Showing papers by "Children's Hospital Oakland Research Institute published in 1996"
TL;DR: It is concluded that loss of phospholipid asymmetry may occur in small subpopulations of red cells and that fluorescently labeled annexin V can be used to quantify and isolate these cells.
302 citations
Journal Article•
TL;DR: Brain MR imaging showed infarction/ischemia in the absence of a recognized cerebrovascular accident in 13% of patients, suggesting that lesions are present by age 6, however, the increase in the average number of lesions per patient with age may indicate progressive brain injury.
Abstract: PURPOSE To define the spectrum of abnormalities in sickle-cell disease, including infarction, atrophy, and hemorrhage, that are identified by brain MR imaging. METHODS All MR studies included T1, T2, and intermediate pulse sequences. Images were interpreted without knowledge of the clinical history or neurologic examination findings. Brain MR imaging was performed in 312 children with sickle-cell disease. RESULTS Seventy patients (22%) had infarction/ischemia and/or atrophy, infarction/ischemia was noted in 39 children (13%) who had no history of a stroke (the "silent" group). The prevalence rates for silent lesions were 17% for sickle-cell anemia and 3% for hemoglobin sickle-cell disease. For patients with sickle-cell anemia and a history of cerebrovascular accident, infarction/ischemia lesions typically involved both cortex and deep white matter, while silent lesions usually were confined to deep white matter. Within the age range studied, the prevalence of infarction/ischemia did not increase significantly with age, although older patients with lesions had more lesions than did younger patients with lesions. CONCLUSIONS Brain MR imaging showed infarction/ischemia in the absence of a recognized cerebrovascular accident in 13% of patients. The prevalence of these lesions did not increase significantly between the ages of 6 and 14 years, suggesting that lesions are present by age 6. However, the increase in the average number of lesions per patient with age may indicate progressive brain injury.
283 citations
TL;DR: The measurement of urinary free cortisone (UFE) excretion in normals and in patients with disorders of the pituitary‐adrenal axis is validated in an attempt to more accurately measure the activity of 11β‐HSD2 in vivo.
Abstract: OBJECTIVE Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyse the interconversion of cortisol to hormonally inactive cortisone; defects in the 11 beta-HSD2 isoform result in hypertension. The kidney, expressing high levels of 11 beta-HSD2, is the principal source of cortisone in man. We have validated the measurement of urinary free cortisone (UFE) excretion in normals and in patients with disorders of the pitultary-adrenal axis in an attempt to more accurately measure the activity of 11 beta-HSD2 in vivo. SUBJECTS Forty-one normal adults, 12 normal children < 12 years of age, 15 patients with Cushing's syndrome, 12 with hypopitultarism on replacement hydrocortisone, 12 with the syndrome of apparent mineralocorticoid excess (AME) and 7 volunteers consuming liquorice. MEASUREMENTS A complete 24-hour urine collection was analysed by gas chromatography/mass spectrometry for "A-ring' reduced cortisol and cortisone metabolites, i.e. tetrahydrocortisols (THF and allo-THF) and tetrahydrocortisone (THE). In addition, urinary free cortisol (UFF) and urinary free cortisone were quantified using deuterium-labelled internal standards. RESULTS In normal adults and children, UFE excretion exceeded that of UFF (UFF 30.4 +/- 2.4 micrograms/24h (mean +/- SE), UFE 54.6 +/- 4.1 micrograms/24h, adults) (for conversion to nmol/24h multiply E by 2.78 and F by 2.76 respectively). Thus the normal UFF/UFE ratio was 0.54 +/- 0.05 in contrast to the (THF + allo-THF)/THE ratio of 1.21 +/- 0.06. UFE excretion was normal in hypopituitary patients on replacement hydrocortisone. Although UFE was elevated in all forms of Cushing's syndrome, the UFF/UFE ratio was grossly elevated in patients with the ectopic ACTH syndrome (14.0 +/- 6.7, n = 6). UFE was below the lower limit of the assay (< 1 microgram/24h) in most patients with the so-called type 1 variant of AME and significantly reduced in 4 patients described as having the type 2 variant of AME (10.5 +/- 3.5 micrograms/h, P < 0.05) and in 7 volunteers consuming liquorice (26.8 +/- 10.0 micrograms/24h, P < 0.01). In ectopic ACTH syndrome, AME, and liquorice ingestion the UFF/UFE ratio was more deranged than the (THF + allo-THF)/THE ratio. CONCLUSION In normals the discrepant THF + allo-THF/ THE and UFF/UFE ratio suggests that much more of the UFE is derived from the kidney. Reduction in UFE excretion is seen following liquorice ingestion and in both variants of AME, though it is more profound in AME1. The high UFF/UFE ratio in the mineralocorticoid excess state seen in the ectopic ACTH syndrome is compatible with substrate-saturation of renal 11 beta-HSD2. The measurement of UFE and the UFF/UFE ratio is a significant advance in the analysis of human 11 beta-HSD activity in vivo; in particular, the UFF/UFE ratio appears to be a more sensitive index than the (THF + allo-THF)/THE ratio of renal 11 beta-HSD2 activity.
