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.     

Molecular dynamics simulations and coupled nucleotide substitution experiments indicate the nature of A·A base pairing and a putative structure of the coralyne-induced homo-adenine duplex

Coralyne is an alkaloid drug that binds homo-adenine DNA (and RNA) oligonucleotides more tightly than it does Watson–Crick DNA. Hud’s laboratory has shown that poly(dA) in the presence of coralyne forms an anti-parallel duplex, however attempts to determine the structure by NMR spectroscopy and X-ray crystallography have been unsuccessful. Assuming adenine–adenine hydrogen bonding between the two poly(dA) strands, we constructed 40 hypothetical homo-(dA) anti-parallel duplexes and docked coralyne into the six most favorable duplex structures. The two most stable structures had trans glycosidic bonds, but distinct pairing geometries, i.e. either Watson–Crick Hoogsteen (transWH) or Watson–Crick Watson–Crick (transWW) with stability of transWH > transWW. To narrow down the possibilities, 7-deaza adenine base substitutions (dA→7) were engineered into homo-(dA) sequences. These substitutions significantly reduced the thermal stability of the coralyne-induced homo-(dA) structure. These experiments strongly suggest the involvement of N7 in the coralyne-induced A·A base pairs. Moreover, due to the differential effect on melting as a function of the location of the dA→7 mutations, these results are consistent with the N1–N7 base pairing of the transWH pairs. Together, the simulation and base substitution experiments predict that the coralyne-induced homo-(dA) duplex structure adopts the transWH geometry.     

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.     

The effect of sodium, potassium and ammonium ions on the conformation of the dimeric quadruplex formed by the Oxytricha nova telomere repeat oligonucleotide d(G(4)T(4)G(4)).

The DNA sequence d(G(4)T(4)G(4)) [Oxy-1.5] consists of 1.5 units of the repeat in telomeres of Oxytricha nova and has been shown by NMR and X-ray crystallographic analysis to form a dimeric quadruplex structure with four guanine-quartets. However, the structure reported in the X-ray study has a fundamentally different conformation and folding topology compared to the solution structure. In order to elucidate the possible role of different counterions in this discrepancy and to investigate the conformational effects and dynamics of ion binding to G-quadruplex DNA, we compare results from further experiments using a variety of counterions, namely K(+), Na(+)and NH(4)(+). A detailed structure determination of Oxy-1.5 in solution in the presence of K(+)shows the same folding topology as previously reported with the same molecule in the presence of Na(+). Both conformations are symmetric dimeric quadruplexes with T(4)loops which span the diagonal of the end quartets. The stack of quartets shows only small differences in the presence of K(+)versus Na(+)counterions, but the T(4)loops adopt notably distinguishable conformations. Dynamic NMR analysis of the spectra of Oxy-1.5 in mixed Na(+)/K(+)solution reveals that there are at least three K(+)binding sites. Additional experiments in the presence of NH(4)(+)reveal the same topology and loop conformation as in the K(+)form and allow the direct localization of three central ions in the stack of quartets and further show that there are no specific NH(4)(+)binding sites in the T(4)loop. The location of bound NH(4)(+)with respect to the expected coordination sites for Na(+)binding provides a rationale for the difference observed for the structure of the T(4)loop in the Na(+)form, with respect to that observed for the K(+)and NH(4)(+)forms.     

A novel insight into the regulation of light-independent chlorophyll biosynthesis in <Emphasis Type="Italic">Larix decidua</Emphasis> and <Emphasis Type="Italic">Picea abies</Emphasis> seedlings

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment–protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.     

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.     

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.     

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.