The biochemical and ultrastructural effects of tunicamycin and D-glucosamine in L1210 leukemic cells

Tunicamycin was found to specifically inhibit the incorporation of a number of sugars into L1210 leukemia cell glycoproteins. This inhibition of glyco-protein biosynthesis led to a cessation of cell growth which was reversible in a dose-dependent and time-dependent manner. After removal of the antibiotic from L1210 cell cultures resumption of sugar incorporation preceded that of thymidine incorporation and the recovery of cell growth. The treatment of cells with tunicamycin resulted in a significant increase in the intracellular pool of UDP-N-acetylglucosamine which occurred concurrently with alterations in cell ultrastructure including distentions of the endoplasmic reticulum and nuclear membranes. Similar ultrastructural changes and increases in the intracellular pools of UDP-sugars were observed in L1210 cells exposed to 5 mM D-glucosamine, which suggested that the antiproliferative effects of tunicamycin may be related to the accumulation in the endoplasmic reticulum of one or more nucleotide sugar precursors of asparagine-linked glycoprotein biosynthesis. However, the biological effects of tunicamycin could be distinguished from those caused by D-glucosamine. Exposure of L1210 cells to tunicamycin resulted in specific alterations in the biochemical composition of the plasma membrane and in the inhibition of cellular agglutination by wheat germ agglutinin which were not apparent following exposure to equitoxic concentrations of the aminosugar. These studies, together with those which demonstrated that recovery of the cellular capacity to synthesize glycoproteins was obligatory for the recovery of cellular proliferation in tunicamycin-treated cells, suggested that inhibition of the synthesis of glycoproteins was the major factor limiting L1210 leukemic cell proliferation.     

Metal-ion catalysis of aldose-ketose isomerizations in acidic solutions

A comparative study has been made of the ability of metal ions to catalyze the Lobry de Bruyn-Alberda van Ekenstein transformation. An analytical enzymic assay system, which is highly sensitive and selective, was developed to monitor the rates of isomerization of hexose and triose phosphates. The metal ions catalyze the isomerization of hexose phosphates by virtue of their Lewis acid character with the first transition series being most effective. The metal ions also appear to have no ability to direct the course of the reactions. Amino acids also catalyze the isomerization apparently by virtue of the free carboxylate anion.     

Number of Transformable Units Per Cell in Diplococcus pneumoniae

Analysis of frequencies of single and random multiple transformations in Diplococcus pneumoniae showed that there are at least two transformable units per cell of the total population in highly competent cultures. If 100% of the cells are competent in these cases, the units may be interpreted as the strands of one duplex deoxyribonucleic acid recipient chromosome. The theory is developed to allow for extension to more complex situations.     

Comparison of the structure of the immunoglobulins from horse serum

A study of the chemical structure of the horse immunoglobulins IgG and IgA(T) has shown that the amino acid contents of the peptide chains are very similar. These globulins differ most markedly in the products of papain digestion. IgG gives 3.5s products, whereas IgA(T) gives a 5s fraction and smaller components. This difference appears to be associated with the presence of an additional easily reducible disulphide bond in the Fd fragment of the heavy chain. There is two to three times as much carbohydrate in IgA(T) as in IgG. In both, this is in the heavy chain and in IgA(T) more than half is covalently bound to the Fd fragment. The differences in antigenic specificity between horse IgG and IgA(T) appear to be due to structural differences in the Fc fragment.     

Relative nutritional value of ciliate protozoa and algae as food forDaphnia

The relative importance of autotrophic flagellates, desmids, cyanobacteria, and ciliates as food forDaphnia magna was examined using cohort life tables. Each cohort was fed a single food type at a given concentration, and comparisons among each type were made. Algal feeding treatments included three levels of young (7 to 14 days old)Chlamydomonas reinhardi (Chlorophyta, Chlamydomonadacae), two levels of senescent (> 14 days old)C. reinhardi, two levels ofCryptomonas sp. (Chlorophyta, Cryptomonadacae), two levels ofStaurastrum sp. (Chlorophyta, Desmidacae), four levels of young (7 to 15 days old) or senescent (> 15 days old)Microcystis aeruginosa (Cyanophyta, Chlorococcacae), and a no-food treatment. The ciliatesCyclidium sp. andParamecium caudatum were also presented at concentrations of 1 or 10² cells/ml, as well as mixtures ofC. reinhardi (10³/ml) andCyclidium (1/ml) orP. caudatum (1/ml).Daphnia growth, reproduction, and survivorship were highest whenC. reinhardi orCryptomonas were the food source, while those starved or fedM. aeruginosa had shorter survivorship and lower growth and reproduction.Daphnia grew and had high survivorship when fedP. caudatum, but even though eggs were produced, most were aborted after 2 or 3 days.Staurastrum andCyclidium produced intermediate growth and survivorship, but reproduction was seen only in the 10³ Staurastrum/ml treatment. Carbon and nitrogen content were general indicators of nutritional value. However, growth, reproduction, and survivorship were higher in some cohorts fed treatments containing relatively low levels of carbon and nitrogen. Other cohorts were short-lived and did not reproduce, despite being fed much higher levels of carbon and nitrogen. The results also suggest that green algae are nutritionally valuable forDaphnia, whereas cyanobacteria are not. As measured by life-table parameters, the nutritional value of ciliates was variable, with some being poor food sources. Thus, the potential of ciliates as a trophic link between microbial production and higher trophic levels may vary with the ciliate community structure. Our results suggest that ciliates alone were insufficient as a food source to supportDaphnia population growth.     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.