Atypical incorporation of single amino acids into myelin proteins.

—Purified myelin incorporated l -[¹⁴C]leucine and l -[¹⁴C]lysine into myelin proteins in an enzymatic process similar to that of renal brush border membranes. The system was not inhibited by cycloheximide or puromycin or by pretreatment with ribonuclease; the reaction was inhibited by cetophenicol. ATP was an effector, shifting the optimal pH from 7.2 to 8.3. In the presence of ATP, myelin was less dense in a sucrose gradient. Ammonia was released from the membrane during the incorporation of amino acids. Myelin preloaded with cold leucine did not incorporate [¹⁴C]leucine but did incorporate [¹⁴C]lysine; there was no cross inhibition between the two amino acids. The incorporation was into or onto proteins of the Wolfgram proteolipid fraction of myelin. The incorporation was of the high affinity type with a K_m of 10^?7m and was restricted to the natural amino acids.     

Lipid-protein interaction. The incorporation of myelin proteolipid apoprotein into phosphatidylcholine bilayers

Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty-acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc (the onset temperature for gel-to-fluid transition) is also detected by infrared spectroscopy. Studies using steady-state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide, perturbing the phospholipids close to it, but allowing rapid (greater than 10(4) s-1) exchange with all the lipid molecules in the system.     

The in vivo incorporation of linolenic acid into neuronal and glial cells and myelin

Abstract— Neurons, astrocytes, oligodendrocytes, and myelin were prepared from 21-day-old rat brain at various times after intracerebral injection of [1-¹⁴C]linolenate. Comparisons of phospholipid specific radioactivity demonstrated that the oligodendrocytes were much more active than neuronal, astroglial, or myelin fractions. This is consistent with the concept that the oligodendrocyte is responsible for synthesis of the relatively large mass of myelin sheath. Initially the phosphatidylcholine fraction was more active than the phosphatidylethanolamine fraction, but during the 36 h after injection the former decreased in radioactivity while the latter fraction showed an increase. Fatty acid elongation occurred rapidly. Within 2h after injection, 2/3 of the label had been converted to elongated products (20:4. 20:5, 22:5 and 22:6). All three cell types apparently contained the enzymes necessary to incorporate, elongate, and desaturate linolenic acid and this occurred at similar rates in each cell type. No direct precursor-product relationship was found between the lipids of oligodendrocytes and myelin. There was, however, a lag in the appearance of elongated fatty acids in the phosphoglycerides of myelin. indicating that the polyunsaturated fatty acids in myelin were synthesized elsewhere and transported into the myelin sheath.     

The effects of inhibitors of protein synthesis on incorporation of lipids into myelin

Abstract— Following intracranial injections of puromycin, the incorporation of [³H]leucine into brain protein was inhibited by 80 per cent. Conversely, incorporation of [³⁵S]sulphate into sulphatide or [2-³H]glycerol into phosphatidyl choline was not inhibited. Under these conditions, appearance of labelled protein in myelin was inhibited by 90 per cent, while the appearance of newly labelled sulphatide and phosphatidyl choline in myelin membrane was not greatly affected. Experiments with cycloheximide gave similar results with phosphatidyl choline, but incorporation of [³⁵S]sulphate into total sulphatide was decreased by about 30 per cent in animals given cycloheximide. Neither puromycin nor cycloheximide had any inhibitory effect on galactocerebroside sulphotransferase.     

Synthesis of functionalized biphenyl-C-nucleosides and their incorporation into oligodeoxynucleotides

We describe the synthesis of eight novel C-nucleosides in which the nucleobases are replaced by biphenyl residues that carry one or two electron donor (-OCH3,-NH2) or acceptor (-NO2) functional groups in the distal ring. These C-nucleosides were synthesized convergently and in high yields from a common bromophenyl-C-nucleoside precursor via Suzuki coupling with the respective boronic acids or esters. These nucleosides were subsequently converted into the corresponding phosphoramidite building blocks and efficiently incorporated into oligodeoxynucleotides by standard phosphoramidite chemistry.     

