Polymorphism in a ferritin H gene from chromosome 6p

Summary This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.     

Experimental documentation of natural hybridization inCactaceae: Origin of Lloyd's Hedgehog Cactus,Echinocereus ×lloydii

The origin ofEchinocereus ×lloydii Britt. & Rose, pro sp. (Lloyd's Hedgehog Cactus) was investigated using comparative morphology, cytology, biochemistry, and particularly, artificial hybridization. Numerous artificial crosses between the putative parentsE. coccineus Engelm. (a species of claret-up cactus) andE. dasyacanthus Engelm. (Texas Rainbow Cactus) were successful, resulting in the production of hundreds of seeds with hybrid embryos. The F₁ hybrid progeny (i.e., syntheticE. ×lloydii) grew to sexual maturity in about four and one-half years, whereupon successful backcrosses and F₂ generation hybrids were also obtained. The known F₁ hybrids closely approximated naturalE. ×lloydii. The fertility of these syntheticE. ×lloydii was high, like their natural counterparts. The populations ofE. ×lloydii in Pecos County, Texas are inferred to have originated as the result of natural interspecific hybridization. It is assumed thatE. ×lloydii or similar plants may arise wherever the parental taxa grow sympatrically.     

Rates and Patterns of Scndna and Mtdna Divergence within the Drosophila Melanogaster Subgroup

Levels of DNA divergence among the eight species of the Drosophila melanogaster subgroup and D. takahashii have been determined using the technique of DNA-DNA hybridization. Two types of DNA were used: single-copy nuclear DNA (scnDNA) and mitochondrial DNA (mtDNA). The major findings are: (1) A phylogeny has been derived for the group based on scnDNA which is congruent with chromosomal data, morphology, and behavior. The three homosequential species, simulans, sechellia, and mauritiana, are very closely related; the scnDNA divergence indicate the two island species are a monophyletic group. (2) The rates of change of scnDNA and mtDNA are not greatly different; if anything scnDNA evolves faster than mtDNA. (3) The rates of scnDNA evolution are not closely correlated to chromosomal (inversion) evolution. (4) The Drosophila genome appears to consist of two distinct classes of scnDNA with respect to rate of evolutionary change, a very rapidly evolving fraction and a relatively conservative fraction. (5) The absolute rate of change was estimated to be at least 1.7% nucleotide substitution per one million years. (6) DNA distance estimates based on restriction site variation are correlated with distances based on DNA-DNA hybridization, although the correlation is not very strong.     

Synthesis and secretion of mucous glycoprotein by the gill of Mytilus edulis. I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.     

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.     

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.