273 citations
Journal Article•
TL;DR: The inter- and intrafamilial variability in expression of FGFR2 mutations suggests that these three syndromes, presumed to be clinically distinct, are instead representative of a spectrum of related craniosynostotic and digital disorders.
Abstract: Fibroblast growth factor receptor 2 (FGFR2) mutations have been associated with the craniosynostotic conditions Crouzon, Jackson-Weiss, and Pfeiffer syndromes. Previously, mutations were described in the exons IIIa and IIIc, which form the extracellular, third immunoglobulin-like domain (IgIII) and adjacent linker regions, both of which are normally involved in ligand binding. For all three conditions, mutations were found in exon IIIc. Only in Crouzon syndrome were mutations identified in exon IIIa. In this study, 39 cases with one of these three conditions were screened for exon IIIa or IIIc mutations. Eleven mutations are reported in 17 unrelated cases. Mutations in exon IIIa are identified for not only Crouzon but also Jackson-Weiss and Pfeiffer syndromes. Four mutations in either exon IIIa or exon IIIc reported only in Crouzon syndrome are present also in one of the other two syndromes. Two insertions, one in exon IIIa in a Crouzon syndrome patient and the other in exon IIIc in a Pfeiffer syndrome patient, were observed. The latter mutation has the same alternative RNA splicing effect as a reported synonymous mutation for Crouzon syndrome. A missense mutation was detected in one Pfeiffer syndrome family in which two members had craniosynostosis without limb anomalies. The inter- and intrafamilial variability in expression of FGFR2 mutations suggests that these three syndromes, presumed to be clinically distinct, are instead representative of a spectrum of related craniosynostotic and digital disorders.
172 citations
TL;DR: The formation of cholesterol chlorohydrins could be potentially disruptive to cell membranes and result in cell lysis and death, and could also be potential biomarkers for oxidative damage associated with neutrophil/monocyte activation.
128 citations
TL;DR: Results show that GP Ib-IX-von Willebrand factor interactions lead to cytoskeletal reorganization, that the cytoplasmic domain of GP Ib regulates these reorganizations, and that the Cytoplaski domain is absolutely required for attachment of the GP Ib -IX complex to the cytoskeleton.
89 citations
TL;DR: The present study shows that, in intact platelets, αβ forms clusters when occupied by ligand and is selectively moved into the open canalicular system; αβ that has not bound ligand remains diffusely distributed at the periphery of the cell.
75 citations
TL;DR: Hb fluorescence was found to reflect the departure of heme from globin due to a decrease in fluorescent quenching effect by the heme moiety, and hemin was shown to be a more potent catalyst of lipid peroxidation in RBC membrane than nonheme irons.
72 citations
TL;DR: It is found that the larger part of urinary cortisol and cortisone is not free but is released from conjugation by enzymes present in snail digestive juice.
