Comparison of yeast mitochondrial Phe-tRNA synthetase subunits to their cytoplasmic counterparts: Isolation and determination of amino acid compositions

The α and β subunits of yeast mitochondrial Phe-tRNA synthetase are separated and isolated by means of chromatography on DEAE-cellulose, after enzyme alkylation with iodoacetate. The comparison of amino acid compositions of yeast mitochondrial and cytoplasmic native Phe-tRNA synthetases and their components shows significant differences. Results indicate that the two enzymes are coded for by different nuclear genes.     

An Improved Technique for Sequential R-, Q- and C-Banding of Bone Marrow Chromosomes

A procedure for the sequential staining of metaphase cells to provide R-banding, Q-banding and C-banding of chromosomes is described. The combined techniques allow more precise identification of marker chromosomes and more accurate definition of breakpoints in the consistent chromosome translocations that are associated with certain human malignancies.     

Enterobacteriaceae and Aeromonas hydrophila in Minnesota frogs and tadpoles (Rana pipiens)

In 222 Rana pipiens frogs and 34 tadpoles captured in and near Minnesota, Aeromonas hydrophila and 29 species of Enterobacteriaceae, including yersinia enterocolitica and Salmonella arizonae, were isolated from intestines. The prevalence of members of the family Enterobacteriaceae was lowest in frogs captured in early spring and highest in frogs captured in late summer.     

The effects of Cu on the adenylate energy charge of open ocean phytoplankton

The effects of short-term, acute Cu exposure (6 h) on the adenylateenergy charge (EC_A) of open-ocean phytoplankton populations(northeastern equatorial Pacific) were investigated. Energycharge remained at {small tilde}0.77 over the range of Cu additions(0.025 – 5.µg l^–1), even though ¹⁴C uptakeand total adenylate levels (ATP + ADP + AMP) were reduced byas much as 60%. These findings suggest that EC_A alone is nota sensitive indicator of acute sublethal metal effects on phytoplankton. ¹This research was supported by the NSF Biological OceanographyProgram grant #OCE 81-17286.     

Arming α2-macroglobulin with ricin A chain forms a cytotoxic conjugate that inhibits protein synthesis and kills human fibroblasts

A conjugate containing α₂-macroglobulin and highly purified ricin A chain was made using N-succinimidyl-3-(2-pyridyldithio)propionate. Radioimmunoassay indicated that it contained 1.2 mol A chain per mol α₂-macroglobulin. The conjugate inhibited polyuridylic-acid directed translation by rat liver ribosomes and protein synthesis in human fibroblasts. There was a 90 min lag period before the beginning of inhibition in fibroblasts, but complete inhibition could be achieved. By measuring protein synthesis as a function of protein concentration, it was demonstrated that 8.25·10^?9M conjugate was required to inhibit 50% of protein synthesis in 6 h. To achieve the same level of inhibition, 165-times more (1.3·10^?6M) unconjugated A chain was required, and 180-times less ricin (4.6·10^?11M). Ricin was more than 28 000 times more inhibitory than A chain alone. The presence of α₂-macroglobulin did not increase the cytotoxicity of unconjugated A chain, and it even protected the cells to a slight extent. The inhibitory action of the conjugate was blocked by antibodies specific for α₂-macroglobulin or ricin, and it was not prevented by galactose or antibodies specific for ricin B chain. Electron microscopy of the conjugate indirectly labelled with ferritin demonstrated that it was internalized by receptor mediated endocytosis through coated pits. These data indicate that the A chain portion of the conjugate survives the conditions in the lysosomes to the extent that it retains its ability to inactivate cytoplasmic ribosomes.     

Molecular cloning of pea mRNAs encoding a shoot-specific polypeptide and light-induced polypeptides

Summary The molecular cloning of cDNA corresponds to pea seedling mRNA sequences encoding a shoot-specific polypeptide, the small subunit of the ribulose 1,5 biphosphate carboxylase and a component of the light-harvesting chlorophyll a/b complex is described. cDNA prepared from polysomal poly(A)RNA of light-grown shoots was enriched for shoot-specific and light-induced sequences by heterologous liquid hybridization with mercurated polysomal poly(A)RNA of dark-grown roots, followed by sulfhydryl chromatography. Cloned shoot-specific sequences were identified by 2D electrophoretic analysis of hybrid release translation products. The cloned shoot-specific sequence corresponded to a mRNA of 850 nt present both in light-and dark-grown shoots, and produced anin vitro translation product of M_r27 500 and isoelectric point of 4.7.     

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation

Helium-neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He-Ne lasers; we undertook to resolve whether a low-power (7 mW) He-Ne laser could provide sufficient excitation to permit flow cytometric detection of APC-labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T-lymphocytes; APC-4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He-Ne laser; antibody-bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein-labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody-bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He-Ne lasers and APC-antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.     

Development and characterization of monoclonal antibodies to human type III procollagen

Hybridomas which secrete monoclonal antibodies against human type III procollagen have been developed. By an enzyme-linked immunosorbent assay, three of the monoclonal antibodies have been determined to be against non-helical extensions of the molecules while two of the antibodies are against helical portion of the molecules which is sensitive to bacterial collagenase action. These findings have been further confirmed by carrying out immuno-reaction of the pro α-chains transferred on nitrocellulose paper from sodium dodecyl sulfate polyacrylamide gels. These monoclonal antibodies have been found to be suitable reagents for immunohistochemical studies as well as for immunoassays of type III procollagen and collagen.     

Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: reversible inhibition at high magnesium ion concentrations

Magnesium stimulates phosphorylation of the calcium pump protein of the sarcoplasmic reticulum by inorganic phosphate, but the effect is reversed by high [Mg2+]. This reversal is readily explained in terms of the generally accepted existence of two conformational states of the enzyme, E1 and E2. E2 is the form of the enzyme that can be phosphorylated by Pi, and it has one binding site for Mg2+. E1 is the form of the enzyme that has two high-affinity Ca2+ binding sites, and it is phosphorylated by ATP when Ca2+ is bound. Mg2+ can bind weakly to the two Ca2+ sites and to a third site known to be present on E1; this stabilizes E1 at the expense of E2 when [Mg2+] is large. Stabilization of E1 at pH 6.2 and 25 degrees C was found to be a highly cooperative function of [Mg2+] and was not prevented by increasing [Pi]. The latter result requires the existence of a binding site for Pi on E1, with an affinity for Pi comparable to that of E2. Cooperativity with respect to [Mg2+] requires that E2 is the stable state of the enzyme in the absence of ligands, with an equilibrium constant [E2]/[E1] on the order of 10(3) or higher at pH 6.2 and 25 degrees C.