High-resolution proton magnetic resonance studies of the 3'-terminal colicin fragment of 16 S ribosomal RNA from Escherichia coli. Assignment of iminoproton resonances by nuclear Overhauser effect experiments and the influence of adenine dimethylation on the hairpin conformation

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Spontaneous development of anti-hepatitis B virus envelope (anti-idiotypic) antibodies in animals immunized with human liver endonexin II or with the F(ab')2 fragment of anti-human liver endonexin II immunoglobulin G: evidence for a receptor-ligand-like relationship between small hepatitis B surface antigen and endonexin II.

In a previous study, we have identified endonexin II (E-II) on human liver plasma membranes as a specific, Ca(2+)-dependent, small hepatitis B surface antigen (HBsAg)-binding protein. In this article, we describe the spontaneous development of anti-HBs antibodies in rabbits immunized with native or recombinant human liver E-II and in chickens immunized with the F(ab')2 fragment of rabbit anti-human liver E-II immunoglobulin G. Anti-HBs activity was not observed in rabbits immunized with rat liver E-II. Cross-reactivity of anti-E-II antibodies to HBsAg epitopes was excluded, since anti-HBs and anti-E-II activities can be separated by E-II affinity chromatography. The existence of an anti-idiotypic antibody is further demonstrated by competitive binding of human liver E-II and this antibody (Ab2) to small HBsAg, suggesting that Ab2 mimics a specific E-II epitope that interacts with small HBsAg. In addition, it was demonstrated that anti-HBs antibodies developed in rabbits after immunization with intact human liver E-II or in chickens after immunization with F(ab')2 fragments of rabbit anti-human liver E-II immunoglobulin G recognize the same epitopes on small HBsAg. These findings strongly indicate that human liver E-II is a very specific small HBsAg-binding protein and support the assumption that human liver E-II is the hepatitis B virus receptor protein.     

Selection for non-diapause inAmblyseius cucumeris andAmblyseius barkeri and exploration of the effectiveness of selected strains for thrips control

In Europe and North America the western flower thrips,Frankliniella occidentalis, is an important pest in various greenhouse crops, such as sweet pepper and cucumber. Two species of predatory mite are commercially applied for biological control of this pest:Amblyseius cucumeris andA. barkeri. Thrips control is generally successful from March onwards. During winter, however, thrips control by these predatory mites is less effective. An important reason for this is that the commercially applied strains of both mite species enter reproductive diapause under short-day photoperiods, whereas the western flower thrips does not enter diapause. In this paper we report on selection experiments for non-diapause in strains of both mite species, aimed at obtaining predators that do not enter diapause under light- and temperature conditions prevailing in winter. Additional experiments were done to estimate the potential of the selected lines as control agents ofF. occidentalis. Selection for non-diapause proved highly successful in both predatory mite species. In a New Zealand strain ofA. cucumeris diapause incidence decreased from 41% to 0% in about ten generations; in a Dutch strain ofA. barkeri diapause incidence decreased from 67% to 0% in about six generations. Furthermore, selection for non-diapause had no influence on predator performance, measured as predation rate and oviposition rate on a diet of first instar thirps larvae. Rates of predation and oviposition were the same for selected and unselected lines in both species; rates of predation and oviposition were higher forA. cucumeris than forA. barkeri. After 18 months under non-diapause conditions, no less than 92% of a sample of the selected non-diapause line ofA. cucumeris did not enter diapause when tested under diapause-inducing conditions. This indicates that ‘non-diapause’ is a stable trait in these predatory mites. Finally, a small-scale greenhouse experiment in a sweet pepper crop showed that the selected non-diapause line ofA. cucumeris established successfully under diapause-inducing short-day conditions.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Failure to find sex differences in testosterone activated aggression in two strains of rats