69 citations
TL;DR: The results suggest that essential to the regulation of FAS transcription by cAMP is the interaction of an inverted CCAAT box motif with a constitutively produced trans-acting factor that either itself undergoes modification in response to cAMP or associates with a protein that is produced or modified bycAMP exposure.
69 citations
TL;DR: Formalin-fixed, paraffin-embedded samples from 19 cases of osteosarcoma are analyzed for molecular changes at the RB locus using polymerase chain reaction amplification of polymorphic short tandem repeat sequences (microsatellite repeats).
Abstract: Studies of osteosarcoma cell lines or frozen tissue have detected loss of heterozygosity (LOH) at the retinoblastoma (RB) locus by Southern blot analysis or restriction fragment length polymorphism. Most archived clinical specimens cannot be analyzed by these techniques. We analyzed formalin-fixed, paraffin-embedded samples from 19 cases of osteosarcoma for molecular changes at the RB locus using polymerase chain reaction amplification of polymorphic short tandem repeat sequences (microsatellite repeats). Four repeat sequences, two within and two flanking the RB gene, were analyzed. Fourteen of 18 informative cases (78%) showed molecular changes at the RB locus. LOH was identified in 13 cases (72%). Unexpectedly, microsatellite instability (MI) was found in eight cases (44%). All of the cases of MI involved alterations of more than one repeat unit, and six of eight were associated with LOH. LOH was identified at three unlinked loci in one case and at a single locus in another Microsatellite analysis of archival tissue yields prevalence rates of LOH comparable to those found by other methods and has the added advantage of showing MI. The ability to use formalin-fixed, paraffin-embedded tissue extends genetic analysis to routinely processed surgical material and may permit molecular confirmation of challenging cases of osteosarcoma.
TL;DR: The results of this study demonstrate that this community in northeastern Brazil is highly endemic for E. histolytica with infection rates similar to other developing nations.
Abstract: Infection with the human pathogenic parasite Entamoeba histolytica has not been well-characterized in northeastern Brazil. In this study, the prevalence of E. histolytica infection in a slum in northeastern Brazil was assayed using an enzyme-linked immunosorbent assay (ELISA) for antibodies against the galactose/N-acetyl-D-galactosamine (Gal/GalNAc)-inhibitable adherence lectin of E. histolytica. Sera from a total of 335 individuals were examined for anti-Gal/GalNAc lectin antibodies. The overall seropositivity was 24.7%; 29.4% of females and 19.4% of males were positive. Among different age groups there was a peak of 40% positivity in the 6-14-year-old age group. There was also familial clustering of seropositivity. To examine colonization, stool samples from 155 people were examined microscopically for the presence of the parasite. Fourteen of 155 stools (9.0%) were identified as containing E. histolytica or nonpathogenic E. dispar. These 14 positive stools were analyzed with an ELISA that detects Gal/GalNAc lectin antigen and can distinguish between E. histolytica and E. dispar. Four stools (29%) were positive for E. histolytica and the remaining 10 were identified as E. dispar-positive. Although the overall colonization rate by microscopy was only 9%, with a third identified as E. histolytica, up to 40% of older children develop serologic evidence of having experienced pathogenic E. histolytica infection. The results of this study demonstrate that this community in northeastern Brazil is highly endemic for E. histolytica with infection rates similar to other developing nations.
TL;DR: Fatty acid bromohydrins could contribute to the antimicrobial activity and inflammatory tissue damage by eosinophils and neutrophils, and could potentially be useful specific markers for HOBr production in vivo.
TL;DR: The reduction of diversity for the HLA class II alleles in the Colombian Indians is suggestive of a population bottleneck during the colonization of the Americans, with little to no subsequent admixture with neighboring African Colombian populations in the last approximately 300 years.
