilogram of body weight per day. Globin-chain ratios, proportions of ...
467 citations
TL;DR: The platelet cytoskeleton contains two actin filament-based components, which fill the cytoplasm and mediate contractile events and the membrane skeleton, which coats the plasma membrane and regulates properties of the membrane such as its contours and stability.
Abstract: The platelet cytoskeleton contains two actin filament-based components. One is the cytoplasmic actin filaments which fill the cytoplasm and mediate contractile events. The other is the membrane skeleton, which coats the plasma membrane and regulates properties of the membrane such as its contours and stability. In the unstimulated platelet, only 30-40% of the actin is polymerized into filaments; the rest is thought to be prevented from polymerizing by the association of thymosin beta 4 with monomeric actin and by the association of gelsolin with the barbed ends of pre-existing actin filaments. When platelets are activated, there is a rapid increase in actin polymerization; new filaments fill the extending filopodia and form a network at the periphery of the platelet. As a result of activation, myosin binds to cytoplasmic actin filaments, causing them to move towards the center of the platelet. As platelets aggregate, additional cytoskeletal reorganizations occur: GP IIb-IIIa associates with adhesive ligand in a platelet aggregate; this results in the association of GP IIb-IIIa, membrane skeleton proteins, and signaling molecules with cytoplasmic actin. Future studies should help to elucidate the significance of the cytoskeleton in regulating signal transduction events in platelets.
248 citations
TL;DR: The use of GC/MS and microbore HPLC/electrospray mass spectrometry for clinical studies in hypertension and mineralocorticoid research is described and this method is able to distinguish almost all steroid related disorders.
240 citations
TL;DR: The results imply that the membrane skeleton may play an important role in binding signaling molecules at sites of integrin-cytoskeleton interactions and in mediating signal transduction events in platelets.
232 citations
TL;DR: Entrapment of purified alpha-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo.
Abstract: While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-hemoglobin chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-hemoglobin chains, purified alpha-hemoglobin chains were entrapped within normal erythrocytes. These "model" beta-thalassemic cells generated significantly (P < 0.001) greater amounts of methemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of alpha-hemoglobin chain autoxidation and appearance of membrane-bound globin. In addition, alpha-hemoglobin chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001) enhanced in the alpha-hemoglobin chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha-hemoglobin chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped alpha-hemoglobin chains. In summary, entrapment of purified alpha-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess alpha-hemoglobin chains can be examined.
198 citations
TL;DR: If a semi-synthetic high-fat diet could elicit the same extent of diet-induced atherosclerosis as that observed in mice fed a natural ingredient highfat diet and to discover strain-specific plasma and liver lipid variants for future genetic characterization, nine inbred strains of mice were evaluated.
Abstract: Nine inbred strains of mice, which are progenitors of recombinant inbred sets, were evaluated for aortic lesion formation and plasma and liver lipid levels. This survey was done to determine if a semi-synthetic high-fat diet could elicit the same extent of diet-induced atherosclerosis as that observed in mice fed a natural ingredient highfat diet and to discover strain-specific plasma and liver lipid variants for future genetic characterization. Evaluation of aortic lesions after 18 wk of diet consumption showed that strains C57BL/6J, C57L/J, SWR/J and SM/J were susceptible to atherosclerosis and that A/J, AKR/J, C3H/HeJ, DBA/2J and SJL/J were relatively resistant. High-density lipoprotein cholesterol (HDL-C) levels were negatively correlated to lesion formation. Susceptible strains had decreased HDL-C levels when switched from chow to the semi-synthetic high-fat, high cholesterol diet, whereas resistant strains either showed no change or a slight increase in HDL-C levels. The exception to this pattern was found in SM mice, which were susceptible to aortic lesion formation but maintained the same HDL-C level on both chow and high-fat diets. HDL size differed among the strains, and levels of plasma apolipoprotein A-I and A-II correlated with HDL-C levels. Liver damage was not correlated to HDL-C levels or to susceptibility to atherosclerosis. Mice from strain A, which are resistant to atherosclerosis, had evidence of liver damage as observed by elevated levels of plasma alanine aminotransferase activity, by liver histology, by increased liver weight and by exceptionally high hepatic cholesterol content. For all strains, the levels of liver cholesterol and triglycerides were inversely correlated with each other; phospholipids did not vary greatly among strains. No remarkable differences in hepatic fatty acid profile were noted among the strains fed the atherogenic diet, but the fatty acid profile did differ considerably from that found in the diet itself.
