Search PubMed⌕ Search

Biomedical subjects

R M Jack

Publications and source records attributed to R M Jack.

At least 19 recordsLinked to original sources
PubMed1998

Ahomocysteinemia in molybdenum cofactor deficiency.

We report an infant with molybdenum cofactor deficiency (MCD) and a unique clinical presentation of hemiplegia, hypotonia, dystonia, and bilateral basal ganglia changes. Biochemistry revealed absent serum homocysteine, low concentrations of plasma cystine, high levels of urinary S-sulfocysteine and sulfite, and high levels of oxypurines in serum and urine. The depletion of cysteine and cystine through reaction with sulfite suggests that other thiols and thiol-dependent proteins may be similarly depleted. Ahomocysteinemia may be a clue to the mechanism of cytotoxicity in MCD.

Brain↗
PubMed1997

Fetal origin of amniotic fluid polymorphonuclear leukocytes.

OBJECTIVE: Although polymorphonuclear leukocytes are the inflammatory cells most frequently recovered from the amniotic cavity in cases of suspected intrauterine infection, the source of these cells has not been definitively determined. We took advantage of the gender difference between the mother and her male fetus, and we report four cases in which amniotic fluid polymorphonuclear leukocytes were identified as fetal by fluorescence in situ hybridization with probes specific for X and Y chromosomes. Fetal membranes were intact at the time amniotic fluid was obtained in all cases. STUDY DESIGN: Amniotic fluid was obtained from women with male fetuses in premature labor with clinical or laboratory evidence of infection. Cytospin preparations of amniotic fluid samples with polymorphonuclear leukocytes were prepared and sequentially stained with fluorescent reagents. To determine which cells were polymorphonuclear leukocytes, all replicate samples were stained with the fluorescent nuclear stain 4'-6-diamidino-2-phenyl-indole. This allowed definition of the characteristic multilobed polymorphonuclear leukocytes nuclear morphologic features. The sample was then probed with a rhodamine-labeled probe specific for the X chromosome and a fluorescein-labeled probe specific for the Y chromosome to assess whether the polymorphonuclear leukocytes were male or female. RESULTS: Ninety percent to 99% of polymorphonuclear leukocytes identified by normal multiple lobed nuclear morphologic study on 4'-6-diamidino-2-phenyl-indole staining had an X and Y chromosome and were therefore fetal cells. CONCLUSIONS: These data demonstrate a fetal response during intraamniotic infection. Further investigation of the roles for maternal and fetal polymorphonuclear leukocytes in chorioamnionitis may provide valuable information about the critical interaction of the two immune responses in this setting.

Amniotic Fluid↗
PubMed1997

Complement receptor type 1 (CR1, CD35) is a receptor for C1q.

Molecular definition of the cellular receptor for the collagen domain of C1q has been elusive. We now report that C1q binds specifically to human CR1 (CD35), the leukocyte C3b/C4b receptor, and the receptor on erythrocytes for opsonized immune complexes. Biotinylated or radioiodinated C1q (*C1q) bound specifically to transfected K562 cells expressing cell surface CR1 and to immobilized recombinant soluble CR1 (rsCR1). *C1q binding to rsCR1 was completely inhibited by unlabeled C1q and the collagen domain of C1q and was partially inhibited by C3b dimers. Kinetic analysis in physiologic saline of the interaction of unlabeled C1q with immobilized rsCR1 using surface plasmon resonance yielded an apparent equilibrium dissociation constant (K[eq2]) of 3.9 nM. Thus, CR1 is a cellular C1q receptor that recognizes all three complement opsonins, namely, C1q, C3b, and C4b.

Binding Sites↗
PubMed1997

Lipid transfer from human epithelial cells to Pneumocystis carinii in vitro.

Pneumocystis carinii lipids are similar to host lipids, but it is not known if some of these lipids are acquired from host cells. The ability of P. carinii to incorporate a fluorescent fatty acid analogue (Bodipy-C12) was analyzed, the metabolism of the incorporated lipid by P. carinii was characterized, and lipid transfer from human alveolar epithelial cells (A549) to P. carinii was investigated. Both P. carinii and A549 cells incorporated exogenous Bodipy-C12 in a concentration-dependent manner. Biochemical analysis of labeled P. carinii revealed incorporation of Bodipy-C12 into complex lipid classes. Incubation of unlabeled P. carinii with Bodipy-C12-labeled A549 cells demonstrated lipid transfer to P. carinii, a process facilitated by attachment. These data suggest that P. carinii can incorporate and modify an exogenous fluorescent lipid. The observed transfer of lipid from A549 cells to P. carinii provides important insight into the interaction of this organism with alveolar epithelial cells.