Synthesis and incorporation of [6,7]-selenatryptophan into dihydrofolate reductase

Until recently, the only selenium containing amino acid which could be used to completely substitute for a wild type amino acid was selenomethionine (SeMet). In the last decade the preparation of SeMet containing proteins has proved to be valuable tools in the determination of three-dimensional structure by multiwavelength anomalous diffraction (MAD) techniques. The potential utility of a selenium containing tryptophan analog, beta-seleno[3,2-b]pyrrolyl-L-alanine ([4,5]selenatryptophan), has recently been demonstrated in the literature. This finding shows promise for the bioincorporation of its positional isomer, beta-selenolo[2,3-b]pyrrolyl-L-alanine ([6,7]selenatryptophan), thereby adding to the essential arsenal of selenium-containing amino acids for use in the characterization of proteins. The synthesis of [6,7]selenatryptophan by enzymatic biotransformation with tryptophan synthase from selenolo[2,3-b]pyrrole was carried out as well as its characterization by NMR spectroscopy and thin layer chromatography. Selenatryptophyl dihydrofolate reductase ([6,7]SeTrp-DHFR) was then synthesized in vivo, purified, and found to exhibit no perturbations to enzymatic activity.     

Synthesis of alanyl nucleobase amino acids and their incorporation into proteins

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.     

In vivo incorporation of different amino acids into electrophoretically characteristic polypeptides synthesized by HeLa cell mitochondria

The $\text{in vivo}$ incorporation of each of the twenty common amino acids into electrophoretically characteristic polypeptides synthesized by HeLa cell mitochondria has been investigated. Under labeling conditions which allow translation only on mitochondrial ribosomes, incorporation of all the amino acids, except aspartic acid, cysteine, glutamic acid and glycine, has been detected. These exceptions are probably due to problems related to amino acid pool size and/or equilibration.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

Synthesis of an amphiphilic tetraantennary mannosyl conjugate and incorporation into liposome carriers

We have synthesized a novel conjugate (Man(4)K(3)DOG) composed of a tetramannosyl head group connected, via a polyethylene glycol spacer, to a lipid moiety. This amphiphilic molecule was easily incorporated into the bilayers of liposomes. As expected from the clustering effect, such multivalent mannose residues when exposed on the surface of the vesicles showed much higher binding affinity for Concanavalin A than their monomannosyl analogue. Mannosylated liposomes prepared with the tetravalent antenna could be promising carriers for e.g., loading dendritic cells with antigens for vaccination purposes.     

Synthesis of 2'-modified nucleotides and their incorporation into hammerhead ribozymes.

Several 2'-modified ribonucleoside phosphoramidites have been prepared for structure-activity studies of the hammerhead ribozyme. The aim of these studies was to design and synthesize catalytically active and nuclease-resistant ribozymes. Synthetic schemes for stereoselective synthesis of the R isomer of 2'-deoxy-2'-C-allyl uridine and cytidine phosphoramidites, based on the Keck allylation procedure, were developed. Protection of the 2'-amino group in 2'-deoxy-2'-aminouridine was optimized and a method for the convenient preparation of 5'-O-dimethoxytrityl-2'-deoxy-2'-phthalimidouridine 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was developed. During the attempted preparation of the 2'-O-t-butyldimethylsilyl-3'-O-phosphoramidite of arabinouridine a reversed regioselectivity in the silylation reaction, compared with the published procedure, was observed, as well as the unexpected formation of the 2,2'-anhydronucleoside. A possible mechanism for this cyclization is proposed. The synthesis of 2'-deoxy-2'-methylene and 2'-deoxy-2'-difluoromethylene uridine phosphoramidites is described. Based on a '5-ribose' model for essential 2'-hydroxyls in the hammerhead ribozyme these 2'-modified monomers were incorporated at positions U4 and/or U7 of the catalytic core. A number of these ribozymes had almost wild-type catalytic activity and improved stability in human serum, compared with an all-RNA molecule.     

Molecular mechanisms of acrolein-mediated myelin destruction in CNS trauma and disease

Abstract

Myelin is a critical component of the nervous system facilitating efficient propagation of electrical signals and thus communication between the central and peripheral nervous systems and the organ systems that they innervate throughout the body. In instances of neurotrauma and neurodegenerative disease, injury to myelin is a prominent pathological feature responsible for conduction deficits, and leaves axons vulnerable to damage from noxious compounds. Although the pathological mechanisms underlying myelin loss have yet to be fully characterized, oxidative stress (OS) appears to play a prominent role. Specifically, acrolein, a neurotoxic aldehyde that is both a product and an instigator of OS, has been observed in studies to elicit demyelination through calcium-independent and -dependent mechanisms and also by affecting glutamate uptake and promoting excitotoxicity. Furthermore, pharmacological scavenging of acrolein has demonstrated a neuroprotective effect in animal disease models, by conserving myelin's structural integrity and alleviating functional deficits. This evidence indicates that acrolein may be a key culprit of myelin damage while acrolein scavenging could potentially be a promising therapeutic approach for patients suffering from nervous system trauma and disease.     

