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Biomedical subjects

F A Garver

Publications and source records attributed to F A Garver.

At least 37 records · Page 2Linked to original sources
PubMed1985

Partial characterization of an erythropoiesis inhibitory factor.

An inhibitory factor of erythropoiesis, obtained from normal human urine, is indicated to be a complex of a fragment of alpha 1-acid glycoprotein and prostaglandin F2 alpha. Immunoelectrophoresis reveals two protein components in the EIF complex which separate during acrylamide gel electrophoresis. A gamma-globulin (MW 185,000) is a carrier of the complex. A fragment of alpha 1-acid glycoprotein (MW 9300) retains the inhibitory factor, PGF2 alpha. Noncovalent forces bind the PGF2 alpha to the protein, and PGF2 alpha can be extracted with benzene.

Blood Proteins↗
PubMed1985

Identification and quantification of Hb C with an enzyme-linked immunosorbent assay.

A highly specific enzyme-linked immunosorbent assay (ELISA) was developed for the rapid identification and quantification of hemoglobin C in hemolysates. The procedure involves coating the surface of microtiter wells with Hb C and then addition of monospecific rabbit antibodies that recognize the unique beta 6 GLU----LYS substitution in Hb C. Next, an antibody to rabbit gamma-globulin conjugated with alkaline phosphatase is added, followed by substrate; a yellow color is formed due to the enzymatic hydrolysis of the substrate, which can be measured spectrophotometrically. For quantification purposes, a hemolysate containing Hb C is introduced just prior to the addition of the Hb C antibody. This results in blocking the attachment of the anti-Hb C to the Hb C coated to the plastic surface. Upon addition of anti-rabbit gamma-globulin conjugate and substrate, there is a consequent reduction or elimination of color formation. Since the degree of diminution of color formation is dose-dependent, standard curves can be developed for quantification of Hb C in unknowns. Of the total hemoglobin, the amounts of Hb C in heterozygotes averaged 27.3 +/- 5.7% by ELISA and 25.1 +/- 3.9% by radioimmunoassay (RIA). In SC individuals the corresponding values were 30.2 +/- 10.1% by ELISA and 24.7 +/- 10.9% by RIA. In homozygotes, Hb C values averaged 83.2 +/- 4.2% by ELISA and 85.0 +/- 6.6% by RIA. Subjects with Hb C beta(+)-thalassemia had 66.5 +/- 3.7% Hb C as measured by ELISA and 63.5 +/- 9.1% as determined by RIA. The ELISA procedure offers distinct advantages for Hb C identification and quantification over other techniques in parameters such as specificity, sensitivity, and rapidity.

Enzyme-Linked Immunosorbent Assay↗
PubMed1984

Immunochemical identification of normal and variant hemoglobins after electrophoretic separation and transfer to nitrocellulose membranes.

A highly specific method for the conclusive identification of normal and variant human hemoglobins is described in this communication. The method employs the standard electrophoretic technique in combination with the immunoblot technique using monospecific antisera raised in rabbits. Various hemoglobins such as Hb, S, C, A2, and F were separated on an alkaline polyacrylamide gel or a cellulose-acetate membrane, and transferred to a nitrocellulose membrane and the individual hemoglobins were identified by the immunoblot procedure. With this method hemoglobins with similar or identical electrophoretic mobilities can be definitively identified with the use of monospecific antisera.

Electrophoresis, Cellulose Acetate↗
PubMed1984

Increased HbF in sickle cell anemia is determined by a factor linked to the beta S gene from one parent.

Members of 7 large families, containing 20 patients with sickle cell anemia (SS) characterized by high levels of fetal hemoglobin (HbF), were studied using immunofluorescence to count F cells and a radioimmunoassay to measure small amounts of HbF. In five of these families, one of the sickle cell trait (AS) parents had a much higher HbF and F-cell count than the other; in one family, both parents had a marked increase in HbF and F cells; in the remaining family, HbF and F cells were at borderline values in both parents. Seven of 14 AS siblings, but only 1 of 8 normal hemoglobin (AA) siblings, also had HbF and F-cell counts above the "normal" range. It seems that a factor for increased F cells, linked to the beta S gene of one parent, is segregating in these families and is responsible for the greatly increased HbF and F cells in the SS subjects. HbF per F cell in AS parents and siblings was the same as that of normal AA subjects, whereas in the SS offspring it was greatly increased, suggesting that it was the result of marrow hyperplasia associated with their hemolytic anemia. The similarity of this "increased F-cell gene" to heterocellular hereditary persistence of fetal hemoglobin (HPFH). Swiss type, is discussed, and it is suggested that it may control the persistent synthesis of HbF in sickle cell anemia by its presence in early infancy.