137 citations
TL;DR: Hamstrings may be important hip extensors in some cerebral palsy patients with crouch gait; however, other deformities contributing to crouch need to be considered before isolated hamstring lengthening is performed in these patients.
Abstract: After observing patients with increased anterior pelvic tilt following medial hamstring lengthening in cerebral palsy crouch gait, we became concerned that the hamstrings may be functionally important hip extensors. To evaluate this, we studied the three-dimensional motion of the hip and knee, calculated hamstring muscle length, and evaluated dynamic electromyography (EMG) of the medial hamstrings in 16 patients with diplegic cerebral palsy and crouch gait to determine if the hamstrings were extending the hip. Twelve of 16 patients exhibited marked prolongation of electrical activity in the medial hamstrings, and in eight of these 12, the hamstrings were contracting concentrically, thus aiding in hip extension during gait. Hamstrings may be important hip extensors in some cerebral palsy patients with crouch gait; however, other deformities contributing to crouch (such as hip flexion contracture) need to be considered before isolated hamstring lengthening is performed in these patients.
108 citations
TL;DR: It is proposed that the altered molecular conformation of oxidized phospholipids facilitates access to the sn-2 ester bond, thereby ensuring their preferential hydrolysis in the presence of a phospholIPase A2.
Abstract: Cleavage of oxidized fatty acids by phospholipase A2 has been implicated as the first step in the repair mechanism for oxidative damage to membrane phospholipids. However, the mechanism by which this enzyme preferentially hydrolyzes oxidized fatty acyl chains is poorly understood. Using a lipid monolayer technique, we found that the molecular surface areas of 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)-phosphatidylcholine (PLPC-OOH) and 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) were increased by as much as 50% relative to the parent nonoxidized 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC). These experimental data directly indicate a drastically changed molecular conformation of oxidized phospholipids in which the hydroperoxy or hydroxy group in the sn-2 fatty acid is close to the lipid-water interface. Phospholipases A2 from porcine pancreas and from bee venom were shown to break down PLPC-OOH and PLPC-OH monolayers much faster than PLPC monolayers. In all cases, the presence of serum albumin in the subphase enhanced monolayer breakdown by extracting hydrolysis products from the monolayer, but monolayer breakdown was always much faster for oxidized than for nonoxidized PLPC. This did not appear to be due to change in the extent of monolayer penetration by phospholipase A2, since enzyme-monolayer interaction studies revealed essentially identical penetration behavior of bee venom phospholipase A2 with PLPC, PLPC-OOH, and PLPC-OH monolayers. We propose that the altered molecular conformation of oxidized phospholipids facilitates access to the sn-2 ester bond, thereby ensuring their preferential hydrolysis in the presence of a phospholipase A2.
100 citations
TL;DR: In infants in whom diphtheria-tetanus-pertussis (DTP) vaccination is deferred because of medical contraindications, vaccination with the PRP-OMPC conjugate would appear to be preferable to HbOC because of the ability of the former to elicit antibody responses in the absence of diphTheria toxoid vaccination.
86 citations
TL;DR: Examination of the completed domain map for the animal fatty acid synthase indicates that the catalytic domains are clustered in two groups separated by a central structural core: the ketoacyl synthase, malonyl/acetyltransferase, and dehydrase in the amino-terminal half and the enoyl reductase, ketoreductase, acyl carrier protein, and thioesterase in the carboxyl- terminus of the transferase domain.
85 citations
TL;DR: These studies show that unsaturated phospholipids and cholesterol can be profoundly modified by reaction with hypochlorous acid, and warrants further investigation to define the role of lipid modifications in neutrophil-mediated membrane disruption.
