The periodate oxidation of amino acids with reference to studies on glycoproteins

1. All α-amino acids are oxidized by periodate, but at different rates. 2. The rates of oxidation of individual α-amino acids vary with pH. In general, oxidation proceeds more rapidly at alkaline pH. 3. Serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine and histidine are rapidly and extensively oxidized by periodate. 4. Cysteine, cystine, methionine, tryptophan, tyrosine and histidine are oxidized by periodate when they are substituted in the carboxyl and amino groups, as in a polypeptide chain.     

Studies on a glycopeptide from ovalbumin

1. The structure of the carbohydrate component of the glycopeptide isolated from the proteolytic digest of ovalbumin has been investigated by chemical and enzymic methods. 2. The results are consistent with the presence of a single carbohydrate prosthetic group, linked through its reducing end group to the peptide chain. 3. Further, all the 2-amino-2-deoxy-d-glucose units appear to be in the N-acylated form, the phenolic hydroxyl group of tyrosine is free and the ω-carboxyl group of aspartic acid is substituted. 4. The carbohydrate component has a branched-chain structure, the two non-reducing ends being terminated by a d-mannopyranosyl and a 2-acetamido-2-deoxy-d-glucopyranosyl residue respectively. 5. The terminal d-mannopyranosyl unit is probably linked through at least one other d-mannopyranosyl residue to the remainder of the carbohydrate.     

Circular pancreatic trypsin inhibitor. A novel substrate for studies on intracellular proteolysis

Several recent studies indicate that substrates for ubiquitin-dependent proteolysis must possess unblocked alpha-amino termini. To examine further the importance of free amino groups for proteolytic susceptibility we selected pancreatic trypsin inhibitor (PTI) as a test substrate. PTI can be circularized to form cPTI, a molecule that lacks alpha-amino groups in the absence of an endoproteolytic cleavage. We compared the breakdown of radioiodinated PTI and cPTI in rabbit reticulocyte lysate and found that cPTI was not stabilized relative to PTI. In addition, proteolysis of PTI or cPTI was not inhibited upon conversion of their lysine residues to homoarginine. However, neither degradation of PTI nor cPTI required ATP, and ubiquitin conjugation to either molecule was minor relative to known substrates of the ubiquitin pathway. Thus, PTI and cPTI are cleaved by an ATP-independent endoprotease(s) that does not require the substrate to be ubiquitinated. Such an activity was identified in low salt fractions obtained upon DEAE chromatography of reticulocyte lysate. The ubiquitin/ATP-dependent protease and another large multisubunit protease, both of which elute from DEAE-Fractogel at higher salt concentrations, do not degrade PTI or cPTI. Although monomeric PTI was rapidly degraded in reticulocyte lysate, cross-linked PTI molecules were stable both in reticulocyte lysate and following introduction into cultured cells using red blood cell-mediated microinjection. Thus, increased rates of turnover do not necessarily correlate with greater molecular mass of the substrate.     

Deamination of mammalian glutamate receptor RNA by Xenopus dsRNA adenosine deaminase: similarities to in vivo RNA editing.

Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD) converts adenosines to inosines within dsRNA. A great deal of evidence suggests that dsRAD or a related enzyme edits mammalian glutamate receptor mRNA in vivo. Here we map the deamination sites that occur in a truncated glutamate receptor-B (gluR-B) mRNA after incubation with pure Xenopus dsRAD. We find remarkable similarities, as well as distinct differences, between the observed deamination sites and the sites reported to be edited within RNAs isolated from mammalian brain. For example, although deamination at the biologically relevant Q/R editing site occurs, it occurs much less frequently than editing at this site in vivo. We hypothesize that the similarities between the deamination and editing patterns exist because the deamination specificity that is intrinsic to dsRAD is involved in selecting editing sites in vivo. We propose that the observed differences are due to the absence of accessory factors that play indirect roles in vivo, such as binding to and occluding certain sites from dsRAD, or promoting the RNA structure required for correct and efficient editing. The work reported here also suggests that dsRAD is capable of much more selectivity than previously thought; a minimal number of deamination sites (average < or = 5) were found in each gluR-B RNA. We speculate that the observed selectivity is due to the various structural elements (mismatches, bulges, loops) that periodically interrupt the base paired region required for editing.     

Fine-resolution mapping of spontaneous and double-strand break-induced gene conversion tracts in Saccharomyces cerevisiae reveals reversible mitotic conversion polarity.

Spontaneous and double-strand break (DSB)-induced gene conversion was examined in alleles of the Saccharomyces cerevisiae ura3 gene containing nine phenotypically silent markers and an HO nuclease recognition site. Conversions of these alleles, carried on ARS1/CEN4 plasmids, involved interactions with heteroalleles on chromosome V and were stimulated by DSBs created at HO sites. Crossovers that integrate plasmids into chromosomes were not detected since the resultant dicentric chromosomes would be lethal. Converted alleles in shuttle plasmids were easily transferred to Escherichia coli and analyzed for marker conversion, facilitating the characterization of more than 400 independent products from five crosses. This analysis revealed several new features of gene conversions. The average length of DSB-induced conversion tracts was 200 to 300 bp, although about 20% were very short (less than 53 bp). About 20% of spontaneous tracts also were also less than 53 bp, but spontaneous tracts were on average about 40% longer than DSB-induced tracts. Most tracts were continuous, but 3% had discontinuous conversion patterns, indicating that extensive heteroduplex DNA is formed during at least this fraction of events. Mismatches in heteroduplex DNA were repaired in both directions, and repair tracts as short as 44 bp were observed. Surprisingly, most DSB-induced gene conversion tracts were unidirectional and exhibited a reversible polarity that depended on the locations of DSBs and frameshift mutations in recipient and donor alleles.     