Adolescent↗
PubMed1984

Identification and quantification of hemoglobins A2 and Barts with an enzyme-labeled immunosorbent assay.

The presence and quantity of hemoglobins Barts and A2 in hemolysates from normal donors and individuals with alpha- and beta-thalassemia trait, respectively, were determined with an enzyme-labeled immunosorbent assay (ELISA). This technique requires the incorporation of monospecific antisera capable of specifically reacting only with these hemoglobins, e.g., with the delta chain of Hb A2 and gamma 4 chains of Hb Barts. By the ELISA, the mean percentage of Hb Barts in hemolysates from normal persons and persons with alpha-thalassemia was 0.25 (SD 0.07) and 6.1 (SD 0.40), respectively. Corresponding values for Hb A2 in hemolysates from normals and persons with beta-thalassemia were 3.1 (SD 0.22) and 5.9 (SD 0.21), respectively. The results obtained by the ELISA procedure were in good agreement with those determined by radioimmunoassay or microcolumn chromatography. The ELISA technique is more sensitive and specific than biochemical assays currently used to measure these hemoglobins and can detect 250 ng of Hb Barts in 100 micrograms of hemoglobin or 50 ng of Hb A2 in 5 micrograms of hemoglobin.

Adult↗
PubMed1983

Chronic lymphatic leukemia evolving into chronic myelocytic leukemia.

A diagnosis of chronic lymphatic leukemia (CLL) was made in an 83-year-old man on the basis of marked lymphocytosis (131.1 X 10(9)/L) and infiltration of the marrow (77%) by small lymphoid cells. The hemoglobin was 11.8 g/dL and the platelet count was 427.5 X 10(9)/L. With minimal and unsustained treatment, abrupt resolution of the lymphocytosis occurred with reciprocal emergence of a classic picture of chronic myelogenous leukemia (CML). During the hematologic evolution, blood lymphoid cells were isolated for surface marker, cytogenetic, and functional studies. E-rosette receptors, Fc-receptors, and membrane immunoglobulins were found on only 7.5%, 7.4%, and 10.0% of these cells. These cells also failed to express a CLL-associated antigen we have detected on approximately equal to 95% of the cells of all CLL patients studied, regardless of cell phenotype. In addition, the patient's lymphoid cells responded poorly to leucoagglutinin (LPHA) stimulation, showed increased spontaneous metabolic activity and exhibited the Philadelphia chromosome. These observations suggest that the patient's transient initial lymphocytosis was not due to CLL, but perhaps represented myeloid precursors in circulation prior to terminal differentiation.

Aged↗
PubMed1983

Isolation of amniotic fluid erythrocytes and their possible use in prenatal diagnosis.

Many of the components of amniotic fluid have been found to be valuable in prenatal diagnosis; however, the presence of erythrocytes is usually considered undesirable. The authors have used buoyant density centrifugation on 96 amniotic fluid specimens from 70 subjects to isolate small numbers of erythrocytes from a majority of these specimens. Through immunofluorescence, these specimens were found to have higher levels of fetal hemoglobin-containing cells than the adult, indicating that the erythrocytes were at least in part fetal in origin. Thus, erythrocytes present in amniotic fluid could also be used in prenatal diagnosis.

Amniocentesis↗
PubMed1983

The modeled structure of the IgG Gar VL region and its implications for anti-flavin and anti-DNP fine specificities.

The variable domain (V) sequence of the lambda-light chain of the Gar IgG2 human myeloma protein was determined from enzymatically generated peptides that were isolated, characterized, and ordered by overlap and/or by subgroup homology. The sequence consisted of the amino terminal 107 residues of the light chain, and shared subgroup V lambda III-specific residues as well as a pattern of CDR deletions unique to most V lambda III sequences. The structural role of the light V region in the high affinity for flavins and the low affinity for 2,4-dinitrophenyl haptens characteristic of the intact IgG molecule was coarsely assessed by way of model building and by comparison with the refined combining site model of MOPC 315, an Ig with relative affinity values for DNP and riboflavin the converse of those observed in Gar.