TL;DR: Comparison of the diacyl species of PC, PE and PI in bloodstream trypanosomes showed major differences in the relative amounts of individual molecular species between the different classes but not striking changes in the degree of saturation or overall chain length, in contrast, in procyclic Trypanosoma brucei.
TL;DR: The results demonstrate that the primary effect of PMA on Cl secretion is not inhibition of apical Cl channels but inhibition of basolateral K channels, which is 10-fold greater in Cl media than in gluconate media.
Abstract: Pretreating confluent T84 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits adenosine 3',5'-cyclic monophosphate (cAMP)- and carbachol-induced Cl secretion Both a sustained short-circuit current (Isc), seen after the addition of 50 microM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and 100 microM 3-isobutyl-1-methylxanthine (IBMX), and a transient current, seen after the subsequent addition of 100 microM carbachol, are inhibited by 80% following pretreatment with 100 nM PMA for 2 h Pretreatment with PMA has no effect on the level of cystic fibrosis transmembrane conductance regulator protein or apical cAMP-dependent Cl conductance Carbachol does not induce an increase in apical Cl conductance Basolateral K conductance was measured in monolayers treated with apical nystatin and exposed to a K gradient Agonist-independent K conductance is 10-fold greater in Cl media than in gluconate media Pretreatment with PMA inhibits agonist-independent K conductance by 57% in Cl media but stimulates K conductance by 19-fold in gluconate media The addition of carbachol induces a transient increase in basolateral K conductance, and pretreatment with PMA inhibits the agonist-dependent K conductance by 66% in Cl media and by 92% in gluconate media In Cl media, serosal barium, at 3 mM, inhibits agonist-independent K conductance but has no significant effect on the carbachol-induced conductance In nonpermeabilized monolayers, serosal barium inhibits the cAMP-dependent Isc by 56% but has no effect on the carbachol-induced Isc These results demonstrate that the primary effect of PMA on Cl secretion is not inhibition of apical Cl channels but inhibition of basolateral K channels(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Fundamental differences in both variable region content and antibody quality elicited by different Hib PS conjugate vaccines are indicated.
Abstract: Haemophilus influenzae b polysaccharide (Hib PS)-protein conjugate vaccines differ chemically and immunologically. To determine whether anti-Hib PS variable region expression might differ according to vaccine formulation, infants were vaccinated at 2, 4, and 6 mo of age with Hib PS coupled to either meningococcal outer membrane protein complex (Hib PS-OMPC) or tetanus toxoid (Hib PS-T), or Hib PS oligomers coupled to a mutant diphtheria toxin (Oligo-CRM). Two anti-Hib PS idiotypes were measured in sera obtained after the third injection: HibId-1, expressed by anti-Hib PS antibodies having the kappa II-A2 variable region, and HibId-2, a newly defined cross-reactive idiotype associated with a subset of anti-Hib PS antibodies having lambda VII variable regions. HibId-1 was present in 33, 68, and 64% of infants given either Hib PS-OMPC, Oligo-CRM, or Hib PS-T, respectively (P < 0.001). The respective values for HibId-2 were 47, 18, and 10% (P = 0.001). Subjects who were vaccinated with Hib PS-OMPC or Hib PS-T and who produced detectable HibId-1-positive antibody, had significantly higher mean antibody avidity than subjects who did not produce HibId-1 positive antibodies. In contrast, Oligo-CRM evoked high avidity anti-Hib PS antibodies, irrespective of the idiotypic profile. These findings indicate fundamental differences in both variable region content and antibody quality elicited by different Hib PS conjugate vaccines.
TL;DR: Circular dichroism spectra of normal beta A and beta Willamette show very little difference under a variety of solvent conditions, indicating that fragmentation differences in their respective CAD mass spectra are substantially governed by primary rather than secondary structure.
