Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol l-serine Pyruvate aminotransferase by dibutyryl adenosine 3′,5′-monophosphate

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine: pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have no effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serine aminotransferase diminishes with the age of animals. Increase in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyrl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.     

Restriction fragment length polymorphism (RFLP) analysis of bovine nuclear protein genes

Summary We have recently cloned both the bovine protamine (Krawetz et al. 1987, DNA 6: 47–57) and high mobility group (HMG-1) cDNAs (Pentecost and Dixon 1984, Bioscience Reports 4: 49–57). They have been used as probes for Restriction Fragment Length Polymorphism analysis of male-female pairs of different species and breeds, within the genus Bos. Utilizing this approach we have studied inheritance, chromosomal location and gene copy number of the bovine protamine and HMG-1 genes. This revealed that these nuclear protein genes are highly conserved suggesting that selective pressure has maintained their gene structures during evolution. A polymorphic Taq 1 restriction fragment was identified that was shown to be a heritable marker. These genes are not sex-linked and are present in a single copy for protamine and at least two copies for the HMG-1.     

Sperm ultrastructure of five species of the Neotropical ant genus Pseudomyrmex (Hymenoptera: Formicidae)

The seminal vesicles of adult males of five species of Pseudomyrmex were prepared for light and transmission electron microscopy. The Pseudomyrmex spermatozoa are long and slender with similar morphology. The head region has an acrosome and a nucleus. In all the studied species, two morphologically distinct types of acrosomal vesicles were observed, a long structure, as observed in all known ants, and a pear‐shaped one, never before observed in ants. The nucleus is elongated and both condensed and loose chromatin are present. The flagellum has an axoneme, a centriolar adjunct, two mitochondrial derivatives and two accessory bodies. The centriolar, the mitochondrial derivatives and the accessory bodies are similar to observations in most ant species that have been studied. The axoneme presents an uncommon 9 + 9 + 1 microtubule arrangement and the central microtubule has 13 protofilaments. The acrosomal dimorphism and the different levels of chromatin organization are exclusive characteristics of Pseudomyrmex. Furthermore, the 9 + 9 + 1 microtubule arrangement is different from all Hymenoptera, as well as from most insects, which present a 9 + 9 + 2 arrangement. These new morphological characters that are specific to Pseudomyrmex, are valuable synapomorphies of the genus and can be used in taxonomic characterization of the Pseudomyrmecinae subfamily and in phylogenetic analyses in Formicidae family.     

Pre‐copulation intervals,copulation frequencies,and initial progeny sex ratios in two biotypes of whitefly,Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) is a species complex, and its systematic classification requires controlled crossing experiments among its genetic groups. Accurate information on pre‐copulation intervals, copulation frequencies, and initial frequency of egg fertilization of newly emerged adults is critical for designing procedures for collecting the virgin adults necessary for these experiments. In the literature, considerable variation is reported between B. tabaci populations, with respect to the length of the pre‐copulation interval and the initial frequency of egg fertilization. Here, we used a video‐recording method to observe continuously the copulation behaviour of the Mediterranean/Asia Minor/Africa (B biotype) and the Asia II (ZHJ1 biotype) groups of B. tabaci. We also recorded the initial frequency of egg fertilization, as determined by the sex of the progeny. When adults were caged in female–male pairs on leaves of cotton plants, the earliest copulation events occurred 2–6 h after emergence; at 12 h after emergence 56–84% of the females had copulated at least once, and nearly all (92–100%) had copulated at least once by 36 h after emergence. Both females and males copulated repeatedly. Approximately 80 and 20% of copulation events occurred during the photophase and scotophase, respectively. By 72 h post‐emergence, the females of the B and ZHJ1 biotypes had copulated on average 6.1 and 3.9 times, respectively. When adults were caged in groups on plants 1–13 h after emergence, 30–35% of the eggs deposited during this period were fertilized, and approximately 90% of females were fertilized by the end of the 13 h. Although timing of copulation differed in detail between the two genetic groups, the results demonstrate that B. tabaci adults can start to copulate as early as 2–6 h post‐emergence and the majority of females can become fertilized on the day that they emerge.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Sensitivity of the cell cycle to TGFβ1 does not correlate with transformation of a rat liver epithelial cell line

The effects of TGF1 on cell cycle events in a rat liver derived epithelial cell line (BL9) and in two in vitro transformants of this line were studied by flow cytometry. Using either ethidium bromide staining or the incorporation of bromodeoxyuridine to evaluate DNA synthesis it was shown that TGF1 prevented the entry of G0/G1 phase BL9 cells into S phase. TGF1 did not exert its inhibitory effect(s) on DNA synthesis by the modulation of early events in the cell cycle. The tumorigenic transformed BL9 cell lines gave contrasting responses to the effects of TGF1. DNA synthesis in a BL9 cell line derived by transfection with an active N-ras oncogene was unaffected by TFG1 and thus appeared refractory to its growth controlling effects. On the other hand cells from a BL9 cell line derived by in vitro transformation with activated aflatoxin B₁ retained their sensitivity to the effects of TGF1. Thus the loss of the inhibitory effect of TGF1 on DNA synthesis is not obligatory for the malignant transformation of rat liver epithelial cells.Abbreviations TGF1 transforming growth factor 1 - BSA bovine serum albumin - FBS foetal bovine serum - BrdUrd bromodeoxyuridine - PI propidium iodide - PBS phosphate buffered saline