Synthesis,characterization, and electrocatalytic behaviour of hydrothermally grown nanostructured La2O3 and La2O3/K-complex

Rare earth metals play a conspicuous role in magnetic resonance imaging (MRI) for detecting cancerous cells. The alkali metal potassium is a neurotransmitter in the sodium–potassium pump in biomedical sciences. This unique property of rare earth metals and potassium drew our attention to carry forward this study. Therefore, in this work, previously synthesized potassium (K) complexes formed by the reflux of 4-N,N-dimethylaminobenzoic acid (DBA) and potassium hydroxide in methanol, and named [(μ2–4-N,N-dimethylaminobenzoate-κO)(μ2–4-N,N-dimethylaminobenzoic acid-κO)(4-N,N-dimethylaminobenzoic acid-κO) potassium(I) coordination polymer)] were treated hydrothermally with La₂O₃ nanomaterials to obtain a nanohybrid La₂O₃/K-complex. After that, the K-complex was analyzed using single-crystal X-ray diffraction and ¹H and ¹³C NMR spectroscopy. In addition, the structural and morphological properties of the as-prepared nanostructured La₂O₃/K-complex were also characterized, which involved an investigation using X-ray diffraction (XRD)spectroscopy, Fourier transform infrared (FTIR) spectroscopy, atomic force spectroscopy (AFM), transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) analysis. After this, the electrochemical redox behaviour of the synthesized nanohybrid material was studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Therefore, the results from these studies revealed that the as-prepared material was a La₂O₃/K-complex that has a promising future role in sensing various analytes, as it showed effective electrocatalytic behaviour.     

Mismatch Repair by Efficient Nick-Directed, and Less Efficient Mismatch-Specific, Mechanisms in Homologous Recombination Intermediates in Chinese Hamster Ovary Cells

Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in ~10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A -> G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was >80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks.     

α-d-allopyranosyl β-d-fructofuranoside (allo-sucrose) and its derivatives

Reduction of 3-ketosucrose (1) with sodium borohydride gave mainly α-d-allopyranosyl β-d-fructofuranoside (2) characterized as its octabenzoate. Using sodium borodeuteride, [3-²H]allo-sucrose (5) and [3-²H]sucrose (6) were obtained in the ratio 12:1. The mixture was fractionated on Dowex-50 X8 resin (Ca²⁺ form), and the [3-²H] derivatives were isolated as their octa-acetates. Inspection of the ¹³C-n.m.r. spectra of 5 and 6 enabled the C-3 signals to be assigned. allo-Sucrose (2) was more readily obtained by oxidation of sucrose with dimethyl sulphoxide-acetic anhydride followed by reduction with sodium borohydride and fractionation on Dowex-50 X8 (Ca²⁺) resin. Tritylation of 2 followed by acetylation gave, after chromatography, the 6,1′,6′-tritrityl ether (9, 10%), the 6,6′-ditrityl ether (10, 26%), and a mixture of monotrityl ethers (20%). Hydrogenolysis of 9 and 10 gave the penta-acetate and hexa-acetate, respectively, with no detectable migration of AcO-4. Treatment of 2 with sulphuryl chloride at -50° gave the 6,6′-dichloride.     

The synthesis of 2,1′-anhydro-,2,1′:3,6-dianhydro-, and 2,1′:3,6: 3′,6′-trianhydro-sucrose

1′-O-Mesyl-6,6′-di-O-tritylsucrose and the corresponding 1′-O-tosyl derivative were prepared from 6,6′-di-O-tritylsucrose by selective sulphonylation. Both sulphonates underwent intramolecular cyclisation reactions, to give 2,1′-anhydrosucrose in high yields rather than the isomeric 1′,4′-anhydride. Sequential benzoylation, detritylation, and mesylation of the 2,1′-anhydride afforded 2,1′-anhydro-6,6′-di-O-mesylsucrose tetrabenzoate which, in the presence of base, gave 2,1′:3,6:3′,6′-trianhydrosucrose that was not identical with the product previously claimed to have this structure. Several derivatives of 2,1′-anhydrosucrose were prepared possessing different functional groups at either the 6,6′- or 4,6′-positions. Dimolar mesitylene-sulphonylation of 3,3′,4′6′-tetra-O-acetylsucrose gave the 6,1′-disulphonate, which, in the presence of alkali, gave 2,1′:3,6-dianhydrosucrose, which was transformed into the 2,1′:3,6:3′,6′-trianhydride by sequential bromination at C-6′ (carbon tetrabromide-triphenylphosphine) and base-catalysed cyclisation. Treatment of 3,3′,4′,6′-tetra-O-benzoylsucrose with sulphuryl chloride furnished the 4,6,1′-trichloro derivative, which, on alkaline hydrolysis, was converted into 2,1′:3,6-dianhydro-4-chloro-4-deoxy-galacto-sucrose.