Abstract: Electrospray ionization collisionally activated dissociation (CAD) mass spectra of multiply charged human hemoglobin beta-chain variant proteins (146 amino acid residues, 15.9 kDa), generated in the atmospheric pressure/vacuum interface and in the collision quadrupole of a triple-quadrupole mass spectrometer, are shown and compared. Several series of structurally informative singly and multiply charged b- and y-mode product ions are observed, with cleavage of the Thr 50-Pro 51 CO-NH bond to produce the complementary y96 and b50 sequence ions as the most favored fragmentation pathway. The eight different beta-globin variants studied differ by a single amino acid substitution and can be differentiated from the observed m/z shifts of the assigned product ions. The overall fragmentation patterns for the variant polypeptides are very similar, with the exception of the Willamette form, in which Arg is substituted for Pro- 51, and multiply charged y96 product ions are not observed. Circular dichroism spectra of normal beta A and beta Willamette show very little difference under a variety of solvent conditions, indicating that fragmentation differences in their respective CAD mass spectra are substantially governed by primary rather than secondary structure.
TL;DR: Analysis of NADPH/NADPt ratio in acatalasemic mouse erythrocytes demonstrated that NADPH maintenance alone was not sufficient to explain oxidant resistance, and that catalase activity was required, and elucidates the importance of NAD PH in the maintenance of normal catal enzyme activity.
TL;DR: Microscale analysis of a variant hemoglobin (Hb) has been achieved by combination of high performance liquid chromatography (HPLC) and electrospray mass spectrometry (ESMS) and the method should be almost universally applicable and allow characterization of almost all variant Hbs.
Abstract: Microscale analysis of a variant hemoglobin (Hb) has been achieved by combination of high performance liquid chromatography (HPLC) and electrospray mass spectrometry (ESMS) and the method should be almost universally applicable. We have eliminated preparative scale HPLC of globin chains and semi-preparative HPLC of proteolytic digests which had been used prior to mass spectrometry. Use of microbore HPLC columns reduced the time required for analysis substantially and solvent usage by 100x. Molecular masses of intact globins and masses and sequence information of tryptic peptides could be obtained without collecting and separately analyzing chromatographic fractions.As an example of the use of these methods, we report the characterization of an unknown hemoglobinopathy case that was finally authenticated as Hb P-Galveston {βl17(G19)His->Arg], using the following sequence of analyses: 1) ESMS of complete hemolysate, 2) analytical HPLC of globin chains, 3) combined microbore HPLC/ESMS of globin chains to det...
TL;DR: Results indicate that, in the insect cell host, all seven catalytic components of the 2505-residue recombinant fatty acid synthase fold correctly, the acyl-carrier-protein domain is appropriately phosphopantetheinylated post-translationally, and the multifunctional polypeptide forms catalytically competent dimers.
Abstract: A cDNA encoding the 2505-residue multifunctional rat fatty acid synthase has been constructed and expressed as a catalytically active protein in Spodoptera frugiperda (Sf9) cells using Autographa californica nuclear polyhedrosis virus (baculovirus). The 7.5 kb cDNA was engineered by the amplification and sequential splicing together of seven fragments contained in overlapping cDNAs that collectively spanned the entire rat fatty acid synthase coding sequence. The full-length cDNA was cloned into a baculoviral transfer vector and used together with linearized baculoviral DNA to co-transfect Sf9 cells. Recombinant viral clones were purified and identified by Western blotting. The recombinant fatty acid synthase was expressed maximally 2 days after infection of the Sf9 cells, constituting up to 20% of the soluble cytoplasm, and could be conveniently separated from the insect host fatty acid synthase by high-performance anion-exchange chromatography. The catalytic properties of the purified recombinant fatty acid synthase are indistinguishable from those of the best preparations of the natural protein obtained from rat liver. These results indicate that, in the insect cell host, all seven catalytic components of the 2505-residue recombinant fatty acid synthase fold correctly, the acyl-carrier-protein domain is appropriately phosphopantetheinylated post-translationally, and the multifunctional polypeptide forms catalytically competent dimers. Thus the baculoviral system appears to be well suited for the expression of specific fatty acid synthase mutants that can be used to explore the mechanism by which the seven domains of this multifunctional homodimer co-operate in the biosynthesis of fatty acids.
