The solution structure of ribosomal protein S17E from Methanobacterium thermoautotrophicum: a structural homolog of the FF domain

The ribosomal protein S17E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. S17E is a 62-residue protein conserved in archaea and eukaryotes and has no counterparts in bacteria. Mammalian S17E is a phosphoprotein component of eukaryotic ribosomes. Archaeal S17E proteins range from 59 to 79 amino acids, and are about half the length of the eukaryotic homologs which have an additional C-terminal region. Here we report the three-dimensional solution structure of S17E. S17E folds into a small three-helix bundle strikingly similar to the FF domain of human HYPA/FBP11, a novel phosphopeptide-binding fold. S17E bears a conserved positively charged surface acting as a robust scaffold for molecular recognition. The structure of M. thermoautotrophicum S17E provides a template for homology modeling of eukaryotic S17E proteins in the family.     

Mutation of regulatory serines of rat tyrosine hydroxylase to glutamate: effects on enzyme stability and activity

Tyrosine hydroxylase is phosphorylated at four serine residues in its amino-terminus by multiple kinases. Phosphorylation of serine 40 by cAMP-dependent protein kinase results in alleviation of dopamine inhibition [J. Biol. Chem. 267 (1992) 12639]. The other serines are at positions 8, 19, and 31. The effect of phosphorylation at these serines has been investigated using mutated forms of tyrosine hydroxylase containing glutamates at the positions of the serines. The S8E, S19E, and S31E tyrosine hydroxylase variants have similar steady-state kinetic parameters and similar binding affinity for catecholamines to wild-type enzyme. The S8E, S19E, S31E, and S40E variants differ in stability at elevated temperatures. The S40E variant is the least stable, while the others are all more stable than wild-type enzyme. The increased stability of S8E, S19E, and S31E tyrosine hydroxylases may be one of the physiological effects of phosphorylation. It may also have implications for the interpretation of activities of heterogeneous mixtures of tyrosine hydroxylase which have been phosphorylated.     

The origin of oestrone sulphate in normal and malignant breast tissues in postmenopausal women.

Conversion of oestrone sulphate to oestrone has been suggested to make a major contribution to the level of oestrone found in breast tissues. In order to examine the ability of breast tissues to take up oestrone sulphate (E1S), 3H E1S or E1-35S was infused into postmenopausal women with advanced breast cancer. For 3 subjects infusion of 3H E1S was repeated after treatment with Danazol, a potential inhibitor of oestrone sulphatase activity. After infusion of 3H E1S significant levels of 3H E1S were detected in normal and malignant breast tissues (tissue: plasma ratios 0.14 +/- 0.13 and 0.24 +/- 0.12 respectively, mean +/- S.D., n = 5). Similar 3H E1S tissue: plasma ratios were detected after infusions of 3H E1 indicating that the 3H E1S detected in breast tissues after infusion of 3H E1S may have originated from the hydrolysis of 3H E1S in tissues other than the breast, with subsequent uptake and sulphation in breast tissues. After infusion of E1-35S no significant levels of radioactivity were detectable in normal or malignant breast tissues. Treatment with Danazol had no significant effect on tissue levels of 3H E1S or on the CRE1S E1 or MCR-E1S. It is concluded that oestrone sulphate, as such, is not taken up by breast tissues and that any contribution that oestrone sulphate makes to the oestrogen content of breast tissues will depend upon prior hydrolysis.     

Metabolism of estrogens and thier sulfates by mouse kidney tissue in vitro.

Whole kidney tissue from mouse was incubated with labelled estrone 3-sulfate (E13S), 17beta-estradiol 3-sulfate (17betaE23S), estrone (E1), and 17beta-estradiol (17betaE2). Considerable reduction of E13S and E1 occurred. E13S gave rise primarily to 17alpha-estradiol 3-sulfate (17 alphaE23S) together with lesser amounts of 17betaE23S. By incubating [3H-35S]E13S with the tissue it was confirmed that formation of the diol sulfates was direct, without accompanying hydrolysis and reconjugation. Conversion of E1 was mainly to 17betaE2 with, on the average, lesser amounts of 17alphaE2. A small degree of direct conversion of 17betaE23S to E13S was found. 17beta-Estradiol was converted to a limited extent to E1 and to much smaller amounts of 17alphaE2.     

