Olga O. Blumenfeld

Studies of an unusual hemoglobin in patients with diabetes mellitus

The properties of an unusual hemoglobin found in patients with diabetes mellitus resembled those of hemoglobin A1c prepared from normal subjects. A two-fold increase of hemoglobin A1c was found in diabetic patients. Structural studies suggest the possibility that an amino sugar is bound to hemoglobin A1c in diabetic patients.

Collagen chain mRNAs in isolated heart cells from young and adult rats

Collagen is the predominant component of the extracellular matrix of the heart, where it is organized in a hierarchy of structures. To establish the cellular origin of the various collagen types, type I-procollagen alpha 2 chain and types III and IV collagen mRNAs were examined in preparations of myocytes and non-myocyte heart cells freshly isolated from rats 1 to 6 months old. The cardiomyocytes appeared morphologically intact and functionally competent. Fibroblast-like cells predominated in the non-myocyte cell fractions but endothelial and smooth muscle cells were also present. RNA from whole ventricular tissue served as a control. Northern and dot blot analyses were used to establish the presence or absence of mRNAs. In RNA prepared from whole ventricular tissue, the mRNAs for alpha-, beta-, and gamma-actin isotypes were detected whereas mRNA for alpha-actin was found in myocytes and those for beta- and gamma-actins were found in non-myocyte cells, confirming further the nature of the cell populations. Procollagen types I and III mRNAs were not detected in the total RNA of cardiomyocytes but mRNA for type IV collagen was present. The mRNAs for all three collagen types were present in the non-myocyte cells. These results suggest that in the rat heart the non-myocyte cells, probably fibroblasts, are responsible for interstitial collagen production. Both cell populations may engage in the formation of basement membrane collagen type IV.

DETERMINATION OF CARBONYL COMPOUNDS WITH N-METHYL BENZOTHIAZOLONE HYDRAZONE.

Abstract N -Methyl benzothiazolone hydrazone reacts with various carbonyl compounds. The azine and “osazine” derivatives formed under defined conditions have characteristic spectra which permit the identification of saturated and unsaturated aldehydes and ketones, keto acids, and many other related compounds in a simple spectrophotometric assay. The azines of aldehydes can be further reacted with the oxidized form of MBTH to give rise to tetraazopentamethine cyanine dyes used in a colorimetric procedure. The sensitivity of both methods is such that microgram amounts of a carbonyl compound can be identified and measured rapidly and accurately. Since carbohydrates having pyranose structures are unreactive in the colorimetric and spectrophotometric methods, many aldehydes and ketones can be measured in their presence.

Localization of types I, III and IV collagen mRNAs in rat heart cells by in situ hybridization

Previous studies investigating the cellular origins of several collagens in young adult rat hearts (Eghbali et al., 1988) demonstrated that the mRNAs for types I and III collagen occurred in non-myocyte cells, mostly fibroblasts, whereas the mRNA for type IV collagen was observed in both myocytes and non-myocyte cells. In the present study, cellular localization of collagen mRNAs has been achieved by in situ hybridization in rat heart tissue and in isolated heart cells. Frozen tissue sections, isolated cardiomyocytes, cultured neonatal cardiomyocytes and fibroblasts were hybridized with DNA probes for type-specific collagens, actin, and myosin heavy chain. Silver grains were visualized by dark field imaging. In heart sections, types I and III mRNAs were observed predominantly adjacent to myocytes and in the interstitium, where fibroblasts are known to be present. In contrast, type IV collagen mRNA was identified both within the myocytes and the interstitium. In freshly isolated adult cardiomyocytes and in cultured neonatal cardiomyocytes, collagen type IV mRNA was observed but type I collagen mRNA was not. In cultured neonatal fibroblasts, both types IV and I collagen mRNAs were abundant.

Morphology, composition, and function of struts between cardiac myocytes of rat and hamster

SummaryThe morphology, composition, and function of struts that interconnect the lateral surfaces of cardiomyocytes were examined in the hearts of rats and hamsters. Methods included brightfield and fluorescent light microscopy, secondary and backscatter scanning electron microscopy, and transmission electron microscopy in conjunction with silver stain, cationic dye, and antibody to type-I collagen. These studies reveal a twisted, beaded appearance and a complex substructure of collagen fibrils embedded in a ground substance that has a positive reaction with cationic dye. A hierarchy of patterns of branching and attachment was seen among intercellular struts ranging in diameter from 0.1 μm to several urn. The hypothesis that struts tether not only the surfaces but the contractile lattices of laterally adjacent myocytes is supported by the following: (a) the attachments of struts to the collagen weave of the sarcolemma, often lateral to the level of Z bands, (b) the presence of collagen type I in a composite material arrangement, (c) the relative dispositions and configurational changes of struts and myocyte surfaces in various physiological states and induced, non-physiological perturbations of cardiac muscle, (d) the corrugated sarcolemmas with infoldings near Z bands, and (e) the continuity of intracellular filaments from Z bands to the inner aspect of the sarcolemma in relaxed and contracted myocytes. Implications of struts acting as tethers and sites for storage of energy in the motions of myocytes during the cardiac cycle are discussed.

