Carbohydrate composition of purified serum glycoproteins in Mucolipidosis II and Mucolipidosis III

Summary Mucolipidosis II (I-cell disease) and Mucolipidosis III (ML III) are inherited disorders in which the molecular defect may involve an abnormality in a common post-translational modification step (possibly glycosylation) shared by lysosomal hydrolases. We tested whether such an alteration might be a generalized defect in glycoprotein biosynthesis and, thus, be reflected in an abnormal carbohydrate composition of non-lysosomal glycoproteins. The apoprotein of low density lipoprotein (apo-LDL) and immunoglobulin G (IgG) were purified to apparent homogeneity. Gas liquid chromatographic (glc) analysis of the carbohydrate content of these glycoproteins from ML II, ML III and normal sera revealed no differences in the relative ratios and total amounts of mannose, galactose, N-acetylglucosamine and sialic acid. These results suggest that if the postulated post-translational defect in these disorders involves changes in carbohydrate composition, it is not a general defect in glycosylation and may be specific for lysosomal hydrolases.     

Carbohydrate composition of purified human liver α-L-Fucosidase

Summary Human liver -L-fucosidase was purified to apparent homogeneity and analyzed for carbohydrate content primarily by gas-liquid chromatography (glc). The enzyme is about 7% carbohydrate by weight and contains the following sugars (residues per 50,000 molecular weight subunit): mannose (8.3), glucosamine (4.3) (presumably N-acetylated), sialic acid (1.6) and glucose (1.6). Galactose (0.8) and L-fucose (1.8) were also found but their presence may be due to artifacts of the purification procedure.     

Leaf and tepal anatomy, plastid ultrastructure and chlorophyll content in Galanthus nivalis L. and Leucojum aestivum L.

The leaf structure of Galanthus nivalis L. (snowdrop) and Leucojum aestivum L. (snowflake) is characterized by a homogeneous mesophyll tissue. The dominant characters of the leaves are cavities with mucose substance. There is a striking difference between these plants tepal anatomy. A central cavity occurs only in snowdrop tepals. Plastids from white parts of the tepals have a poorly developed membrane system. Leaves and green parts of tepals of both species possess amoeboid chloroplasts and contain chlorophyll a and b. The chlorophyll content in tepals is lower than in leaves, but the chlorophyll a:b ratio is always 2:1. Both, snowdrop and snowflake are from the family Amaryllidaceae, but their ecology is different. This paper presents common features related to systematic relatedness and differences induced by ecological factors.     

The Prebiotic Provenance of Semi-Aqueous Solvents

Origins of Life and Evolution of Biospheres - The numerous and varied roles of phosphorylated organic molecules in biochemistry suggest they may have been important to the origin of life. The...     

Addressing the problems of base pairing and strand cyclization in template-directed synthesis: a case for the utility and necessity of 'molecular midwives' and reversible backbone linkages for the origin of proto-RNA

Nucleic acid synthesis is precisely controlled in living organisms by highly evolved protein enzymes. The remarkable fidelity of information transfer realized between template and product strands is the result of both the spatial selectivity of the polymerase active site for Watson-Crick base pairs at the point of nucleotide coupling and subsequent proof-reading mechanisms. In the absence of naturally derived polymerases, in vitro template-directed synthesis by means of chemically activated mononucleotides has proven remarkably inefficient and error-prone. Nevertheless, the spontaneous emergence of RNA polymers and their protein-free replication is frequently taken as a prerequisite for the hypothetical 'RNA world'. We present two specific difficulties that face the de novo synthesis of RNA-like polymers in a prebiotic (enzyme-free) environment: nucleoside base selection and intramolecular strand cyclization. These two problems are inherent to the assumption that RNA formed de novo from pre-existing, chemically-activated mononucleotides in solution. As a possible resolution to these problems, we present arguments and experimental support for our hypothesis that small molecules (referred to as 'molecular midwives') and alternative backbone linkages (under equilibrium control) facilitated the emergence of the first RNA-like polymers of life.     

A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses

Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAV(UK)deltaS2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAV(UK)deltaS2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAV(PV). This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAV(PV) by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID(50)], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID(50). In contrast, na?ve equids subjected to the low- or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure.     