西藏林芝真蚋亚属三新种（双翅目：蚋科）

本文记述西藏林芝真蚋亚属Ｅｕｓｉｍｕｌｉｕｍ三种：凸端真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉｍｕｌｉｕｍ）ｃｏｎｃａｖｕｓｔｙｌｕｍｓｐ．ｎｏｖ．、林芝真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉｍｕｌｉｕｍ）ｌｉｎｇｚｉｅｎｓｅｓｐ．ｎｏｖ．、裂缘真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉｍｕｌｉｕｍ）ｓｃｈｉｚｏｌｏｍｕｎｓｐ．ｎｏｖ 。     

17-oxidoreduction of 17beta estradiol, estrone and their 3-sulfates by kidney slices from guinea pig and human.

Slices of whole kidney and kidney cortex from the female guinea pig catalyzed a marked reduction of estrone 3-sulfate (E13S) and estrone (E1) to 17beta-estradiol 3-sulfate (E23S) and 17beta-estradiol (E2), respectively, as well as the reverse (dehydrogenation) reactions. Slices of medulla did not appear active in E23S-E13S interconversion but did possess the ability to interconvert E2 and E1, besides possessing considerable sulfatase activity. The use of [3H-55S]E13S and [3H-55S]E23S as substrates, together with a demonstrated lack of estrogen sulfate synthesis by the tissue slices, provided ample evidence that the intact sulfates were involved in direct oxidoreduction. Slices of human kidney cortex catalyzed the reduction of E13S to a very limited extent. Slices of whole kidney and of cortex from guinea pig formed small amounts of estrogen glucuronide(s).     

Interaction between concurrent strength and endurance training

To assess the effects of concurrent strength (S) and endurance (E) training on S and E development, one group (4 young men and 4 young women) trained one leg for S and the other leg for S and E (S+E). A second group (4 men, 4 women) trained one leg for E and the other leg for E and S (E+S). E training consisted of five 3-min bouts on a cycle ergometer at a power output corresponding to that requiring 90-100% of oxygen uptake during maximal exercise (VO2 max). S training consisted of six sets of 15-20 repetitions with the heaviest possible weight on a leg press (combined hip and knee extension) weight machine. Training was done 3 days/wk for 22 wk. Needle biopsy samples from vastus lateralis were taken before and after training and were examined for histochemical, biochemical, and ultrastructural adaptations. The nominal S and E training programs were "hybrids", having more similarities as training stimuli than differences; thus S made increases (P less than 0.05) similar to those of S+E in E-related measures of VO2max (S, S+E: 8%, 8%), repetitions with the pretraining maximal single leg press lift [1 repetition maximum (RM)] (27%, 24%), and percent of slow-twitch fibers (15%, 8%); and S made significant, although smaller, increases in repetitions with 80% 1 RM (81%, 152%) and citrate synthase (CS) activity (22%, 51%). Similarly, E increased knee extensor area [computed tomography (CT) scans] as much as E+S (14%, 21%) and made significant, although smaller, increases in leg press 1 RM (20%, 34%) and thigh girth (3.4%, 4.8%). When a presumably stronger stimulus for an adaptation was added to a weaker one, some additive effects occurred (i.e., increases in 1 RM and thigh girth that were greater in E+S than E; increases in CS activity and repetitions with 80% 1 RM that were greater in S+E than S). When a weaker, although effective, stimulus was added to a stronger one, addition generally did not occur. Concurrent S and E training did not interfere with S or E development in comparison to S or E training alone.     

Specific radioimmunoassay of estrone sulfate. Application to measurement in male plasma

We described a new, specific and easy to use radioimmunoassay (RIA) of estrone sulfate (E1S) in males. After synthesis of an E1S-6-(O-carboxymethyl) oxime hapten then coupling to BSA, we obtained a specific anti-E1S antiserum. Although the cross-reactivity of DHEAS with our anti-E1S antiserum was low (CR=0.002%), we confirmed the absolute necessity of separating plasma DHEAS from plasma E1S, before E1S RIA, because in plasma, DHEAS is present at levels 3-6000-fold higher than E1S, which generally is ignored. Thus, we elicited an easy separation of DHEAS from E1S, by a fast chromatography on in-house minicolumns. This new RIA, was applied to the determination of E1S plasma normal values in males. In 27 young men (<35 years), mean+/-S.D. were 1.97nmol/l+/-1.07nmol/l and in 63 untreated healthy aged men (>55 years), 1.80nmol/l+/-1.21nmol/l. No significant difference was seen between young and older subjects. The ranges of E1S plasma levels in these subjects were rather large and the ratios between the highest and the lowest E1S plasma levels were seven in the young group and 23.4 in the older group. No decrease of E1S plasma levels was observed with ages. Contrary to large interindividual E1S plasma level variations, the intraindividual variations have been found to be no significant. Correlations between E1S and unconjugated estrogens, E2 and E1 were 0.22 (P=0.016) and 0.51 (0.001), respectively.     

Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization

Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid species.     

Raised Erythrocyte Sedimentation Rate in Asymptomatic Hyperlipidaemia

In 101 subjectively healthy persons, who at a routine health examination were found to be markedly hyperlipidaemic, the erythrocyte sedimentation rate (E.S.R.) was significantly raised. The E.S.R. was raised above 25 mm/hr in 44, 64, and 23% respectively of subjects with type II A, II B, and IV hyperlipoproteinaemia in comparison to only 6% in sex- and age-matched normolipidaemic controls.Several explanations for this E.S.R. elevation have been studied. The levels of plasma lipoproteins do not appear themselves to influence the E.S.R., as among other things there was no difference in plasma concentrations of cholesterol and triglycerides in hyperlipidaemic persons with high and low E.S.R. The concentration of lysolecithin, which may decrease the E.S.R. was the same in the hyperlipidaemic persons with high E.S.R. as in those with low. However, the incidence of ischaemic exercise E.C.G. changes was 38% in the hyperlipidaemic subjects with E.S.R. below 24 mm/hr and 72% in those with E.S.R. above 25 mm/hr. For the time being one possible reason for the raised E.S.R. thus seems to be that the hyperlipidaemia causes silent vascular disease that in its turn produces the E.S.R. elevations.     

Estrogen defines the dynamics and destination of transactivated EGF receptor in breast cancer cells: Role of S1P3 receptor and Cdc42

Sphingosine-1-phosphate (S1P) receptors mediate transactivation of epidermal growth factor receptor (EGFR) by estrogen (E2). Here we report that the amount of intracellular EGFR remains elevated after stimulation of MCF-7 cells with E2 and S1P, although membrane-localized EGFR and S1P3 receptors are quickly internalized. Co-localization of internalized EGFR and LAMP-2 was lower in cells treated with E2/S1P, suggesting that endosomal EGFR might be directed for recycling instead of degradation. In addition, we found that E2/S1P activated Cdc42 and that knockdown of Cdc42 restores fast EGFR degradation after E2/S1P stimulation. Inhibition of S1P3 receptors prevented E2-induced activation of Cdc42, supporting the important role of the S1P receptor in E2 signaling. This is a novel mechanism further defining the effect of E2/S1P on the EGFR transactivation in breast cancer cells.     

Mono- or double-site phosphorylation distinctly regulates the proapoptotic function of Bax

Bax is the major multidomain proapoptotic molecule that is required for apoptosis. It has been reported that phosphorylation of Bax at serine(S) 163 or S184 activates or inactivates its proapoptotic function, respectively. To uncover the mechanism(s) by which phosphorylation regulates the proapoptotic function of Bax, a series of serine (S)→ alanine/glutamate (A/E) Bax mutants, including S163A, S184A, S163E, S184E, S163E/S184A (EA), S163A/S184E (AE), S163A/S184A (AA) and S163E/S184E (EE), were created to abrogate or mimic, respectively, either single or double-site phosphorylation. The compound Bax mutants (i.e. EA and AE) can flesh out the functional contribution of individual phosphorylation site(s). WT and each of these Bax mutants were overexpressed in Bax(-/-) MEF or lung cancer H157 cells and the proapoptotic activities were compared. Intriguingly, expression of any of Bax mutants containing the mutation S→A at S184 (i.e. S184A, EA or AA) represents more potent proapoptotic activity as compared to WT Bax in association with increased 6A7 epitope conformational change, mitochondrial localization/insertion and prolonged half-life. In contrast, all Bax mutants containing the mutation S→E at S184 (i.e. S184E, AE or EE) have a mobility-shift and fail to insert into mitochondrial membranes with decreased protein stability and less apoptotic activity. Unexpectedly, mutation either S→A or S→E at S163 site does not significantly affect the proapoptotic activity of Bax. These findings indicate that S184 but not S163 is the major phosphorylation site for functional regulation of Bax's activity. Therefore, manipulation of the phosphorylation status of Bax at S184 may represent a novel strategy for cancer treatment.     

