Actin acetylation in Drosophila tissue culture cells

In a permanent cell line derived from Drosophila embryos, cytoplasmic actin is produced as an unstable precursor, which is subsequently converted to a stable form. This conversion results in a reduction in isoelectric point, with no apparent change in molecular weight. The conversion involves an enzymatic acetylation, and results in an insensitivity to aminopeptidase digestion, suggesting N-terminal blockage. Both the acetylated and unacetylated actins can participate in the assembly of F-actin, but with different efficiencies.This work was supported by a grant from the NIH (GM 22866).     

Human erythrocyte galactosyltransferase. Characterization, membrane association and sidedness of active site

Human erythrocyte UDPgalactose : 2-acetamido-2-deoxy-alpha-D-galactopyranosylpeptide galactose beta(1 lead to 3) transferase (Galactosyltransferase) has been characterized in terms of detergent and metal ion requirements. Michaelis constants for donor and acceptor substrates, inhibition constant for N-acetylgalactosamine, pH optimum and ionic strength effects. The assay thus optimized permits initial velocity measurements. Galactosyltransferase was shown to be membrane-bound by demonstrating its association with erythrocyte ghosts after high and low ionic strength treatments to remove weakly-associated proteins. In the absence of detergents, no activity was detectable in sealed ghosts and inside-out vesicles derived from erythrocyte membranes. Enzyme activation by detergents paralleled solubilization of membrane proteins. Both latency and solubilization studies indicated a substrate inaccessible active site for the enzyme in situ in the membrane. Galactosyltransferase activity in resealed ghosts, leaky ghosts and inside-out vesicles was resistant to the action of trypsin, chymotrypsin or pronase applied as single agents. A mixture of these proteases, however, strongly reduced the enzyme activity in inside-out vesicles and leaky ghosts, indicating a cytosolic orientation for the active site of the galactosyltransferase.     

Alcohol-induced changes in the phospholipid molecular species of Escherichia coli.

In Escherichia coli, the additon of ethanol resulted in the synthesis of an increased proportion of phospholipids containing two unsaturated fatty acids. The addition of hexanol resulted in the opposite effect, an increase in the proportion of monounsaturated molecular species. The alcohol-induced changes were quantitatively similar to those caused by changing growth temperature. These results suggest that both adaptation to temperature and alcohol-induced changes in lipid composition share some common regulatory features.     

Isolation and characterization of beef thyroid giant RNA]

Total HMW-RNAs were prepared by three different methods (method with phenol, method with NaClO4, method without phenol using Ultrogel AcA 22 filtration). Giant RNAs were obtained in the void volume by filtration on Sepharose 2B. The giant RNAs/total HMW-RNA ratio is higher (6.77%) with the gel filtration method than with phenol or NaClO4 methods (1.41% and 1.00% respectively). The nucleotide composition of these RNAs is DNA-like and the sedimentation constants are approximately 70-100 S.     

Synthesis of poly(adenosine diphosphate ribose) in synchronized Chinese hamster cells.

Chinese hamster ovary cells were synchronized by mitotic selection and used to study the relation of poly(adenosine diphosphate ribose) synthesis to DNA synthesis and the different phases of the cell cycle. DNA synthesis was measured in cells rendered permeable to exogenously supplied nucleotides. Poly(ADPR) synthesis was also measured in permeable cells in the presence of both minimum and maximum DNA damage. The maximum DNA damage was produced by treating the cells with saturating concentrations of DNase. As anticipated, the DNA synthesis complex showed its maximum activity during S phase and showed 4–5-fold less activity during the other phases of the cell cycle. The basal level of poly(ADPR) synthesis was elevated during G1, fell to its lowest level during S phase, then increased during G2 and rose to its highest level during G1. The DNase responsive activity of poly(ADPR) synthesis was relatively constant thru the cell cycle but showed a peak at the end of S phase; then the activity decreased during the subsequent G2-M period.     

E rosette formation at 37°:A property of mitogen-stimulated human peripheral blood lymphocytes

Peripheral blood lymphocytes from healthy humans formed stable E rosettes with sheep erythrocytes (SRBC) at 37°C after culture with phytohemagglutinin or the divalent cation ionophore A23187. Cells manifesting this phenomenon exhibited “blast” morphology, appeared by 16 hr of culture, increased dramatically in percentage and absolute number by 62 hr, and persisted in large numbers for the duration of culture (182 hr). Unstimulated lymphocytes formed rosettes at 4°C but not at 37°C. Increased “stickiness” due to surface-bound lectin mitogen was not the cause of rosette formation at 37°C.Formation of E rosettes at 37°C has previously been considered a property of lymphocytes less differentiated than the circulating T cell (e.g., thymocytes, leukemic lymphoblasts). The present findings indicate that this property can be “reexpressed” during blastogenesis in culture.This observation also demonstrates technical problems associated with the use of SRBC to quantitate lymphocytes with complement receptors (B cells) by the EAC rosette assay in culture. False positives resulted from 37°C E rosette formation, but this was overcome by replacing the SRBC with guinea pig erythrocytes in the EAC assay.     

Pentobarbital anesthesia: Lack of effect on venous prostaglandins in dogs

Venous prostaglandins A, E, and F were determined by radioimmunoassay in 10 dogs before and one hour after administration of sodium pentobarbital (35 mg/Kg, iv). In the conscious state, PGA was 0.34 ± 0.04 ng/ml (mean ± SE), PGE 0.20 ± 0.01 ng/ml, and PGF 0.25 ± 0.03 ng/ml. During pentobarbital anesthesia, these levels were unchanged (p >0.05). Thus, pentobarbital anesthesia had no effect on peripheral venous prostaglandin levels.