Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function

The glycoprotein hormone erythropoietin (Ep), the primary regulator of erythropoiesis, is synthesized by the kidney and secreted as the mature protein with three N-linked and one O-linked oligosaccharide chains. To investigate the role(s) of each carbohydrate moiety in the biosynthesis and function of Ep, we have used oligonucleotide-directed mutagenesis of a cDNA for human Ep to alter the amino acids at each of the carbohydrate attachment sites. Each mutated cDNA construct was expressed in stably transfected sublines of a kidney cell line, baby hamster kidney. We show, by preventing attachment of N-linked carbohydrate at asparagines 38 or 83, or preventing O-linked glycosylation at serine 126, that glycosylation of each of these specific sites is critical for proper biosynthesis and secretion of Ep. Fractionation of cellular extracts demonstrated that the mutant proteins lacking glycosylation at each of these three sites, (38, 83, and 126) were associated mainly with membrane components or were degraded rapidly. Less than 10% of these three mutant proteins were processed properly and secreted from the cells. The Ep protein lacking N-linked glycosylation at asparagine 24 is synthesized and secreted as efficiently as native Ep. The carbohydrates at positions 24 and 38 may be involved in the biological activity of Ep, since the absence of either of the oligosaccharide side chains at these positions reduced the hormone's biological activity.     

Examination of the secondary structure of the kringle 4 domain of human plasminogen

The structure of a small region of human plasminogen (F4), consisting of amino acid residues Val354-Ala439 and containing its kringle 4 (K4) domain (residues Cys357-Cys434), has been predicted from Chou-Fasman calculations and hydropathy profiles, and compared to circular dichroism (CD) measurements on the isolated fragment. Calculations, by the Chou-Fasman method, of the probabilities of various types of secondary structures that exist in this region reveal that no helical structures are present. Of the total of 86 amino acid residues present in this K4-containing peptide region, 37% can adopt conformations of beta-pleated sheets, 48% of the amino acids can exist in beta-turns, and 15% of the residues can be present as coils. The structure of F4 in dilute aqueous solution has been experimentally evaluated by CD measurements. At pH = 7.4, in dilute salt solutions, a total of 64% beta-structures, 30% beta-turns, and 6% coiled structures is estimated to be present in this peptide region. Consideration of the marginal stability of many of the conformational regions of F4, as predicted by Chou-Fasman calculations, suggests that secondary structural flexibility is present in this fragment, which could result in ready adoption of new conformations. The hydropathy profile of F4 has been determined and suggests that this polypeptide is highly hydrophilic, especially in the regions of residues His387-Tyr396 and Cys406-Lys413. Thus, it appears as though a large portion of the surface of F4 can be exposed to solvent in its native conformation.     

The effect of sulfide and an increased food supply on the meiofauna and macrofauna at the East Flower Garden brine seep

A sulfurous brine seep at the East Flower Garden Bank, northwest Gulf of Mexico, produces conditions conducive to the growth of a luxuriant prokaryotic biota. Hydrodynamic cropping continually harvests this biota and distributes it to sandy-bottom and hard-bank benthic communities downstream of the seep. Consequently, both macro- and meiofaunal abundances are dramatically increased above the regional norm in parts of the seep system. When sulfide is present, the lower Bilaterian groups belonging to the meiofauna dominate the community; without sulfide, macrofaunal groups, particularly crustaceans, dominate the community. Outside the influence of the seep, meiofaunal copepods predominate. Changes in taxonomic composition and abundance indicate that the sandy-bottom benthos at 70–80 m depth at the East Flower Garden bank is foodlimited and that, under these conditions, meiofauna, particularly the higher Bilaterian groups, dominate the community numerically. Perhaps, under food-limiting conditions, meiofauna compete favorably with macrofauna for food.     

Polymorphic phase behavior of cardiolipin derivatives studied by 31P NMR and X-ray diffraction

The polymorphic phase behavior of cardiolipin (diphosphatidylglycerol) analogues with two to five chains per phospholipid head group, namely, dilysocardiolipin, monolysocardiolipin, cardiolipin, and acylcardiolipin, respectively, has been studied by 31P NMR and X-ray diffraction. Dilysocardiolipin dispersions at low salt concentration are micellar, and a transition to a lamellar phase takes place between 1 and 2 M NaCl. From light-scattering measurements, it is also found that a transition takes place from the micellar state with a midpoint at 5.2 mM CaCl2, 0.95 M HClO4, and 1.5 M NaCl. Monolysocardiolipin dispersions are lamellar throughout the concentration range from zero to saturated NaCl. Cardiolipin dispersions undergo a transition from a lamellar to an inverted hexagonal phase between 1 and 2 M NaCl. Acylcardiolipin dispersions are in an inverted hexagonal phase throughout the concentration range from zero to saturated NaCl. The chemical shift anisotropies of both phosphate groups in dilysocardiolipin and of one of the phosphate groups in monolysocardiolipin are drastically reduced in the lamellar phase, indicating a different conformation of the phosphatidyl head group from that normally found in diacyl phospholipid bilayers. The results provide strong support for the "shape" concept of lipid polymorphism when viewed in its most general form including configurational entropy, hydrophobic effects, etc. and indicate the importance of head-group interactions in determining the lipid phase behavior.     

Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.     

A plasmodial alpha-tubulin cDNA from Physarum polycephalum. Nucleotide sequence and comparative analysis

As the first step towards correlating structure and function of tubulin in the slime mold Physarum polycephalum we have elucidated the nucleotide sequence of a cDNA that appears to code for all but the last 25 to 30 C-terminal amino acids of a plasmodial alpha-tubulin. Differences in amino acid sequence from those of other alpha-tubulins are distributed fairly evenly throughout the sequence, although a relatively extensive conserved region is found in position 396 to 426 near the C terminus. A small region in position 298 to 307 contains a cluster of amino acid residues unique to Physarum alpha-tubulin. The sequence is 70% homologous to two yeast alpha-tubulins and about 83% homologous to five animal alpha-tubulins. A comparison of the homologies of all the known alpha-tubulins indicates that a large decrease in the accepted point mutation rate has occurred during the evolution of the metazoa, suggesting a major functional specialization of microtubules.     

Replication of cytomegalovirus in human arterial smooth muscle cells.

Cytomegalovirus (CMV) strain AD-169 replicated in smooth muscle cell (SMC) cultures derived from human umbilical arteries, producing enveloped infectious virions. However, unlike the effects of CMV on fully permissive human lung fibroblasts, the effects of strain AD-169 on SMC cultures were delayed and prolonged, resulting in extended survival of a fraction of the starting population. This period of survival did not exceed the life-span of the control SMC cultures. Infectious CMV continued to be isolated from the surviving SMC cultures after extinction of the original inoculum by dilution and after treatment of the cultures with CMV neutralizing antibody. The implications of these findings for the pathogenesis of atherosclerosis are discussed.     

Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.