Functional consequences of phosphomimetic mutations at key cAMP-dependent protein kinase phosphorylation sites in the type 1 inositol 1,4,5-trisphosphate receptor

Regulation of Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca(2+) signals. In this study, regulation of Ca(2+) release by phosphorylation of type 1 InsP(3)R (InsP(3)R-1) was investigated by constructing "phosphomimetic" charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP(3)R-1 splice variants. Ca(2+) release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP(3)R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP(3)R-1, InsP(3)-induced Ca(2+) release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca(2+) release through the S2- S1589E/S1755E InsP(3)R-1 was enhanced approximately 8-fold over wild type and approximately 50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP(3)R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca(2+) release was enhanced approximately 3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca(2+) release. The sensitivity of Ca(2+) release in cells expressing S2+ S1755E InsP(3)R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP(3)R-1. In contrast, mutation of S2+ S1589E InsP(3)R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP(3)R-1 resulted in robust Ca(2+) oscillations when cells were stimulated with concentrations of alpha-IgM antibody that were threshold for stimulation in S2- wild type InsP(3)R-1-expressing cells. However, at higher concentrations of alpha-IgM antibody, Ca(2+) oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP(3)R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.     

Detergent modification of myosin function and structure in solution

Rabbit skeletal muscle myosin and myosin subfragment 1 (S1) MgATPase activities were increased 2-3-fold by the addition of a variety of molecules that contained single straight saturated 12-16-carbon chains. The nonionic detergent dodecyl nonaoxyethylene ether (C12E9) increased the activity of S1 to 50% of maximum at a free C12E9 concentration of 27 +/- 9 microM. The activation was reversible and was not due to chemical modification of S1 amino acid side chains. The Vmax for actin-activated S1 MgATPase activity was increased 3-fold by C12E9. The apparent association constant for S1 binding to pure F-actin was reduced 3-fold by C12E9. The [C12E9] dependencies of the increase in S1 and acto-S1 MgATPase activities and of the decrease in acto-S1 binding were equal, within experimental uncertainty, suggesting that a single detergent-induced S1 conformational change is sufficient to explain the results. The stoichiometry of C12E9 bound to S1 in the S1-C12E9 complex was estimated, by the S1 concentration dependence of the C12E9 activation midpoint and by the light-scattering increase when S1 and detergent were mixed, to be 7 and 57 C12E9 molecules per S1, respectively. The results are discussed in relation to possible structural aspects of the mechanism of action for S1 and acto-S1 MgATPase activities.     

Induction of tumor-reactive CTL by C-side chain variants of the CTL epitope HER-2/neu protooncogene (369-377) selected by molecular modeling of the peptide: HLA-A2 complex

To design side chain variants for modulation of immunogenicity, we modeled the complex of the HLA-A2 molecule with an immunodominant peptide, E75, from the HER-2/neu protooncogene protein recognized by CTL. We identified the side chain orientation of E75. We modified E75 at the central Ser(5) (E75 wild-type), which points upward, by removing successively the HO (variant S5A) and the CH2-OH (variant S5G). Replacement of the OH with an aminopropyl (CH2)3-NH3 (variant S5K) maintained a similar upward orientation of the side chain. S5A and S5G were stronger stimulators while S5K was a weaker stimulator than E75 for induction of lytic function, indicating that the OH group and its extension hindered TCR activation. S5K-CTL survived longer than did CTL induced by E75 and the variants S5A and S5G, which became apoptotic after restimulation with the inducer. S5K-CTL also recognized E75 endogenously presented by the tumor by IFN-gamma production and specific cytolysis. S5K-CTL expanded at stimulation with E75 or with E75 plus agonistic anti-Fas mAb. Compared with S5K-CTL that had been restimulated with the inducer S5K, S5K-CTL stimulated with wild-type E75 expressed higher levels of E75(+) TCR and BCL-2. Activation of human tumor-reactive CTL by weaker agonists than the nominal Ag, followed by expansion with the nominal Ag, is a novel approach to antitumor CTL development. Fine tuning of activation of tumor-reactive CTL by weak agonists, designed by molecular modeling, may circumvent cell death or tolerization induced by tumor Ag, and thus, may provide a novel approach to the rational design of human cancer vaccines.     

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.     

Five new polar sterols from the black coral Antipathes subpinnata.

In addition to the known (20S,22E)-cholesta-1,4,22-triene-16 beta,18, 20-triol-3-one, (20S,22E)-24-methyl-cholesta-1,4,22-triene-16 beta,18,20-triol-3-one, and (20S,22E)-24-methylcholesta-4,22-diene-16 beta,18,20-triol-3-one, the methanol extract of the Mediterranean anthozoan Antipathes subpinnata was shown to contain five new sterols: (20S,22E)-cholesta-1,4,22-triene-18,20-diol-3-one, (20S,22E)-24-methylcholesta-1,4,22-triene-18,20-diol-3-one, (20S)-cholest-4-ene-16 beta,18,20-triol-3-one, (20S,22E)-cholesta-4,22-diene- 16 beta,18,20-triol-3-one, and (20S,22E)-24-methylcholesta-4,22-diene-16 beta,18,20-triol-3-one, whose stereostructures were elucidated on the basis of physicochemical evidence. The previously unassigned chirality at C-20 of the known sterols has been also established as S.     