TL;DR: Co-chromatography of human, orangutan, and macaque luteal phase urine samples demonstrated the presence of a steroid conjugate peak in all three species that was identified as being androsterone and etiocholanolone glucuronides.
Abstract: In vivo studies using carbon 14 labeled estradiol (E2) and progesterone (Po) were performed to characterize the time course and metabolic fate of circulating E2 and Po. Co-chromatography of human, orangutan, and macaque luteal phase urine samples demonstrated the presence of a steroid conjugate peak in all three species that was identified as being androsterone and etiocholanolone glucuronides. An enzyme immunoassay for urinary metabolites of Po was developed subsequently for Macaca spp. using a monoclonal antibody that cross-reacted with both C-19 and C-21 metabolites.
Journal Article•
TL;DR: Dramatic changes in VL region utilization occur in the human antibody response to this Hib PS conjugate vaccine as a function of age, and age-related differences in V region expression may affect the ability of vaccines to confer protective immunity at different ages.
Abstract: The human L chain antibody repertoire specific for the Haemophilus influenzae type b (Hib) polysaccharide (PS) is composed of kappa I, kappa II, kappa III, kappa IV, and lambda L chain V regions, but the most commonly occurring VL is encoded by the unmutated V kappa II-A2 gene. To determine whether this VL repertoire is influenced by age, we used idiotypic probes to monitor V kappa II-A2, V lambda, and V kappa III usage in the antibody response to an Hib PS-protein conjugate vaccine. A single dose of a vaccine consisting of Hib PS coupled to an outer membrane protein complex of Neisseria meningitidis was administered. Adults (n = 35), 18-month-old infants (n = 35), and 2-month-old infants (n = 46), all with > or = 0.9 microgram/ml anti-Hib PS antibodies, were tested for VL region markers in postvaccination sera. V kappa III anti-Hib PS antibodies were not detected in any of the 2-month-old infants but were detected in 29% of the 18-month-old infants and 69% of the adults (p 0.15 microgram/ml) in 45% of these infants. Hibld-1, an idiotope expressed by anti-Hib PS antibodies having the kappa II-A2 V region, was present in postvaccination sera of 66% of the adults and 80% of the 18-month-old infants but was less frequent in the 2-month-old infants (35%, p < 0.001). In contrast, Hibld-2, which is an idiotope expressed by a subset of V lambda VII anti-Hib PS antibodies, was rare or infrequent in adults and 18-month-old infants (0% and 6%, respectively) but was present in 43% of 2-month-old infants (p < 0.001). Our findings demonstrate that dramatic changes in VL region utilization occur in the human antibody response to this Hib PS conjugate vaccine as a function of age. Because previous studies have shown that V region usage correlates with antibody fine specificity and avidity for Hib PS, these age-related differences in V region expression may affect the ability of vaccines to confer protective immunity at different ages.
TL;DR: Authentic taxanes and extracts from cell cultures derived from various yew tree species have been analyzed by microbore high-performance liquid chromatography-electrospray mass spectrometry and excellent spectra with dominant protonated molecules at low nozzle-to-skimmer bias value (45 V) were obtained.
TL;DR: An initial trial with the parent compound, delivered as Arginine Butyrate, has demonstrated rapid stimulation of fetal globin expression to levels that have been shown to ameliorate sickle cell disease and the β thalassemias.
