The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-lH-isoindol-1-one (BMS-200150). The similarity of the IC50 for inhibition of bovine MTPmediated TG transfer (0.6 ,iM) Genetic studies (1)(2)(3)(4) have demonstrated that an absence of microsomal triglyceride (TG) transfer protein (MTP) causes abetalipoproteinemia, a disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing plasma lipoproteins. These studies indicate that MTP is required for the production of the apoB containing lipoproteins, very low density lipoproteins and chylomicrons by the liver and intestine. The requirement for MTP for lipoprotein production is further supported by studies using cell lines that are not of hepatic or intestinal origin. When a truncated form of apoB representing 53% of the full-length apoB-100 is expressed in HeLa cells, virtually no apoB is secreted (5). However, when MTP is coexpressed with apoB, apoB is secreted as a lipoprotein particle. Similar findings have been observed in COS cells (6).MTP is found in the lumen of microsomes isolated from liver and intestine (7). The protein purified from bovine liver is a heterodimer consisting of the multifunctional enzyme, protein disulfide isomerase, and a unique, large 97-kDa subunit (8-10). In vitro, MTP A mixture of compound A (62.5 mmol) and Zn dust (438 mmol) in AcOH (250 ml) was heated at reflux for 18 h, cooled to room temperature, filtered through Celite (Aldrich), and concentrated. The residue was dissolved in CH2Cl2 (500 ml), washed with saturated NaHCO3 (2 x 100 ml) and brine (100 ml), dried over MgSO4, and concentrated. The crude product was recrystallized from i-PrOH to yield 2,3-dihydro-2-[1-

The CYCI-239-0 mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu-replacement of the normal -Ala-Gly-sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-l-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYCI+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYCI-239-0 mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYCI mRNA diminishes translation by inhibiting elongation.During the past 20 years, the CYCI gene and its gene product iso-l-cytochrome c in the yeast Saccharomyces cerevisiae have been the subject of numerous studies. Mutationally altered forms of at least partially functional iso-icytochromes c have been obtained from over 500 intragenic revertants of various cycl mutants. The vast majority of these altered iso-1-cytochromes c are found at a level corresponding to the level of normal iso-l-cytochrome c in wild-type strains. Thus, most changes of the amino acid sequence do not appreciably affect the level of the protein.One type of exception is the revertants that contain AUG initiator codons situated at certain abnormal positions (25, 26, 33; S. B. Baim, Ph.D. Thesis, University of Rochester, Rochester, N.Y., 1984; S. Baim and F. Sherman, unpublished data). In this investigation we describe another type of exception, the cycl-239 revertants which contain only 20o of the normal level of iso-1-cytochrome c and contain a sequence which can accommodate the formation of a hairpin structure within the translated region of the mRNA.MATERIALS AND METHODS Genetic analysis. The symbol CYCI+ or CYCI denotes the wild-type structural gene that encodes iso-l-cytochrome c. Mutations of this gene that cause a deficiency in either the amount or activity are designated by the symbol cycl followed by the allele number, e.g., cycl-17, cycl-239, etc. Intragenic revertants of specific cycl mutants in which the amount or activity of iso-l-cytochrome c has been at least partially restored are designated by the gene symbol CYCI followed by the allele number of the mutation from which they were derived and by capital letters to designate independent revertants. For example, CYCI-239-A, CYCI-239-B, etc., denote intragenic revertants of cycl-239.Conventional Zaret and Sherman (38,39). Briefly, logarithmic-phase cells from an overnight culture were inoculated at various densities into YPD (1% yeast extract [Difco], 2% Bacto-Peptone, and 2% glucose) medium. The cultures were shaken vigorously at 30°C for 18 to 24 hours. The cells were harvested at the appropriate densities and disrupted with glass beads. The lysate was adjusted to 1% sodium dodecyl sulfate and extracted several times with phenol-chloroform-isoamyl alcohol (25:24...

No abstract

The synthesis and structure/activity studies of the effect of varying the "B" group in a series of oxazolidinone antibacterials (I) are described. Two synthetic routes were used: (1) alkylation of aniline with glycidol followed by dialkyl carbonate heterocyclization to afford I (A = H, B = OH), whose arene ring was further elaborated by using electrophilic aromatic substitution methodology; (2) cycloaddition of substituted aryl isocyanates with epoxides to give A and B with a variety of values. I with B = OH or Br were converted to other "B" functionalities by using SN2 methodology. Antibacterial evaluation of compounds I with A = acetyl, isopropyl, methylthio, methylsulfinyl, methylsulfonyl, and sulfonamido and a variety of different "B" groups against Staphylococcus aureus and Enterococcus faecalis concluded that the compounds with B = aminoacyl, and particularly acetamido, were the most active of those examined in each A series, possessing MICs in the range of 0.5-4 micrograms/mL for the most active compounds described.