Abstract: HLA class II variation was analyzed in nine Native American populations of Colombia using PCR/SSOP typing methods. Under the auspices of the Expedition Humana, approximately 30 unrelated native Colombia Indian samples each from the Tule (NW Pacific Coast), Kogui (Sierra Nevada). Ijka (Sierra Nevada), Ingano (Amazonas), Coreguaje (Amazonas), Nukak (Amazonas), Waunana (Pacific), Embera (Pacific) and Sikuani (Northeastern Plains) were collected and analyzed at the DRBI, DQA1, DQB1 and DPB1 loci. The number of different DRB1, DQA1, DQB1 and DPB1 alleles in the Colombian Indians is markedly reduced in comparison with neighboring African Colombian populations, which exhibit a very high degree of class II variability, as discussed in an accompanying paper. In the Colombian Amerindian groups, DR2 (DRB1*1602), DR4 (DRB1*0407, *0404, *0403 AND *0411), DR6 (DRB1*1402) and DR8 (DRB1*0802) comprise > 95% of all DRB1 alleles. We also found an absence of DR3 in all populations, and DR1, DR7 and DR9 allelic groups were either very rare or absent. Each Colombian Amerindian population has a predominant DRB1 allele (f = approximately 0.22-0.65) and DRB1-DQA1-DQB1 haplotype. Several novel DR-DQ haplotypes were also found. At the DPB1 locus, DPB1*0402 (f = 0.28-0.82), *1401 (f = 0.03-0.45), and *3501 (f = 0.03-0.27), were the three most prevalent alleles, each population maintaining one of these three alleles as the predominant (f > 0.26) DPB1 allele. The reduction of diversity for the HLA class II alleles in the Colombian Indians is suggestive of a population bottleneck during the colonization of the Americans, with little to no subsequent admixture with neighboring African Colombian populations in the last approximately 300 years.
TL;DR: Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation, which indicates that rFvIII-reactive T lymphocyte are present in the peripheral circulation of some inhibitor-positive hemophilia A patients.
Abstract: In this study we sought to determine whether factor VIII-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 micrograms of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production.
TL;DR: It is observed that the cysteine 447 was protected against alkylation under these conditions, whereas cysteines 381, 417, and 530 were fully derivatized.
TL;DR: The formation of catalytically-active heterodimers from pairs of inactive, complementary homodimer affords a useful method for testing the validity of the current model for the multifunctional complex.
Abstract: The animal fatty acid synthase is a dimer of identical, multifunctional 272 kDa subunits oriented antiparallel such that two centers for fatty acid synthesis are formed at the subunit interface. In order to clarify the interdomain and intersubunit communications necessary for the operation of the two centers, we have explored the possibility of reassembling catalytically-active fatty acid synthase heterodimers from pairs of inactive dimers carrying mutations in different functional domains. To this end, rat fatty acid synthase mutants, defective in either the beta-ketoacyl synthase, C161T or K326A (KS- FAS), or the acyl carrier protein, S2151A (ACP- FAS), domains, were engineered by site-directed mutagenesis, expressed in insect Sf9 cells using a baculovirus expression system, and purified. A novel procedure was devised to facilitate rapid production and isolation of a population of mixed mutant dimers that had undergone randomization of its constituent subunits. Homodimeric mutants (KS- FAS/KS- FAS and ACP- FAS/ACP- FAS) and KS- FAS heterodimers consisting of paired C161T and K326A mutant subunits were unable to synthesize fatty acids, confirming the essential nature of residues C161, K326, and S2151A. However, KS- FAS/ACP- FAS heterodimers regained partial activity. Formation of these heterodimers necessitated prior dissociation and reassociation of the homodimers, indicating that the rate of spontaneous exchange of subunits in the dimer is negligible. The formation of catalytically-active heterodimers from pairs of inactive, complementary homodimers affords a useful method for testing the validity of the current model for the multifunctional complex.
TL;DR: The hyposmotic shock procedure successfully permeabilized apical membranes of primary cultures of nasal epithelial cells from a patient with cystic fibrosis and of JME/CF 15 cells, a cell line derived from CF bronchial epithelium.