Amino Acid Sequence↗
PubMed1981

Effects of dexamethasone on fetal hemoglobin synthesis in peripheral blood erythroid burst-forming units.

Colonies derived from erythroid burst-forming units (BFU-E) synthesize fetal hemoglobin (HbF) in amounts that far exceed in vivo levels. There is some evidence that HbF synthesis is controlled at the level of a primitive erythroid precursor cell. Dexamethasone may potentiate the development of BFU-E. Since a means of augmenting HbF production in sickle cell anemia or severe beta-thalassemia would be of great therapeutic value, we studied the effects of dexamethasone on HbF and gamma-globin chain synthesis in BFU-E from patients with sickle cell anemia and controls. HbF was measured by radioimmunoassay of BFU-E lysate and gamma-chain synthesis by the incorporation of 3H-leucine into globin, which was then purified by gel filtration and column chromatography. Dexamethasone (10(-9) M) produced an increase in the number of BFU-E in 16 of 19 subjects when compared with numbers of BFU-E cultured with only erythropoietin. The individual BFU-E were larger and contained more subcolonies. Dexamethasone did not increase HbF or gamma-chain synthesis, and there was no relationship between proliferation of BFU-E and augmented HbF production. Thus, although dexamethasone augmented the development of erythroid bursts, there was no increment in HbF.

Anemia, Sickle Cell↗
PubMed1981

Localization of the carbohydrate units in a human immunoglobulin light chain, protein Sm lambda.

The carbohydrate structure and complete amino acid sequence of a human lambda-type immunoglobulin light chain, protein Sm lambda has been determined. The protein was isolated from the urine of a patient with a plasma cell dyscrasia resembling gamma-heavy-chain disease. 13 tryptic peptides covering the entire polypeptide chain of 135 residues were isolated from the aminoethylated protein, and 15 chymotryptic peptides, accounting for 131 residues, were recovered from the carboxymethylated protein. The sequence of 18 of these peptides was partially or completely determined by the Edman-dansyl technique or C-terminal analysis, permitting the establishment of the complete primary structure of the polypeptide chain. The sequences established that this light chain possessed an intramolecular deletion of 81 amino acid residues. The N-terminal 30 residues showed considerable homology with other lambda chains of subgroup II. The defect began at position 31, in the first hypervariable region, and encompassed the remainder of the variable region through position 109. The constant region was fully intact and normal synthesis recommenced with a glutaminyl residue at position 110, the first residue of the constant region. This light chain contained carbohydrate in the hypervariable region just preceding the deletion. The precise number and locations of the oligosaccharide chains were established by amino acid sequence analysis of glycopeptides isolated from proteolytic hydrolysates by chromatography on Bio-Gel P-6 columns. These studies showed that protein Sm lambda contains one N-glycosidically-linked chain attached to asparagine-25 and one O-glycosidically-linked oligosaccharide chain attached to serine-21. The structures of the oligosaccharide chains were determined by methylation analysis, gas chromatography and hydrolysis with specific glycosidases. The structure of the N-glycosidically-linked chain was NeuAc(alpha 2 leads to 6)Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 2)Man(alpha 1 leads to 6)[NeuAc(alpha 2 leads to 6)Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 2)Man(alpha 1 leads to 3)]Man(beta 1 leads to 4)GlcNAc(beta 1 leads to 4)[Fuc alpha 1 leads to 6]GlcNAc leads to Asn. The second O-glycosidically-linked chain was a disialylated tetrasaccharide with the structure, Neu(alpha 2 leads to 3)Gal(beta 1 leads to 3)[NeuAc(alpha 2 leads to 6)GalNAc leads to Ser. This mucin-type disialylated tetrasaccharide in close proximity to N-asparagine-linked chains has not been previously observed in the oligosaccharide chains of immunoglobulins.

Amino Acid Sequence↗
PubMed1981

Inhibition of Friend virus (FVP)-induced murine erythropoiesis with prostaglandin (PGF2 alpha): potentiation and inhibition of erythropoietin and prevention of both with PGD2.