Integration and transcription of group C human adenovirus sequences in the DNA of five lines of transformed rat cells

We have used the Southern blotting technique to analyze the integration patterns of human adenovirus sequences in the DNA of four rat cell lines, F17, 8617, T2C4 and F4, which were transformed by Ad-2 virus, and 5RK clone 6, which was transformed by Ad-5 HindIII-G fragment. We have also analyzed the Ad-specific messenger RNAs synthesized in these cell lines, in 293 cells (an Ad-5 transformed human cell line), and in Ad-2 early infected human KB cells, using the RNA geltransfer hybridization technique. We were interested in whether the Ad sequences are integrated, what the integration patterns are, whether the transforming region is present in an intact form, and whether the transforming region and other early regions are expressed at the mRNA level.Our results show that the integration patterns of Ad sequences range from simple to quite complex. Cells from line 8617 contain a single copy of right-end sequences flanked by left-end sequences. T2C4 cells have four different left-end sequences and two different right-end sequences. 5RK cells contain multiple different pieces of left-end sequences. In agreement with the results of Sambrook et al. (1979a,b), F17 cells contain a single copy of the left 17% of the genome, and F4 cells contain multiple copies of the right 5% of the genome fused to the left ~ 68% of the genome. The complete Ad genome is not present in any of the cell lines, and different regions may not be equimolar. There are no specific sites on the cellular or viral genome at which integration occurs. In 8617, F17 and F4 cells the Ad-2 sequences appear to be located close together on a single chromosome, suggesting that the Ad sequences in these cells arose from a single integration event. F17, 8617, T2C4, F4, and probably 5RK, cells all have an intact early region E1a (map position 1·3–4·6); F17, 8617, T2C4 and F4 cells also have E1b (m.p. 4·6–11·2) intact. E1a and E1b are the regions responsible for transformation. 8617 cells also have an intact early region E4 (m.p. 99-91·5) and T2C4 cells have an intact early region E3 (m.p. 76–86).Ad-2 early infected KB cells were shown to synthesize major E1a-specific mRNAs of 13 S, 12 S and 9 S, and major E1b-specific mRNAs of 22 S and 13 S. All the transformed cells synthesize the E1a 13 S and E1a 12 S mRNAs, and all cells except 5RK synthesize the E1b 22 S and E1b 13 S mRNAs. Early infected KB cells synthesize E3-specific mRNAs of 26 S, 24 S, 22 S, 19 S, 12 S and 9 S: T2C4 cells synthesize the major 22 S and 19 S RNA species, and possibly the less pronounced E3 mRNAs. Early infected cells and 8617 cells synthesize E4-specific mRNAs of 19 S, 17 S, 14 S, 12 S, 11 S, 9 S and 8 S. 8617 cells also synthesize E4 mRNAs of about 23 to 24 S and 21 S. F4 cells synthesize 24 S and 19 S hybrid mRNAs that contain both E4 and E1a sequences: these RNAs arise because F4 cells contain a portion of the E4 region fused to the left end (m.p. 0) of the genome.Our results, as well as those from other laboratories, are consistent with the idea that the transformed phenotype of Ad transformed cells is maintained by expression of Ad genes in E1a and E1b.     

Elasticities in variable environments: properties and implications

Elasticities in stochastic matrix models are used to understand both population and evolutionary dynamics. We examine three such elasticities: stochastic elasticity E(ij)(S) with respect to the (i, j) matrix element, the elasticity E(ij)(S mu) with respect to the mean mu(ij) of the matrix element, and the elasticity E(ij)(S sigma) with respect to the variability sigma(ij) of the matrix element. We show that the stochastic elasticity E(S) does not accurately describe the effect of variability; one should use E(S sigma) and E(S mu). We establish two general properties of these elasticities: a sum rule that connects them and a limit on the sum of the E(S sigma). We discuss the implications of these properties for the analysis of buffering and selection on the average rates versus the variability of rates.     

Mechanism of group IVA cytosolic phospholipase A(2) activation by phosphorylation

Group IVA cytosolic phospholipase A2 (cPLA2) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser505 and Ser727 activates cPLA2, we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA2. The difference in in vitro membrane affinity between wild type (or S505E) and S505A increased with the decrease in Ca2+ concentration, reaching >60-fold at 2.5 microm Ca2+. When HEK293 cells transfected with cPLA2 and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 microm Ca2+, whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile399, Leu400, and Leu552) indicate that a main role of the Ser505 phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA2 in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA2 activation by calcium and phosphorylation.