Abstract: We describe a simple hyposmotic shock procedure whereby the apical membrane of airway epithelium can be made transiently leaky to proteins and other macromolecules. Bovine or human tracheal epithelial cells were grown as confluent polarized cell sheets on porous inserts. While physiological saline was maintained on the basolateral surface, the mucosal surface was exposed to water. This led to marked increases in the uptake of [14C]mannitol across both apical and basolateral membranes. On restoring saline to the mucosal surface, the [14C]mannitol permeability returned to preexposure levels with a half-life of approximately 5 min. Mucosal water also increased efflux of lactate dehydrogenase and the uptakes of fluorescent albumin and dextran (2,000 kDa). Water-induced increases in mannitol permeability were similar at 4 and 37 degrees C, suggesting that pinocytosis was not the mechanism. Detailed time courses of the uptake of dextran and the loss of lactate dehydrogenase and 36Cl showed that the bulk of the permeability increase occurred during the first 2- to 4-min exposure to water. Transepithelial resistance was reversibly decreased by exposure to water, but short-circuit current responses to transport blockers and secretagogues remained qualitatively normal. The hyposmotic shock procedure also successfully permeabilized apical membranes of primary cultures of nasal epithelial cells from a patient with cystic fibrosis (CF) and of JME/CF 15 cells, a cell line derived from CF bronchial epithelium. This simple and efficient procedure may prove useful in studies on the cell and molecular biology of airway and other epithelia.
TL;DR: Analysis of regional Colombian African American HLA population genetics is discussed with respect to the Colombian Amerindian HLA genetics described in an accompanying paper.
Abstract: PCR/SSOP typing methods were used to analyze the HLA Class II DRB1, DQA1, DQB1 and DPB1 loci of samples from three African American populations of Colombia. Forty samples from the Cauca (Pacific), and twenty samples each from the Choco (North Pacific Coast) and the Providencia (Caribbean island) populations, were collected and the Class II loci analyzed under the auspices of the Expedicion Humana. Despite the limited number of samples analyzed, the African Colombian populations exhibit a very high degree of class II polymorphism. A great diversity of DRB1 alleles was found, with representatives from all serological classes, including 19 DRB1 alleles in the Providencia, 16 in the Cauca and 14 in the Choco groups. In addition, a novel DQB1*02 allele (*0203) was found in two individuals from the Cauca population of the Pacific Coast. The sequence of the DQB1*0203 allele, associated with DR3, differs from DQB1*0201 by only one nucleotide substitution (C-->A) in the second position of codon 57, resulting in an Ala to Asp change. The addition of DQB1*0203 brings the total number of DQB1 alleles identified to date to 26. HLA class II diversity is much greater in these African Colombian populations than that seen in nearby Amerindian populations. Analysis of regional Colombian African American HLA population genetics is discussed with respect to the Colombian Amerindian HLA genetics described in an accompanying paper.
TL;DR: Kinetic properties of the refolded recombinant enzyme were indistinguishable from those of a transferase preparation derived from the natural fatty acid synthase by limited proteolysis, indicating that the transferase domain is capable of folding correctly as an independent protein.
TL;DR: The results show that an enzyme specific for the formation of PC from lysoPC can be isolated in PC vesicles with a designed phospholipid molecular species composition and that the lipid environment plays an important role in the regulation of the enzyme's affinity for its substrates.
Abstract: The enzyme acyl coenzyme A:1-acyllysophosphatidylcholine acyltransferase (acyl-CoA:lysoPC acyltransferase) can be isolated in newly formed phosphatidylcholine (PC) vesicles by solubilization of rat liver microsomes with the two substrates lysoPC and acyl-CoA. In this study, we sought to optimize the conditions for the formation of PC vesicles and analyzed the lipid composition and enzyme activity of the newly formed vesicles. Analysis of PC vesicles formed by incubation of the microsomal preparation with 1-(C16:0)lysoPC and C18:1CoA, C18:2CoA, or C20:4CoA showed that the optimal protein:lysoPC ratio was 1:5 (by weight) and the optimal lysoPC:acyl-CoA ratio was 1:1 (molar amounts). PC formation increased with incubation time; after 20 h of incubation at 37 °C, approximately 75% of the lysoPC was converted to PC in the incubation mixture. The phospholipid molecular species composition of the vesicles reflected almost exclusively the substrates used; the vesicles contained approximately 33% of the total acyl...