P-Selectin (CD62/GMP140/PADGEM) is an inducible cell-surface glycoprotein expressed by endothelial cells and platelets following stimulation by inflammatory mediators such as thrombin, histamine, or peroxides. P-Selectin mediates the binding of leukocytes to activated vascular endothelium at sites of inflammation and plays a role in mediating the binding of activated platelets to leukocytes and the vascular cell wall. The adhesive function of P-selectin is mediated by its calcium-dependent (or C-type) lectin domain, which is known to bind to carbohydrate ligands including fucosyl-N-acetyllactosamine (Lex, CD15), sialyl-Lex, and 3-sulfated galactosylceramides (sulfatides). Sulfatides can efficiently block P-selectin/myeloid cell binding in vitro and are excreted at high levels by activated granulocytes. These observations led to the hypothesis that sulfatide may play a role in facilitating the disengagement of CD62, allowing the efficient exit of granulocytes from the blood stream at sites of inflammation. In this report, we extend our previous mutagenesis analysis of the P-selectin binding site [Hollenbaugh, D., Bajorath, J., Stenkamp, R., & Aruffo, A. (1993) Biochemistry 32, 2960] and show that replacement of Tyr48 with Ser or Lys113 with Arg results in P-selectin mutants that, although correctly folded, do not bind to HL60 cells. These results suggest that the conservation of charged and hydrogen-bonding site chains is not sufficient to maintain the P-selectin function and that the exact stereochemistry provided by the side chains of residues lining the P-selectin binding pocket is critical for P-selectin binding.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

A homologous in vitro method was developed from Tetrahymena for ribosomal A-site binding of aminoacyl-tRNA to poly(uridylic acid)-programmed ribosomes with very low error frequency. The reaction mixture pH was the crucial factor in the stable A-site association of aminoacyl-tRNA with high fidelity. At a pH greater than 7.1, endogenous activity translocated A-site-bound aminoacyl-tRNA to the P site. If translocation was allowed to occur, a near-cognate amino-acyl-tRNA, Leu-tRNA, could stably bind to the ribosome by translocation to the ribosomal P site. Near-cognate aminoacyl-tRNA did not stably bind to either site when translocation was blocked. Misreading antibiotics stimulated the stable association of near-cognate aminoacyl-tRNA to the ribosomal A site, thereby increasing the error frequency by several orders of magnitude. Ribosome binding of total aminoacyl-tRNA near equilibrium was not inhibited by misreading antibiotics; however, initial rate kinetics of the binding reaction were dramatically altered such that a 6-fold rate increase was observed with paromomycin or hygromycin B. The rate increase was evident with both cognate and near-cognate aminoacyl-tRNAs. Several antibiotics were tested for misreading potency by the ribosome binding method. We found gentamicin G418 greater than paromomycin greater than neomycin greater than hygromycin B greater than streptomycin in the potentiation of misreading. Tetracycline group antibiotics effectively inhibited A-site aminoacyl-tRNA binding without promoting misreading.

Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics. Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 ,uM, respectively, inhibited growth by 50%. A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T. thermophila. Analysis of polysome profiles from paromomycin-and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo [14C]lysine incorporation. In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes. In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin. We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation. Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes. In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B.Aminoglycoside antibiotics are well-established inhibitors of growth, stimulators of misreading, and inhibitors of protein synthesis in procaryotes and eucaryotes (18). Vazquez and co-workers have studied the in vitro activities of many aminoglycoside antibiotics in eucaryotic model systems derived from yeasts and mammalian cells (20 and references therein). Of the aminoglycoside antibiotics tested, those active in inhibiting protein synthesis were usually found to inhibit the polypeptide chain elongation phase (20). Model systems to study the interaction of antibiotics with the ribosome at separate stages of the elongation cycle have proven to be powerful tools in the elucidation of antibiotic action on ribosomes. Furthermore, these studies have greatly enhanced the understanding of the structure-function relationships of ribosomes (8,15).Eucaryotic protein synthesis has not, however, received as much attention as have procaryotic systems, and the majority of current concepts about the structure of eucaryotic ribosomes have been derived by analogy with the extensive information available from Escherichia coli ribosomes. Model systems derived from different eucaryotic sources do not have uniform responses to a particular aminoglycoside, as is exemplified by the failure of paromomycin (PM) to inhibit the cytoplasmic protein synthesis of yeasts (14), mammalian cells (21), and wheat germ (22). In contrast, cytoplasmic protein synthesis in the lower eucaryote Tet...