The effect of testosterone (testosterone propionate: TP) on intraspecific aggression in males and females of two strains of rats—WEzob and S3—was examined. Pairs of these rats, gonadectomized and treated either with oil or with testosterone propionate (TP), were tested in three different combinations: OIL against OIL, OIL against TP, and TP against TP-treated animals. Subsequently the effects of TP treatment of the subject and for the opponents interaction with sex and strain on the occurrence of diverse social + aggression behavioral parameters were determined. The results of the S3 strain indicate that testosterone treatment of either the subject or the opponent stimulates aggression in both males and females. No sex difference could be determined with respect to the incidence of aggression. In the WEzob strain a stimulatory effect of TP was shown in females but not in males. The absence of a clear stimulatory effect of TP in WEzob males in terms of changes in the total time spent on aggression, however, could wrongly suggest that TP does not affect aggression in these animals. The possibility of TP having an effect on these males in terms of increasing the intensity of fighting is discussed.     

The use of oral prostaglandin E2 in the management of intrauterine fetal death

12 otherwise healthy patients with intrauterine fetal death 1 to 6 weeks earlier were treated with oral prostaglandin E₂. 9 of the 12 patients delivered within 48 hours after treatment began. 2 others delivered within 48 hours after unsuccessful treatment ceased. In a third patient the cervix relaxed after treatment, and the uterine contents were removed by curettage. No serious complications, such as hemorrhage occurred. The uterus seemed surprisingly responsive to oral prostaglandin E₂ in cases of intrauterine fetal death.     

Chaotropic resolution of high molecular weight (type I) NADH dehydrogenase, and reassociation of flavin-rich (type II) and flavin-poor subunits.

1. Type-I NADH dehydrogenase (Complex I) was solubilized and dissociated into subunits by NaClO4. NADH slows the dissociation. On subsequent stepwise addition of (NH4)2SO4 the dissociation is partly reversed, as is to be expected from the opposing effects of ClO-4 and SO-24, which are on the salting-in and salting-out sides, respectively, of the lyotropic series. 2. In consequence, the aggregates of subunits that are separated by (NH4)2-SO4 fractionation consist of randomly associated subunits as well as fragments of Type I enzyme. The fraction precipitating at 27% satd. (NH4)2SO4 is flavin-poor, that remaining soluble at 55% satd. (NH4)2SO4 flavin-rich and those separating between 27 and 55% satd. (NH4)2SO4 intermediate in composition. 3. The fraction remaining soluble at 55% satd. (NH4)2SO4 contains the purified low-molecular-weight iron-sulphur flavoprotein (Type-II dehydrogenase). It is a dimer consisting of one molecule of FMN, one 28-kilodalton and one 56-kilodalton subunit per protomer. Work of others indicates that it contains 4 Fe and 4 acid-labile S atoms per molecule of FMN. Sometimes the fraction remaining soluble at 55% satd. (NH4)2SO4 contained an additional small subunit (12 kilodaltons) and four additional Fe and acid labile S atoms per protomer. The sedimentation coefficients (s020,w) of the two preparations were 5.3 and 6.6 S, respectively, with calculated frictional ratios of 1.5 and 1.24, respectively. 4. The intermediate fractions are mixtures of the various subunits present in Complex I. Specifically a fraction separating at 55% satd. (NH4)2SO4 was found to be a mixture of two fragments, the pure iron-sulphur flavoprotein and a 26-S fragment that contained per protomer four subunits of 12 kilodaltons, one each of 28, 32, 56 and 77 kilodaltons, one molecule of FMN and 20 Fe and acid-labile S atoms. It was probably tetrameric or even larger. 5. The oxidoreductase activity of the intermediate fractions is dependent on the protein concentration, the activity with ferricyanide increasing and that with ferricytochrome c decreasing with increasing protein concentration. This is interpreted as an increased association of subunits present in the intermediate fractions. Similar results are obtained when flavin-rich and flavin-poor fractions are mixed. The association is cooperative. NADH favours the association of the subunits. 6. Association of the subunits is accompanied by a 10-fold increase in k2 (rate constant for intramolecular electron flow), a 10-fold decrease of the accessibility of ferricyanide to the reduced enzyme and a 10(4)-fold decrease of the accessibility of ferricytochrome c. The Ks (NADH) is also decreased. Although the changes are in the direction to be expected from a conversion of Type II enzyme to Type I, the value of k2 is still much less than in the latter enzyme.