TL;DR: Structural confirmation of hemoglobin variants can lead to early diagnosis of hemoglobal hemoglobin-based illnesses as mentioned in this paper, which can be used to diagnose hemoglobin based illnesses early.
Abstract: Structural confirmation of hemoglobin variants can lead to early diagnosis of hemoglobin-based illnesses
TL;DR: Results indicate that presence of α chains favors assembly with β-globin, β-β dimers cannot bind α chains, and that Hb A tetramer formation results in the most thermally stable species.
TL;DR: The findings confirm that low Cp is a characteristic of the acute recovery from major burns and the oxidase assay is shown to be valid for veryLow Cp levels even during high vitamin C provision.
TL;DR: The membrane phospholipid organization in human red blood cells (RBC) is rigidly maintained by a complex system of enzymes but several elements of this system are sensitive to oxidative damage, and the presence of this oxidative stress to the RBC membrane could lead to alterations in membrane lipid organization.
Abstract: The membrane phospholipid organization in human red blood cells (RBC) is rigidly maintained by a complex system of enzymes However, several elements of this system are sensitive to oxidative damage An important component in the destruction of beta-thalassemic RBC is the generation of reactive oxygen species and the release of redox-active iron by the unpaired alpha-hemoglobin chains Consequently, we hypothesized that the presence of this oxidative stress to the RBC membrane could lead to alterations in membrane lipid organization Model beta thalassemic RBC, prepared by the introduction of excess alpha-globin in the cell, have previously been shown to exhibit structural and functional changes almost identical to those observed in beta-thalassemic cells After 24 hr at 37 degrees C, the model beta thalassemic cells exhibited a significant loss of deformability, as measured by ektacytometric analysis, indicative of extensive membrane damage However, a normal steadystate distribution of endogenous phospholipids was found, as evidenced by the accessibility of membrane phospholipids to hydrolysis by phospholipases Similarly, the kinetics of transbilayer movement of spin-labeled phosphatidylserine (PS) and phosphatidylethanolamine (PE) in all samples was in the normal range and was not affected by the presence of excess alpha-globin chains In contrast, a faster rate of spin-labeled phosphatidylcholine (PC) transbilayer movement was observed in these cells While control RBC exhibited a complete loss of their initial (2 mol%) lysophosphatidylcholine (LPC) levels following 24 hr of incubation at 37 degrees C, 15 mol% LPC was still present in model beta-thalassemic cells, suggesting an altered phospholipid molecular species turnover, possibly as a result of an increased repair of oxidatively damaged phospholipids
TL;DR: It is suggested that natural antibodies reacting with Hib CP in healthy Hong Kong Chinese are the product of exposure to some cross‐reactive antigen(s), different from both Hib and E. coli K100 CP.
Abstract: The Chinese population in Hong Kong has a low incidence of invasive Haemophilus influenzae type b(Hib) disease, as well as carriage of the microorganism. Likely stimuli for the natural antibodies to Hib, which might protect against Hib infection, are cross-reactive antigens of bacteria like Escherichia coli K 100. Our aim was to determine the isotype and idiotype distribution and cross-reactivity of natural antibodies against Hib capsular polysaccharide (CP) in healthy Hong Kong Chinese. Titration of 20 sera by ELISA showed IgG antibodies reacting with Hib CP in all individuals. The antibodies were mainly IgG2, and their avidity index ranged widely. Isoelectric focusing (IEF) combined with immunoblotting showed patterns of IgG2 antibody clones against the CP of Hib and E. coli K 100 which were similar in 10 cases. Absorption with Hib CP only eliminated some bands in two sera. Absorption with K 100 CP did not remove any anti-Hib CP bands. In three sera additional clones of antibodies reacting to K 100 CP only, disappeared after absorption with this CP. Spectrotypic analyses of IgG antibodies reacting with anti-Hib idiotype 1 (Id-1) revealed stronger IEF patterns with bands in differing locations compared with anti-Hib CP antibodies. The strong reactivity of serum IgG, IgA and IgM antibodies with monoclonal anti-Hib Id-1 was confirmed by ELISA. This reactivity was not abolished after absorption of the sera with either Hib CP, or K 100 CP. The data indicate a high prevalence of Id-1 among Hong Kong Chinese. However, only one individual had Id-1 antibodies specific for Hib CP, judging from absorption experiments. Others had much lower activity of Id-1 anti-Hib CP antibodies compared with the total IgG Id-1, suggesting that Hong Kong subjects have Id-1-positive antibodies in their serum which are not specific for Hib CP. This is consistent with the nature of Id-1, which is a marker of A2VL region usage rather than a marker of a Hib CP paratope. We suggest that natural antibodies reacting with Hib CP in healthy Hong Kong Chinese are the product of exposure to some cross-reactive antigen(s), different from both Hib and E. coli K 100 CP.