Abstract: The inherited β-hemoglobinopathies (sickle cell disease and β thalassemia) are the result of a mutation in the adult (β) globin gene. The fetal globin chain, encoded by the γ globin genes, can substitute for the mutated or defective β globin chain, but expression of the γ globin gene is developmentally inactivated prior to birth. Reinducing expression of the normal fetal globin genes is a preferred method of ameliorating sickle cell disease and the β thalassemias. Stimulation of as little as 4–8% fetal globin synthesis in the bone marrow can produce >20% fetal hemoglobin in the peripheral circulation, due to enhanced survival of red blood cells containing both sickle and fetal hemoglobin, compared to those containing sickle hemoglobin alone. Butyric acid and butyrate derivatives are generally safe compounds which induce fetal hemoglobin production by stimulating the promoter of the fetal globin genes. An initial trial with the parent compound, delivered as Arginine Butyrate, has demonstrated rapid stimulation of fetal globin expression to levels that have been shown to ameliorate these conditions. Phase 1 trials of an oral butyrate derivative with a long plasma half-life have just begun. These agents now provide a specific new apporach for ameliorating these classic molecular disorders and merit further investigation in larger patient populations.
TL;DR: It is concluded that spectrin plays, at best, only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.
TL;DR: The results suggest that the loss of PtdCho during calcium-loading of human erythrocytes is caused by a previously unrecognized Ptd Cho-hydrolyzing phospholipase D, resulting in direct generation of phosphatidic acid.
Abstract: We have studied the mechanism by which calcium-loading of human erythrocytes stimulates phospholipid turnover and generates diacylglycerol and phosphatidic acid. Using quantitative measurement of individual phospholipid classes, we have demonstrated that the amount of phosphatidic acid generated during calcium-loading of intact red cells exceeds the amount of diacylglycerol formed by phospholipase-C-mediated hydrolysis of the polyphosphoinositol lipids and that addition of the diacylglycerol kinase inhibitor, R59022, only partly inhibited this increase. Thus, in contrast to current explanations, the phosphatidic acid generated following calcium-loading of erythrocytes cannot be solely explained by the action of a polyphosphoinositol-lipid-specific phospholipase C with subsequent phosphorylation of diacylglycerol to phosphatidic acid. Our data demonstrate that calcium-loading of intact erythrocytes, but not of red cell ghost membranes, causes a small but significant decrease in the relative amount of phosphatidylcholine (PtdCho). In order to identify the mechanisms responsible for calcium-mediated hydrolysis of PtdCho, we encapsulated Ptd[Me-14C]Cho-containing rat liver microsomes into erythrocytes and studied the generation of [Me-14C]choline and phospho[Me-14C]choline. We found that choline was the only detectable 14C-labeled product. Furthermore, incubation of erythrocytes with calcium under hypotonic conditions and in the presence of [14C]PtdCho vesicles and ethanol resulted in the formation of [14C]phosphatidylethanol. Together, these results suggest that the loss of PtdCho during calcium-loading of human erythrocytes is caused by a previously unrecognized PtdCho-hydrolyzing phospholipase D, resulting in direct generation of phosphatidic acid. Analysis of the molecular species composition of PtdCho, phosphatidic acid, and diradylglycerol, confirm the simultaneous actions of PtdCho-hydrolyzing and polyphosphoinositol-lipid-hydrolyzing phospholipases in calcium-loaded human erythrocytes.
TL;DR: Twenty involved hips in 16 patients with Legg-Calvé-Perthes disease (LCP) were studied with both plain radiographs and magnetic resonance imaging (MRI) scans to better evaluate the existence of "metaphyseal" changes.
Abstract: Twenty involved hips in 16 patients with Legg-Calve-Perthes disease (LCP) were studied with both plain radiographs and magnetic resonance imaging (MRI) scans to better evaluate the existence of «metaphyseal » changes. Thirty-four sets of radiographs and MRI scans were reviewed in a blinded fashion and compared for the presence and location of these changes. Of 23 hips with plain radiographic metaphyseal changes, 11 showed no such changes on MRI scans (48%). Twelve hips did show MRI changes located in the anterior metaphysis. One hip studied three times had a discrete cystic change located in the central metaphysis. Of 11 hips with no plain radiographic changes in the metaphysis, five showed metaphyseal changes on MRI
TL;DR: A method to elucidate the side-chain structure of novel taxoids was developed through use of electrospray ionization tandem mass spectrometry, which proved valuable in the characterization of new taxoids produced by yew-tree cell cultures.