Animals↗
PubMed1997

D-2-hydroxyglutaric aciduria: hypotonia, cortical blindness, seizures, cardiomyopathy, and cylindrical spirals in skeletal muscle.

An infant girl was demonstrated to have D-2-hydroxyglutaric aciduria, the fifth case described and the first with muscle biopsy of this rare organic aciduria that differs clinically and genetically from the more common L-2-hydroxyglutaric aciduria. Her clinical features included mildly dysmorphic facies, developmental delay, generalized hypotonia, myoclonic seizures, cortical blindness, and dilated cardiomyopathy requiring treatment. Muscle biopsy demonstrated only excessive glycogen histochemically, but ultrastructural examination revealed subsarcolemmal cylindrical spirals and normal mitochondria. Because of the metabolism of D-2-hydroxyglutaric aciduria, we regard valproic acid as contraindicated in the treatment of epilepsy in this disease.

Amino Acid Metabolism, Inborn Errors↗
PubMed1996

Plasma homocysteine and methionine concentrations in children with neural tube defects.

Mild to moderate homocysteinemia in women has been associated with an increased frequency of pregnancies with neural tube defects (NTD). Homocysteinemia is also an independent risk factor for premature vascular disease. In addition to folic acid, supplemental Vitamin B12, Vitamin B6 and betaine may normalize homocysteine metabolism, decrease the risk for NTD formation, and correct related metabolic imbalances in children with NTD. By means of automated amino acid analysis, we assessed total non-fasting homocysteine and methionine in plasma from 24 children with myelomeningocele. This study group (mean age 10.5 +/- 4.9 years) included 12 girls and 12 boys randomly selected from our Birth Defects Clinic. Homocysteine concentrations in our patients (4.7 +/- 1.8 mumol/L) did not differ from those of 20 randomly selected child controls (5.1 +/- 2.6 mumol/L). The mean homocysteine concentration for 36 adult controls (9.3 +/- 3.0 mumol/L) was significantly higher than the mean for either group of children (p < 0.0001). Linear regression analysis revealed negative correlation of total plasma homocysteine with serum folate (r = -0.53; p = 0.01), but not of homocysteine with either methionine or B12. Plasma methionine concentrations from our patients did not differ from adult reference values. Elevated homocysteine in some mothers of children with NTD has been attributed to defective methylation of homocysteine. These preliminary results do not indicate such a defect in the children themselves. A more comprehensive study of homocysteine, methionine and related metabolites in children with NTD and age-matched controls will be required to determine the clinical significance of these findings.

Adolescent↗
PubMed1995

Hemolytically inactive C5b67 complex: an agonist of polymorphonuclear leukocytes.

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

Chemotaxis, Leukocyte↗
PubMed1995

Purification of human lung leukotriene C4 synthase and preparation of a polyclonal antibody.

Leukotriene (LT) C4 synthase is an integral membrane protein that catalyzes the conjugation of LTA4 to reduced glutathione to form LTC4. LTC4 synthase has been cloned and characterized from transformed cell lines, but the protein has not been defined from a tissue source. LTC4 synthase was purified to homogeneity from human lung tissue, utilizing S-hexyl glutathione chromatography followed by LTC4 affinity chromatography. A greater than 100,000-fold purification with a yield of 8 to 25% (n = 4) was achieved. The purified LTC4 synthase migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as an 18-kD protein, and its 19 N-terminal amino acid sequence is identical to that of purified LTC4 synthase from KG-1 myeloid cells or from expression cloning of a KG-1 library in COS cells. Using a rabbit polyclonal IgG raised against purified LTC4 synthase, SDS-PAGE immunoblotting of LTC4 synthase from human lung tissue, eosinophils, KG-1 cells, and platelets showed an 18-kD protein. Immunofluorescence staining of alveolar macrophages in human lung sections with the anti-LTC4 synthase IgG revealed LTC4 synthase to be largely perinuclear in distribution. Thus, LTC4 synthase, the biosynthetic enzyme responsible for the formation of cysteinyl LTs, is present in lung tissue in a form apparently identical to that of hematopoietic cells.

Amino Acid Sequence↗
PubMed1994

Diamniotic placentation associated with omphalopagus conjoined twins: implications for a contemporary model of conjoined twinning.