An erythropoietin-independent virus-induced murine erythroleukemia (FVP) is used to compare the effects of an erythropoiesis inhibitory factor (EIF) isolated from human urine with the effects of prostaglandin F2 alpha. The consequent inhibition of FVP-induced erythropoiesis suggests that EIF and PGF2 alpha have similar effects on the FVP-induced erythropoiesis in mice, and the effect of PGF2 alpha is indirect. The similarity of the actions of EIF and PGF2 alpha may indicate a potential role for prostaglandins in the physiological control of some types of erythrocytosis.

Animals↗
PubMed1980

Immunochemical properties of abnormal hemoglobins C-Harlem (beta 6 Glu replaced by Val, beta 73 Asp replaced by Asn), S (beta 6 Glu replaced by Val), Korle Bu (beta 73 Asp replaced by Asn), Vancouver (beta 73 Asp replaced by Tyr), and Mobile (beta 73 Asp replaced by Val).

Antibodies were made to three mutant hemoglobins, each containing different single amino acid substitutions at beta 73:Hb Korle Bu (Asp replaced by Asn), Hb Mobile (Asp leads to Val), Hb Vancouver (Asp replaced by Tyr); and to one mutant hemoglobin, Hb C-Harlem, containing two substitutions in the beta chain (beta 6 Glu replaced by Val, as in Hb S, and beta 73 Asp replaced by Asn, as in Hb Korle Bu). The antiserum to Hb C-Harlem contained two antibody populations, each specific for one mutant amino acid residue. The antiserum to Hb Vancouver was completely specific for this mutant and did not cross-react with Hb Mobile and Hb Korle Bu; however, antiserum to Hb Korle Bu partially cross-reacted with Hb Mobile and to a smaller degree with Hb Vancouver. Antiserum to Hb Mobile exhibited even less cross-reactivity with Hb Korle Bu and C-Harlem and none with Hb Vancouver. These and previous studies indicate the involvement of at least three independent areas in the beta chain as antigenic determinant sites. It appears that the three mutants at beta 73 elicit the formation of antibodies which have a gradation in their specificity due to the nature of the amino acid sidechain.

Amino Acids↗
PubMed1980

Studies on the Wolfgram high molecular weight CNS myelin proteins: relationship to 2',3'-cyclic nucleotide 3'-phosphodiesterase.

Evidence is presented that the major protein components of the high molecular weight CNS myelin proteins designated as the Wolfgram protein doublet (W1 and W2) contain the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP). CNP is a basic hydrophobic protein containing about 830 to 840 amino acid residues. When electrophoresed on SDS polyacrylamide gels, CNP appears as a protein doublet, separated by a molecular weight difference of about 2500-3000 in bovine, human, rat, guinea pig, and rabbit. A similar protein doublet has been identified as the Wolfgram proteins W2 and W1 in myelin and in the chloroform-methanol-insoluble pellet obtained from myelin. Moreover, the relative Coomassie blue staining intensity of the CNP2 plus CNP1 protein doublet among the species examined was remarkably similar to that observed for electrophoresed myelin and chloroform-methanol-insoluble pellet derived from myelin. Antisera raised against purified bovine CNP recognized the W1 and W2 proteins isolated from bovine and human brain. The amino acid composition of pure bovine CNP is presented and compared with the compositions of several rat and bovine Wolfgram proteins obtained by other investigators. Our electrophoretic, compositional, and immunological data support the contention that the enzyme CNP is a major component of the Wolfgram protein doublet.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1980

The V region sequence of lambda Bence-Jones protein Wh: evidence for separate germ-line sets within lambda-subgroups.

The complete variable region sequence of the glycosylated Bence-Jones protein Wh has revealed that the most hypervariable segments of the first and third complementarity-determing regions (CDR) are identical with those of another glycosylated Bence-Jones protein (Nei) of the same lambda-subgroup. The carbohydrate is attached in both proteins at the identical sequences in the third CDR, and the sequences of the first CDR differ at only one position in fourteen. Because CDR sequence comparisons more sensitively reflect overall germ-line V gene similarities, proteins Wh and Nei could possibly reflect a "set" relationship analogous to the mouse V kappa isotype. Cluster analyses of currently available V lambda sequences supported this suggested relationship.

Amino Acid Sequence↗