Modification and introduction of a specific radioactive label into the erythrocyte membrane sialoglycoproteins

Abstract A specific radioactive label was introduced into the sialoglycoproteins of the erythrocyte membrane by sequential sodium periodate oxidation and tritiated sodium borohydride reduction. This was achieved whether the sialoglycoproteins were isolated, present in situ within the intact erythrocyte, or the isolated erythrocyte membranes. The label is found in the oligosaccharide chains of the sialoglycoproteins predominantly in residues which were formerly those of bound sialic acid. The method appears selective for the sialoglycoproteins and certain, as yet unidentified lipid components.

2 Biochemistry and molecular biology of MNSs blood group antigens

: This chapter has reviewed the nature of antigens of the MNSs blood group system. The structures of the proteins and the molecular features and organization of glycophorin genes were described, emphasizing their domain arrangement and the extensive sequence homology that indicates that their common and variant alleles belong to a single gene family. Methods currently used to examine these antigens are immunoblotting and DNA typing. The majority of variant genes are hybrids of parent glycophorin genes in a variety of arrangements; they contain no other sequences but those of the parent genes. The structures of the hybrids are summarized in Figure 8. Several hybrids appear to have arisen by unequal homologous recombination but others appear to have occurred through gene conversion. In this system the molecular genetic basis for a single variant phenotype may differ, as documented by gene rearrangements that appear to have occurred, as separate events, at different sites in the same intron; this has resulted in protein structures (hence phenotypes) that are identical. For example, unequal homologous recombination occurring within intron 3 can have given rise to only a limited number of phenotypes, namely alpha M-delta S, alpha N-delta S, alpha M-delta S, alpha N-delta S and delta-alpha. In addition, different sites of an exon may have been involved in gene rearrangements through gene conversion leading to nearly identical protein structures, yet different serological phenotypes. Thus, gene conversion could be more significant for generation of antigenic diversification as the number of possible new alleles is quite large. The participation of the HGpE gene in these rearrangements would make this number even larger. New sites and the expressed pseudoexon have created the epitopes of the variant phenotypes, and sequences specific for several variant antisera have been identified. Thus, the molecular basis for several serological reactions involving this system is now better understood.

Isolation of a glycoprotein--glycolipid fraction from human erythrocyte membranes.

Summary An ethanol fractionation procedure was developed for the isolation of glycoproteins from aqueous pyridine solubilized human erythrocyte membranes. The preparation resembles that obtained by extraction with phenol in its amino acid and carbohydrate composition and the presence of antigens. In addition to the glycoproteins the preparation contains glycolipid components.

MNSs Blood Groups and Major Glycophorins

Glycophorins A and B (GPA and GPB) of the erythrocyte membrane are unique compared with membrane glycoproteins of all other cells in that they carry the antigens of a blood group system, the MNSs blood group system, and therefore are readily identifiable in all human populations. The protein and carbohydrate structures of the glycophorins and their disposition in the membrane were among the first to be studied and be firmly established (Huang et al., 1991a). This was made possible because they are the major sialoglycoproteins of the mature erythrocyte, a relatively simple cell lacking all internal organelles; thus, the surface membrane is relatively easy to obtain and yields pure glycophorins using extraction protocols designed for carbohydrate-rich components. Over the years the interest in these molecules was both as the major antigens of the MNSs blood group system and as classical models of integral membrane glycoproteins. More recently, the structure and organization of genes encoding GPA, GPB, and the third member, GPE, were established, and some understanding obtained of factors involved in regulation of their expression in the erythroid cells (Cartron et al., 1990; Cartron and Rahuel, 1992). The nature of glycophorins as blood group antigens and the knowledge that serological variants of the common antigens occur among various populations (Race and Sanger, 1975) prompted investigations of the nature of polymorphism in this family of membrane glycoproteins. These studies revealed a common pattern of molecular mechanisms for protein diversification, and the glycophorin gene family can now serve as a prototype for human gene rearrangements.

Properties of heart fibroblasts of adult rats in culture

SummaryIn the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cell used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate. Direct observation of type IV collagen is consistent with the previous finding of type IV mRNA in cardiac fibroblasts in situ and in freshly isolated populations of these cells.