Abstract: A method to elucidate the side-chain structure of novel taxoids was developed through use of electrospray ionization tandem mass spectrometry. The intact side-chain fragments produced by source fragmentation in the atmosphere-to-vacuum interface were further dissociated in the collision cell of a triple quadrupole mass spectrometer. The distinctive collisionally induced dissociation mass spectra were remarkably informative and gave detailed information on the side-chain functionalities. This technique proved valuable in the characterization of new taxoids produced by yew-tree cell cultures.
TL;DR: The results presented here strongly suggest that M‐CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.
Abstract: The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.
TL;DR: An infant with hemoglobin D Ibadan‐β° thalassemia whose hemoglobinopathy was initially detected by neonatal screening is described, whose previously undescribed condition was confirmed by family studies and by globin chain analysis by mass spectrometric techniques.
Abstract: We describe an infant with hemoglobin D Ibadan-β° thalassemia whose hemoglobinopathy was initially detected by neonatal screening. This previously undescribed condition was confirmed by family studies and by globin chain analysis by mass spectrometric techniques. The case illustrates the importance to neonatal screening programs of confirmatory testing and of linkage with reference laboratories capable of globin chain analysis. Hematologic studies at 36 months of age suggested that the presence of hemoglobin D Ibadan had no deleterious effect on this child with heterozygous β° thalassemia. © 1993 Wiley-Liss, Inc.
TL;DR: The results indicate that the ketoreductase activity per se is unaffected by subunit dissociation and are consistent with a model in which the transfer of substrate from CoA ester to the acyl-carrier-protein domain necessitates juxtaposition of the transferase active-site serine residue of one subunit and the phosphopantetheine moiety of the adjacent subunit.
Abstract: The controversial question as to whether the ketoreductase activity of the animal fatty acid synthase is lost on dissociation of the homodimer has been addressed by using immobilized subunits which cannot reassociate under the conditions of assay. Ketoreductase activity, assessed with the model substrate S-acetoacetyl-N-acetylcysteamine, was identical in immobilized monomers and dimers, exhibiting normal Michaelis-Menten kinetics with Km values in the millimolar range. When acetoacetyl-CoA was used as a substrate, however, biphasic kinetics were observed in the case of the dimer, with estimated Km values in the micro- and milli-molar ranges, but only the high-Km reaction was observed with the monomer. Thus when the ketoreductase activities of the monomer and dimer are assessed with acetoacetyl-CoA at concentrations sufficient to saturate only the low-Km reaction, it appears that the ketoreductase activity towards acetoacetyl-CoA is lost upon dissociation. Reduction of acetoacetyl-CoA via the low-Km pathway is CoA-dependent, indicating that acetoacetyl-CoA can react with the dimer by two mechanisms: a high-Km pathway analogous to that utilized by model substrates and a low-Km pathway in which substrate and product are transferred between acyl-CoA and acyl-enzyme forms. The results indicate that the ketoreductase activity per se is unaffected by subunit dissociation and are consistent with a model in which the transfer of substrate from CoA ester to the acyl-carrier-protein domain necessitates juxtaposition of the transferase active-site serine residue of one subunit and the phosphopantetheine moiety of the adjacent subunit.
TL;DR: A material isolated following pregnenolone incubations with toad inter-renal tissue at 28 degrees C has been identified as a 3 beta-hydroxy-5-ene analogue of aldosterone, which was by far the best precursor.
Abstract: A material isolated following pregnenolone incubations with toad (Bufo arenarum) inter-renal tissue at 28 degrees C has been identified as a 3 beta-hydroxy-5-ene analogue of aldosterone (3 beta, 11 beta, 21-trihydroxy-20-oxo-5-pregnen-18-al). The initial identification was made by enzymic and m.s. methods, and structural confirmation was achieved through comparison with chemically synthesized authentic material. The relative efficacy of corticosterone, 18-hydroxycorticosterone and the 3 beta-hydroxy-5-ene aldosterone analogue as aldosterone precursors was evaluated. In the in vitro situation studied, the 3 beta-hydroxy-5-ene steroid was by far the best precursor.