Characterization of the internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA

Internal initiation of translation on the vesicular stomatitis virus (VSV) phosphoprotein (P) mRNA leads to the synthesis of a second protein [Herman, R. C. (1986) J. Virol. 58, 797-804]. Characterization of this phenomenon shows that initiation at the 5'-proximal and internal AUG codons has different optima for mono- and divalent cations in the reticulocyte lysate. Whereas 5' initiation is stimulated by increasing concentration of K+ over the endogenous level, internal initiation is inhibited. Internal initiation is much less sensitive to the effects of the cap analogue 7mGpppG in both the reticulocyte lysate and the wheat-germ extract under conditions that reduce 5'-proximal initiation to only about 4-5% of the control level. These results imply that 5'-proximal and internal initiations are distinct biochemical processes.     

Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

An abundant, highly conserved tonoplast protein in seeds

We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with M_r 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds.     

DNA interaction, cytotoxicity, and radiosensitization with PtCl4(Nile Blue)2 and PtCl4(Neutral Red)2

Complexes of the positively charged, nuclear staining, quinone-imine dyes Nile Blue and Neutral Red with negatively charged tetrachloroplatinum (II) have been prepared in an effort to form neutral drugs which could gain ready access to the cellular nucleus and deliver significant quantities of the reactive tetrachloroplatinum anion to the vicinity of the DNA. Elemental analysis showed that both the Nile Blue and Neutral Red complexes with tetrachloroplatinum (II) comprised 2 mol of dye and 1 mol of tetrachloroplatinum, forming Pt(Nile Blue)2 and Pt(Neutral Red)2. Exposure of superhelical pBR322 DNA to the complexes or the dyes for 24 h followed by agarose gel electrophoresis showed that Neutral Red and Pt(Neutral Red)2 had little effect on DNA conformation, but that both Nile Blue and Pt(Nile Blue)2 could produce single-strand DNA breaks in a dose-dependent fashion. Studies in exponentially growing asynchronous, hypoxic, and normally oxygenated EMT6 cells at normal pH (7.40) and pH 6.45 demonstrated that neither dye was highly toxic, but that both complexes were capable of producing significant cytotoxicity. Both complexes killed normally oxygenated cells more efficiently than hypoxic cells, but Pt(Neutral Red)2 was more cytotoxic at pH 6.45, while Pt(Nile Blue)2 killed significantly more cells at normal pH. Both complexes decreased the survival of hypoxic EMT6 cells as indicated by the slope of the radiation survival curve [dose modifying factor (DMF) 2.90 for Pt(Nile Blue)2 and 1.45 for Pt(Neutral Red)2]. Studies with the FSaIIC murine tumor showed that both complexes were active radiosensitizing agents in vivo [DMF 1.76 for Pt(Nile Blue)2 and 1.25 for Pt(Neutral Red)2]. These results indicate that these new platinum complexes have characteristics which may make them and similar complexes effective radiosensitizing agents in humans.     

The effect of alkaline borohydride treatment onN-linked carbohydrates of glycoproteins

The effects of treatments of the glycoprotein ribonuclease-B, the proteins ribonuclease-A and myoglobin, and the glyco-amino acid GlcNAc(1-N) Asn with alkalil alkaline sodium borohydride, and aqueous sodium borohydride were systematically studied as a function of the concentration of the reagents, the temperature, and the length of the treatment. High-field¹H-NMR spectroscopy, chromatographic methods and amino-acid analysis were used to characterize products of the treatments of the various compounds. Our results indicate that mild alkaline borohydride treatment, as well as aqueous borohydride treatment alone, is capable of extensively degrading polypeptides and of partially releasing theN-linked glycans from ribonuclease-B. Initially, glycopeptides are produced, the peptide portion of which consists of several amino acids, which are further hydrolyzed to yield a mixture of glyco-asparagines and oligosaccharide-alditols in the ratio of 4:1. Strong alkaline borohydride treatment of ribonuclease-B is capable of completely releasing theN-linked carbohydrates as oligosaccharide-alditols.Abbreviation RNase ribonuclease     

N-succinimidyl methoxyphenylacetic acid ester, an amine-directed chiral derivatizing reagent suitable for enzymatic scale resolutions

A chiral derivatizing reagent, N-succinimidyl-2-(S)-methoxy-2-phenylacetic acid ester (SMPA), directed toward reaction with primary amine-containing compounds has been synthesized and characterized. This reagent is suitable for HPLC resolution from enzymatic-scale reactions where only microgram quantities of chiral products may be obtainable. SMPA derivatization was shown to be effective in the resolution of the enantiomers of a number of different racemic compounds. SMPA was used to resolve the diastereoisomeric derivatives of a previously unknown enzymatically oxygenated product, allowing determination of the stereochemical course of the enzymatic reaction. SMPA is easily prepared from an inexpensive, commercially available, and enantiomerically pure precursor with the formation of a shelf-stable crystalline product which is utilizable in water-containing solutions. In addition to its usefulness for micro-determinations, SMPA is useful for preparative-scale resolutions of enantiomers since the reagent is cleaved from the diastereoisomeric derivative by acid hydrolysis.