Synthesis of low-phenylalanine polypeptides

Low-phenylalanine-peptides for dietotherapy of phenylketonuria (PKU) were prepared from soybean protein isolate. Soluble fraction of soybean protein isolate was hydrolysed by alpha-chymotrypsin then followed by carboxypeptidase-A. Molecular weight distribution and amino acid analysis were made on the resultant polypeptides. The chymotrypsin hydrolysate was divided into two fractions, Fraction I (molecular weight greater than 2500) and Fraction II (molecular weight between 1000 and 2500). The phenylalanine content of Fraction I (3.1%) was lower than that of Fraction II (5%), indicating the nonuniform distribution of phenylalanine in soy bean protein. Carboxypeptidase hydrolysis of Fraction I further reduced the phenylalanine concentration to 2.3%, approximately half of the original concentration in soybean protein isolate.     

Synthesis of 3'-thioribonucleosides and their incorporation into oligoribonucleotides via phosphoramidite chemistry.

Oligoribonucleotides containing 3'-S-phosphorothiolate linkages are valuable probes in nucleic acid biochemistry, but their accessibility has been limited because 3'-thioribonucleoside phosphoramidites have not been available. We synthesized 3'-thioribonucleoside derivatives (C, G, and U) via glycosylations of nucleoside bases with 3-S-thiobenzoyl-5-O-toluoyl-1,2-O-diacetylfuranose 5, which was obtained from 1 ,2-O-isopropylidene-5-O-toluoyl-3-trifluoromethane-sulfonyl-alpha-D-x ylofuranose 2 by SN2 displacement with sodium thiobenzoate. Additionally, a 3'-thioinosine derivative was prepared from inosine via direct modification of the ribose, analogous to the previously reported synthesis of 3'-thioadenosine, except that the intermediate 2',3'-epoxide 9 was first protected as the 5'-O-tert-butyldiphenylsilyl ether prior to subsequent synthetic steps. This hydrophobic silyl group facilitated extraction and isolation of synthetic intermediates. After removal of the protecting groups, the 3'-thionucleosides (C, G, U, and I) were treated with 2,2'-dipyridyl disulfide to protect the free thiol group as a disulfide. The 3'-thionucleosides were converted to the corresponding phosphorothioamidites using procedures analogous to those for standard phosphoramidites. The amino groups of 3'-thiocytidine and 3'-thioguanosine were protected as benzoyl and isobutyryl amides, respectively, and the 5'- and 2'-hydroxyl groups of each nucleoside were protected as dimethoxytrityl and tert-butyldimethylsilyl ethers, respectively. The 3'-thiol group was deprotected by reduction with DTT and phosphitylated to afford analytically pure 3'-S-phosphorothioamidites 15, which were incorporated into oligoribonucleotides by solid-phase synthesis. Chemical assays and mass spectrometry of the synthetic RNA showed that ribose-3'-S-phosphorothiolate linkages were installed correctly and efficiently into RNA oligonucleotides using phosphoramidite chemistry.     

Synthesis of a N-acylsulfamide linked dinucleoside and its incorporation into an oligonucleotide.

A N-acylsulfamide linked thymidine dinucleoside was synthesized and incorporated into an oligonucleotide (ON). The interest is in a linkage analog that has a higher pKa relative to a phosphodiester and when incorporated into ONs is capable of helix formation with complementary RNA. The hybridization property of the resultant ON with RNA was shown to result in significant destabilization.     

Synthesis of 2'-C-alpha-difluoromethylarauridine and its 3'-O-phosphoramidite incorporation into a hammerhead ribozyme

The 2'-C-difluoromethylated nucleoside 4 was synthesized starting from uridine. 4 was then converted to the 3'-O-phosphoramidite derivative 5 and was incorporated into a hammerhead ribozyme (7). The cleavage characteristics of the modified oligonucleotide have been analysed.