We have studied omphalopagus conjoined twins with a diamniotic monochorionic placenta. Although conjoined twins usually present in a single amniotic sac, one other example of diamniotic placenta has been reported in omphalopagus twins [Weston et al., 1990: Am J Med Genet 37:558-561]. Most theories concerning the pathogenesis of conjoined twinning exclude the possibility of diamniotic placentation. However, Spencer [1992: Teratology 45:591-602] recently elaborated a model for conjoined twinning based on duplication of organizing centers (primitive streaks) during gastrulation. We have considered the fate of embryonic membranes according to this model of omphalopagus twinning and show that diamniotic placentation is a predictable outcome.

Abnormalities, Multiple↗
PubMed1994

Regulation of C1q receptor expression on human polymorphonuclear leukocytes.

The C1q receptor (C1qR) is expressed on a variety of cells, including polymorphonuclear leukocytes (PMN), in which stimulation by the C1qR leads to activation as measured by superoxide production. To investigate the expression and modulation of the C1qR on PMN, the binding of biotinylated C1q to PMN in suspension was measured by flow cytometry. Biotinylated C1q bound in a saturable and specific manner to PMN and the use of low ionic strength buffers enhanced binding. Covalent coupling of C1q to Sepharose beads allowed the affinity precipitation of a single 125-kDa band from surface iodinated PMN. The apparent molecular mass of the C1qR increased to 135 kDa upon reduction. Freshly isolated PMN had a uniform expression of C1qRs and phorbol myristate acetate induced a unimodal up-regulation of receptors. The inflammatory peptide FMLP rapidly up-regulated receptors by up to fivefold, and the high level of expression remained constant over 45 min. Taxol inhibited the FMLP induction of C1qR up-regulation, indicating that the ability to move the intracellular stores of C1qR depends on normal microtubule functioning. Thus, the C1qR is a constitutively expressed protein of the human PMN plasma membrane and it is rapidly up-regulated from a discrete intracellular pool of preformed molecules with the same kinetics as CR1 and CR3. It is likely that the C1qR is a component of the PMN complement receptor exocytic vesicle (CREV), in which CR1 and CR3 are also stored.

Carrier Proteins↗
PubMed1993

The role of complement component C3b and its receptors in sperm-oocyte interaction.

Previous studies have shown that human sperm that have undergone the acrosome reaction express a unique tissue-specific variant of the complement component 3 (C3)-binding molecule membrane cofactor protein (MCP, CD46) and that damaged or dead sperm activate the alternative pathway of complement and bind C3 catabolites. In this study we provide evidence that MCP on sperm that have undergone the acrosome reaction specifically binds dimeric C3b and that human sperm acrosomal proteases released during the acrosome reaction directly cleave C3, facilitating its binding to MCP. Furthermore, human and hamster oocytes can activate the alternative pathway of complement and also bind human C3 fragments. Monoclonal antibodies specific for complement receptors type 1 (CD35) and type 3 (CD11b/CD18) bind to the human oocyte plasma membrane, indicating that specific complement-binding molecules may play a role in the attachment of C3 catabolites to oocytes. Subsaturating concentrations of dimeric C3b (0.01-1 microM) promoted penetration of hamster oocytes by human sperm, whereas saturating doses (> 10 microM) inhibited this process. In addition, antibodies to both MCP and C3 significantly inhibited penetration of hamster oocytes by human sperm. These data provide evidence that regulated gamete-induced generation of C3 fragments and the binding of these fragments by selectively expressed receptors on sperm and oocytes may be an initial step in gamete interaction, leading to membrane fusion and fertilization.

Adult↗
PubMed1993

Lipid and membrane protein transfer from human neutrophils to schistosomes is mediated by ligand binding.

Attachment of human neutrophils to schistosomula of Schistosoma mansoni involves leukocyte receptors recognizing carbohydrate, complement and/or IgG ligands on the parasite surface. Here, we examined the transfer of a fluorescent fatty acid analog (BOFA) from human neutrophils to schistosomula coated with concanavalin A (Con A), immune serum or nonimmune serum under co-culture conditions by fluorescence confocal laser scanning microscopy (CLSM). Coating schistosomes with Con A or immune serum and co-culturing them for 24 hours with BOFA-labeled neutrophils resulted in a specific lipid transfer to the surface tegument of the schistosomes. Tegumental labeling was absent when nonimmune serum was used. No significant difference (P < 0.001) was found in the number of neutrophils bound to the worm surface between Con A-coated schistosomes (4.1 +/- 0.345 cells/worm) and worms incubated in immune serum (4.261 +/- 0.362). The number of neutrophils bound to the schistosomula (2.7 +/- 0.223) was significantly reduced in the presence of nonimmune serum (P < 0.0001). The viability of the schistosomula was 98% in nonimmune treated co-cultures, and 91% in cocultures treated with immune serum. HPLC analysis of labeled neutrophils demonstrated that BOFA was incorporated into both phospholipids and neutral lipids, which were almost exclusively triglycerides and, after 18 hours of culture, all of the fatty acid analog was incorporated into complex lipids. Double-label experiments in which schistosomula bearing Con A were first incubated with BOFA-labeled neutrophils and subsequently immunolabeled revealed that the neutrophil membrane proteins, MHC class I, CR1 and CR3 were co-transferred with neutrophil lipids to the parasite tegument.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Purification of human leukotriene C4 synthase.

Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation of LTA4 with reduced glutathione to form LTC4, was purified to homogeneity from the KG-1 myeloid cell line after solubilization of the microsomes utilizing a combination of 0.4% sodium deoxycholate and 0.4% Triton X-102. The solubilized enzyme was then applied to an S-hexyl-glutathione-agarose column that was eluted by the use of 7.5 mM probenecid. After removal of the probenecid by sequential concentration and dilution in an Amicon concentrator, the enzyme was additionally purified and concentrated by binding to and elution from approximately 75 mg of S-hexyl-glutathione-agarose. The enzyme was further resolved by electrophoresis with a nondenaturing Tris-glycine gel, and the LTC4 synthase activity was localized to slices 3 and 4. When the remainder of the eluate from the nondenaturing gel was precipitated by acetone and analyzed by 14% SDS/PAGE with silver staining, a single protein band of 18 kDa was associated with LTC4 synthase activity and was not present in the eluates of slices lacking activity. The overall recovery was 12.5%. In a separate preliminary purification, in which the yield was only approximately 1%, the eluates of the nondenaturing gel had also revealed a single protein of 18 kDa by SDS/PAGE, which was present only in the eluates with LTC4 synthase activity. These data identify LTC4 synthase as a protein of 18 kDa, a size consistent with its membership in the microsomal glutathione S-transferase family.

Chromatography, Affinity↗
PubMed1992

Regulation of MHC class I synthesis and expression by human neutrophils.

Human peripheral blood neutrophils (PMN) treated with granulocyte-macrophage CSF (GM-CSF) increase the amount of class I 42-kDa H chain and 12-kDa L chain, beta 2-microglobulin (beta 2m), that they synthesize by 2.1- and 2.6-fold, respectively. To determine whether the increase in translation was associated with an increase in levels of class I H chain transcript, RNA blot analysis was performed on PMN that had been cultured in the presence of GM-CSF. Under no conditions were there increased levels of class I H chain transcript when class I heterodimer protein synthesis was increased. In addition, there was neither an increase in the synthesis of H chain mRNA, as measured by transcription assay, nor an alteration in the degradation rates of class I H chain transcript in PMN cultured with GM-CSF. In situ hybridization demonstrated that both the percentage of PMN that expressed class I transcript and the relative amounts of transcript per cell in GM-CSF-cultured PMN were the same as those in control PMN. Although there is increased translation of class I heterodimer in PMN treated with GM-CSF, there is no increase in its expression on the plasma membrane. The maintenance of constant levels of class I on the plasma membrane is dependent on continued protein synthesis and is maintained by release of class I heterodimer and free beta 2m into the medium. Heterodimer is released in the context of plasma membrane-derived vesicles, whereas beta 2m is released as a soluble protein. Maintenance of constant levels of class I heterodimer on the plasma membrane is also regulated by constitutive internalization. Up to 30% of class I molecules bearing 125I-Fab-labeled mAb to class I are internalized over 2 h at 37 degrees C. Therefore, inducible synthesis of class I by PMN is likely a consequence of post-transcriptional regulation, whereas the continued synthesis of class I heterodimer is required for maintenance of its expression. Furthermore, there is no increase in class I expression, in spite of increased synthesis, due to the release of class I heterodimer and beta 2m and the internalization of class I heterodimer from the plasma membrane. Thus, PMN are capable of post-transcriptional regulation of protein synthesis and are able to modulate the expression of plasma membrane proteins by regulated expression, release, and internalization.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1992

Peripheral blood neutrophils in chronically neutropenic patients respond to granulocyte-macrophage colony-stimulating factor with a specific increase in CR1 expression and CR1 transcription.