Equine infectious anemia virus envelope evolution in vivo during persistent infection progressively increases resistance to in vitro serum antibody neutralization as a dominant phenotype

Equine infectious anemia virus (EIAV) infection of horses is characterized by well-defined waves of viremia associated with the sequential evolution of distinct viral populations displaying extensive envelope gp90 variation; however, a correlation of in vivo envelope evolution with in vitro serum neutralization phenotype remains undefined. Therefore, the goal of the present study was to utilize a previously defined panel of natural variant EIAV envelope isolates from sequential febrile episodes to characterize the effects of envelope variation during persistent infection on viral neutralization phenotypes and to define the determinants of EIAV envelope neutralization specificity. To assess the neutralization phenotypes of the sequential EIAV envelope variants, we determined the sensitivity of five variant envelopes to neutralization by a longitudinal panel of immune serum from the source infected pony. The results indicated that the evolution of the EIAV envelope sequences observed during sequential febrile episodes produced an increasingly neutralization-resistant phenotype. To further define the envelope determinants of EIAV neutralization specificity, we examined the neutralization properties of a panel of chimeric envelope constructs derived from reciprocal envelope domain exchanges between selected neutralization-sensitive and neutralization-resistant envelope variants. These results indicated that the EIAV gp90 V3 and V4 domains individually conferred serum neutralization resistance while other envelope segments in addition to V3 and V4 were evidently required for conferring total serum neutralization sensitivity. These data clearly demonstrate for the first time the influence of sequential gp90 variation during persistent infection in increasing envelope neutralization resistance, identify the gp90 V3 and V4 domains as the principal determinants of antibody neutralization resistance, and indicate distinct complex cooperative envelope domain interactions in defining sensitivity to serum antibody neutralization.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteine residues is accompanied by the formation of a tight turn of the protein backbone. In nearly 90% of the structures analyzed a type VIII turn was found. The peptide bond between the two cysteines is in a distorted trans conformation, the omega torsion angle ranges from 159 to -133 degrees, with an average value of 171 degrees. The constrained nature of the vicinal disulfide turn and the pronounced difference observed between the oxidized and reduced states, suggests that vicinal disulfides may be employed as a 'redox-activated' conformational switch.     

Intercalation-mediated synthesis and replication: a new approach to the origin of life

We propose that a molecular midwife, a flat molecule approximately 10 Ax10 A with two hydrophobic faces, was essential to the origin of life. This molecule was positively charged, water soluble and did not strongly associate with itself in solution. It may have been a derivative of phthalocyanine that no longer exists on the Earth today, and might have been formed solely from hydrogen cyanide and formaldehyde. The midwife tended to intercalate between side groups (bases, similar to those in RNA) of polymers to form stacks, which incorporated bare bases. The midwife alternated in these stacks with hydrogen-bonded tetrads of bases. Under conditions of low water activity, as in a desert during the day, bare bases in the stacks were joined together by neutral and chemically heterogeneous backbones of no fixed chirality. The components of the backbones were the products of the formose reaction of formaldehyde, and were involved in the reversible formation of N -glycosides and acetals catalysed by divalent metal ions. The final product of this assemblage was a fully intercalated quadruplex of four information-containing polymer strands (four proto -RNA molecules). This process constituted replication of the original polymer that had seeded the formation of the stack. The stack structure ensured that the polymer's base sequence was replicated faithfully despite the lack of both homochirality and chemical homogeneity in the backbone. At night, water from condensing dew would suddenly come in contact with these products, quenching all chemical reactions and releasing midwife molecules and single- or double-stranded proto-RNA. Evaporation of water during the day then gave new stacks containing one or two proto-RNA strands, bare bases, and midwife molecules, which could begin a new replication cycle. Our model also allows for the generation of new stacks and the extension of existing ones, without restricting the base sequence of either, thereby providing a source of genetic information. The proto-RNA replication cycle is driven purely by concentration changes caused by the Sun and the rotation of the Earth. We propose that this system as a whole could have gradually evolved into the RNA World.     

Universal sequence replication, reversible polymerization and early functional biopolymers: a model for the initiation of prebiotic sequence evolution

Many models for the origin of life have focused on understanding how evolution can drive the refinement of a preexisting enzyme, such as the evolution of efficient replicase activity. Here we present a model for what was, arguably, an even earlier stage of chemical evolution, when polymer sequence diversity was generated and sustained before, and during, the onset of functional selection. The model includes regular environmental cycles (e.g. hydration-dehydration cycles) that drive polymers between times of replication and functional activity, which coincide with times of different monomer and polymer diffusivity. Template-directed replication of informational polymers, which takes place during the dehydration stage of each cycle, is considered to be sequence-independent. New sequences are generated by spontaneous polymer formation, and all sequences compete for a finite monomer resource that is recycled via reversible polymerization. Kinetic Monte Carlo simulations demonstrate that this proposed prebiotic scenario provides a robust mechanism for the exploration of sequence space. Introduction of a polymer sequence with monomer synthetase activity illustrates that functional sequences can become established in a preexisting pool of otherwise non-functional sequences. Functional selection does not dominate system dynamics and sequence diversity remains high, permitting the emergence and spread of more than one functional sequence. It is also observed that polymers spontaneously form clusters in simulations where polymers diffuse more slowly than monomers, a feature that is reminiscent of a previous proposal that the earliest stages of life could have been defined by the collective evolution of a system-wide cooperation of polymer aggregates. Overall, the results presented demonstrate the merits of considering plausible prebiotic polymer chemistries and environments that would have allowed for the rapid turnover of monomer resources and for regularly varying monomer/polymer diffusivities.