“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.     

Identification and preliminary characterization of temperature-sensitive mutations affecting HlyB, the translocator required for the secretion of haemolysin (HlyA) from Escherichia coli

We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesis. From random mutagenesis, three mutants, temperature sensitive (Ts) for secretion, were isolated and the DNA sequenced: Glyl0Arg close to the N-terminus, Gly408Asp in a highly conserved small periplasmic loop region PIV, and Pro624Leu in another highly conserved region, within the ATP-binding region. Despite the Ts character of the Gly10 substitution, a derivative of HlyB, in which the first 25 amino acids were replaced by 21 amino acids of the Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispensable for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a conditional lethal mutation in HlyB. We have isolated 4 additional mutations in PIV by directed mutagenesis, giving a total of 5 out of 12 residues substituted in this region, with 4 mutations rendering HlyB defective in secretion. The Pro624 mutation, close to the Walker B-site for ATP binding in the cytoplasmic domain is identical to a mutation in HisP that leads to uncoupling of ATP hydrolysis from the transport of histidine. The expression of a fully functional haemolysin translocation system comprising HlyC,A,B and D increases the sensitivity of E. coli to vancomycin 2.5-fold, compared with cells expressing HlyB and HlyD alone. Thus, active translocation of HlyA renders the cells hyperpermeable to the drug. Mutations in hlyB affecting secretion could be assigned to two classes: those that restore the level of vancomycin resistance to that of E. coli not secreting HlyA and those that still confer hypersensitivity to the drug in the presence of HlyA. We propose that mutations that promote vancomycin resistance will include mutations affecting initial recognition of the secretion signal and therefore activation of a functional transport channel. Mutations that do not alter HlyA-dependent vancomycin sensitivity may, in contrast, affect later steps in the transport process.     

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.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type,Neuronal Subtype,and Ca2+ Signaling

Uptake of Ca²⁺ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca²⁺ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca²⁺ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca²⁺ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca²⁺, as well as diversity of mitochondrial Ca²⁺ uptake mechanisms.     

Legumain forms from plants and animals differ in their specificity

We purified forms of legumain from a plant source (seeds of kidney bean, Phaseolus vulgaris) and a mammal (kidney of pig, Sus scropha) for comparison of their properties. Both forms were found to be stable only under moderately acidic pH conditions, and were maximally active at about pH 6; the plant enzyme was somewhat less stable and had a slightly higher pH optimum. With benzyloxycarbonyl-Xaa-Ala-Asn-aminomethylcoumarylamide substrates, the two forms of legumain showed distinctly different specificities for the P3 residue, the plant legumain preferring amino acids with bulky hydrophobic side chains because of lower Km values. Both forms of legumain were highly specific for hydrolysis of asparaginyl bonds in the arylamide substrates and in neurotensin. Aspartyl bonds were hydrolysed about 100-fold more slowly with lower pH optima. Potential substrates containing other amino acids structurally similar to asparagine were not hydrolysed. There were clear differences in specificity of hydrolysis of protein substrates. The plant legumain differed from pig legumain in its action on tetanus toxoid C-fragment, cleaving at Asn97 but not at Asn337, and produced more extensive digestion of phaseolin. The plant form of legumain was much more weakly inhibited by egg-white cystatin than was the mammalian form.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Response of methanotrophic communities to afforestation and reforestation in New Zealand

Methanotrophs use methane (CH₄) as a carbon source. They are particularly active in temperate forest soils. However, the rate of change of CH₄ oxidation in soil with afforestation or reforestation is poorly understood. Here, soil CH₄ oxidation was examined in New Zealand volcanic soils under regenerating native forests following burning, and in a mature native forest. Results were compared with data for pasture to pine land-use change at nearby sites. We show that following soil disturbance, as little as 47 years may be needed for development of a stable methanotrophic community similar to that in the undisturbed native forest soil. Corresponding soil CH₄-oxidation rates in the regenerating forest soil have the potential to reach those of the mature forest, but climo-edaphic fators appear limiting. The observed changes in CH₄-oxidation rate were directly linked to a prior shift in methanotrophic communities, which suggests microbial control of the terrestrial CH₄ flux and identifies the need to account for this response to afforestation and reforestation in global prediction of CH₄ emission.     

Modelling environmental variation in Young's modulus for Pinus radiata and implications for determination of critical buckling height

• Background and Aims Although density-specific stiffness,E/, (where E is Young's modulus and is wood density) is oftenassumed constant by the elastic similarity model, and in determinationof critical buckling height (H_crit), few studies have testedthis assumption within species. Here this assumption is testedfor Pinus radiata growing across an environmental gradient,and theory is combined with data to develop a model of Young'smodulus. • Methods Analyses use an extensive series of environmentalplots covering the range of climatic and edaphic conditionsover which P. radiata is grown in New Zealand. Reduced majoraxis regression was used to determine scaling exponents betweenlog–log plots of H_crit vs. groundline diameter (D), andE/ vs. D. Path analysis was used to identify significant directand indirect (through stem slenderness) edaphic and climaticinfluences on E. • Key Results Density-specific stiffness exhibited 3-foldvariation. As E/ scaled positively with D, the exponent of 0·95between H_crit and D exceeded the assumed value of 0·67under constant E/. The final path analysis model included meanair temperature in early autumn (T_aut) and slenderness as significant(P < 0·05) positive direct influences on E. Tree leafarea index and T_aut were indirectly associated with E throughtheir significant (P < 0·05) positive direct relationshipwith stem slenderness. Young's modulus was most sensitive toT_aut, followed by stem slenderness then leaf area index, andthe final model explained 76 % of the variance in E. • Conclusions The findings suggest that within speciesE/ variation may influence H_crit and the scaling exponent betweenD and H_crit so important in assumptions regarding allometricrelationships. The model presented may provide a useful meansof determining variation in E, E/ and H_crit across environmentalgradients.