Chronically neutropenic patients from a phase I/II protocol were studied for neutrophil (PMN) abnormalities related to therapeutic use of granulocyte-macrophage colony-stimulating factor (GM-CSF). We analyzed phenotype by flow cytometry to measure indirect immunofluorescent staining and activation of transcription by in situ hybridization. PMN count increased in seven of 17 patients. For the group, PMN expression of complement receptors, CR1 and CR3, increased after GM-CSF administration (P less than .005), while expression of class 1 and FcR III was stable. PMN from both of the patients studied by in situ hybridization demonstrated increased expression of CR1 transcript, which in one case coincided in time and intensity with the course of increased CR1 expression, while in the second case the presence of CR1 mRNA increased but lagged behind the increased CR1 protein expression. Thus, PMN activation was observed after GM-CSF infusion, as indicated by increased complement receptor expression. This effect was due both to translocation of receptors from a preformed intracellular pool to the cell surface, and to transcriptional regulation leading to increased receptor synthesis.

Antigens, Surface↗
PubMed1990

Granulocyte-macrophage colony-stimulating factor increases synthesis and expression of CR1 and CR3 by human peripheral blood neutrophils.

Polymorphonuclear leukocytes (PMN) constitutively synthesize various plasma membrane proteins including CR1(3) (CD35), CR3 (or Mac-1) alpha-chain (CD11b) and MHC class I. PMN are also able to up-regulate rapidly the expression of CR1 and CR3 to the plasma membrane in response to agonists such as FMLP. To determine whether constitutive PMN translation was static or up-regulatable, PMN were cultured in the presence or absence of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) for 8 h. CR1, CR3 and class I proteins immunoprecipitated from lysates of 35S-methionine pulse-labeled PMN were resolved by SDS-PAGE, fluorographed and quantified by densitometry. GM-CSF-treated PMN synthesized 4.5-fold more class I protein, 3.7-fold more CR1, 2.4-fold more CD11b and 3.4-fold more CR3 beta-chain (CD18), compared with untreated control cells. Actinomycin D treatment of replicate samples of PMN decreased the amount of these proteins synthesized by each group of PMN from 30 to 90%, implying that continued translation was required for the increases in protein synthesis. Nascent CR and class I proteins were inserted into the plasma membrane of PMN, thereby supplementing the molecules already expressed on the cell surface. In addition to these longer term effects of GM-CSF, we observed its acute up-regulatory effects on PMN. GM-CSF induced a five- to 12-fold increase in the expression of CR1 and CR3 on the PMN cell surface within 30 min. These increases were both dose- and time-dependent with maximum up-regulation occurring at 25 pM and at 30 min. In contrast to the long term biosynthetic events, this rapid up-regulation was not dependent on protein synthesis but was due instead to mobilization of CR from intracellular compartments similar to those up-regulated by FMLP. These results demonstrate that PMN can respond to microenvironmental stimuli such as GM-CSF both by rapidly up-regulating and increasing translation and expression of functionally important plasma membrane proteins.

CD18 Antigens↗
PubMed1988

Ligand-loaded but not free complement receptors for C3b/C4b and C3d co-cap with cross-linked B cell surface IgM and IgD.

We have performed experiments to investigate possible physical interactions between C receptors (CR) and surface Ig (sIg) on the B cell plasma membrane. These molecules were found to be independent, non-linked, B cell surface structures, because capping CR1, CR2, sIgM, or sIgD with a specific antibody did not affect the distribution of the remainder of these molecules. Both CR1 and CR2, if bound by antibodies that did not independently cap CR, however, became associated with cross-linked sIg because CR that have been bound by intact anti-CR antibodies or their Fab fragments co-capped with sIgM or sIgD that had been bound by divalent anti-IgM or anti-IgD antibody. CR1 that had bound C3b similarly co-capped with sIg when sIg was cross-linked. Ligand-bound or even cross-linked CR did not associate with non-cross-linked sIg because sIgD, bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with CR that had been cross-linked by a sandwich of mouse anti-CR antibody and goat anti-mouse Ig. Other surface molecules, such as B1 and HLA-DR Ag, when bound by specific antibodies, did not cap with cross-linked sIg, and sIgD, when bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with cross-linked sIgM. Interactions between CR and sIg were not mediated by an association with IgG FcR because co-capping of CR and sIg was observed when F(ab')2 fragments of both anti-CR and anti-Ig antibodies were used. These results demonstrate that B cell surface CR can become associated with sIg, but only if sIg is cross-linked and CR is bound by anti-CR antibody or has bound its natural ligand.

Animals↗