The partial acid hydrolysis of a highly dextrorotatory fragment of the cell wall of Aspergillus niger. Isolation of the α-(1→3)-linked dextrin series

1. The cell-wall composition of Aspergillus niger has been investigated. Analysis shows the presence of six sugars, glucose, galactose, mannose, arabinose, glucosamine and galactosamine, all in the d-configuration, except that a small amount of l-galactose may be present. Sixteen common amino acids are also present. 2. The wall consists chiefly of neutral carbohydrate (73-83%) and hexosamine (9-13%), with smaller amounts of lipid (2-7%), protein (0.5-2.5%) and phosphorus (less than 0.1%). The acetyl content (3.0-3.4%) corresponds to 1.0mole/mole of hexosamine nitrogen. 3. A fractionation of the cell-wall complex was achieved, with or without a preliminary phenol extraction, by using n-sodium hydroxide. Though this caused some degradation, 30-60% of the wall could be solubilized (depending on the preparation). Analyses on several fractions suggest that fractionation procedures bring about some separation of components although not in a clear-cut fashion. 4. Cell-wall preparations were shown to yield a fraction having [alpha](D) approx. +240 degrees (in n-sodium hydroxide) and consisting largely of glucose. This was separated into two subfractions, one of which had [alpha](D)+281 degrees (in n-sodium hydroxide) and had properties resembling the polysaccharide nigeran; the other had [alpha](D) +231 degrees (in n-sodium hydroxide). It is suggested that nigeran is a cell-wall component.     

Lipids of isolated neurons

1. Lipids were extracted from neurons isolated from the lateral vestibular nucleus of ox (Bos taurus L.) and the ganglia of Aplysia punctata Cuvier. 2. Thin-layer chromatography of ox-neuron lipid revealed three major fractions corresponding to neutral lipid, phosphatidylethanolamine and phosphatidylserine. Part of the phosphatidylethanolamine was present as the plasmalogen. 3. Aplysia-neuron lipid contained neutral lipid, phosphatidylethanolamine and phosphatidylserine. Both phospholipids appeared to be present predominantly as the plasmalogen form. 4. The fatty acids of alkali-labile lipids of ox neurons were examined by gas–liquid chromatography. The major fatty acids were oleic acid, stearic acid and palmitic acid.     

Immunogenicity and tumor protective activity of B16 melanoma vaccines

The immunogenicity and tumor-protective activity of different vaccines were examined and compared with murine B16 melanoma. All vaccines were prepared from material shed into culture medium by B16 melanoma cells. Vaccine I was generated by concentrating the shed material. Vaccine II was partially purified by precipitating the shed material with 50% ammonium sulfate followed by sephadex G-200 column chromatography. Vaccine III was concentrated shed material that was treated with 0.5% NP-40 and then ultracentrifuged to remove transplantation antigens. Mice were immunized to equal protein concentrations of vaccines weekly for 5 weeks or to control buffer. Antibody, cellular, and tumor-protective immunity to melanoma was measured in all mice 2 weeks following the last immunization. All three vaccine preparations were immunogenic. Vaccine preparation I appeared to be the most immunogenic and the one that most consistently augmented tumor-protective immunity. Augmentation in tumor-protective immunity correlated better with increase in cellular than in humoral immunity to melanoma.     

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.     

An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.