TL;DR: The data demonstrate that ESMS on a quadrupole mass spectrometer of limited mass range has potential utility for studying ligand-binding proteins and in future it might be possible to compare spectra obtained from agonist- and antagonist-bound receptors and determine from subtle changes in protonation state possible differences in the higher order structure of those noncovalent protein complexes.
TL;DR: Six new DPB1 alleles identified by PCR-SSOP methodologies in the course of a retrospective study of the role of HLA matching in the outcome of unrelated donor bone marrow transplantation confirmed that five of these alleles represent novel combinations of previously described sequence motifs in the variable regions ofDPB1.
Abstract: Six new DPB1 alleles were identified by PCR-SSOP methodologies in the course of a retrospective study of the role of HLA matching in the outcome of unrelated donor bone marrow transplantation. Sequencing confirmed that five of these alleles (DPB1*5901, *6801, *7101, *7201, and *7301) represent novel combinations of previously described sequence motifs in the variable regions of DPB1; the sixth (DPB1*7001) appears to result from a novel point mutation. These data support previous observations which suggest that multiple mechanisms, including segmental exchange and mutation, appear to be responsible for generating sequence diversity at the DPB1 locus. The extremely low discrepancy rate of 0.1% between the two laboratories which typed the samples, and the ability to predict the new sequences from probe hybridization patterns, indicate that SSOP is an accurate and efficient method for studying polymorphism at DPB1.
TL;DR: In conclusion, initiation of FAS transcription from a single start site is enhanced by the presence of an adjacent TATA motif, an inverted CCAAT box and an upstream binding site for the transcription factor Sp1; further modulation of transcription is achieved through complex interactions between these promoter elements and an downstream negative regulatory element.
Abstract: The gene for fatty acid synthase (FAS), which contains both GC-rich sequences and a TATA box in its promoter region, is expressed in a tissue-specific manner in response to developmental, nutritional and hormonal signals. Here we report the identification of sequence elements in the 5'-flanking region responsible for modulation of basal promoter activity. Transient transfection of H4IIE hepatoma cells and 3T3-30A5 preadipocytes with plasmids containing the chloroamphenicol acetyltransferase gene driven by FAS promoter sequences of different lengths revealed that two regions between nucleotides -249 and -30 contain elements capable of enhancing transcription. One of these positive regulatory elements was localized to nucleotides -241/-236 using DNase I footprinting, electrophoretic mobility-shift assays and mutagenesis. The sequence element is a typical GC box and the nuclear protein binding to this region appears immunochemically indistinguishable from Sp1. The second positive regulatory element, an inverted CCAAT box, was localized to nucleotides -98/-92 by electrophoretic mobility-shift assays and mutagenesis. A putative negative regulatory element, initially identified by reporter gene transfection experiments, was localized between nucleotides -319 and -301 by DNase I footprinting, electrophoretic mobility-shift assays and deletion mutagenesis; this region consists of 78% G residues. In conclusion, initiation of FAS transcription from a single start site is enhanced by the presence of an adjacent TATA motif, an inverted CCAAT box and an upstream binding site for the transcription factor Sp1; further modulation of transcription is achieved through complex interactions between these promoter elements and an upstream negative regulatory element.