Lipase from Candida paralipolytica

The extracellular lipase from Candida paralipolytica required alkaline earth metal ion as the cofactor*² in the reaction mixture not emulsified but dispersed by shaking, contradicting the fact that it required bile salt or anionic surfactant as the essential activator*³ in the systems emulsified with polyvinyl alcohol, as previously reported.¹) The two kinds of factors necessary to activate both reaction systems respectively were unexchangeable for each other. These facts would be direct evidences of the difference of interfacial nature between two substrate forms prepared from the same substrate.

The zero-order reaction has been observed under the non-emulsified conditions and the activation mechanism by alkaline earth metal ions has been studied partly.     

Analysis of the hexagonal II phase and its relations to lipidic particles and the lamellar phase A freeze-fracture study

Model systems of phosphatidylethanolamine (PE) and cardiolipin (DPG), as pure components and in binary mixtures with phosphatidylcholine (PC) have been morphologically analysed. The relation between the hexagonal_II (H_II) phase and lipidic particles as well as between the H_II phase and the lamellar phase has been studied. Moreover, the periodicity of the various H_II tubes was determined. (1) The periodicity of the H_II phase of cardiolipin is dependent on the cation involved. DPG-Ca exhibits the smallest tube to tube distance when compared to Mg²⁺ and Mn²⁺. Moreover, the DPG-Ca tubes are quite straight, in contrast to the Mg²⁺ and Mn²⁺ tubes, which appear to be frequently curved. (2) H_II tubes with two distinct diameters have been observed in H_II phase containing lipid mixtures. The thickness of the H_II tube is related to the composition of the tube. In the cardiolipin-lecithin system, structural separation of the pure cardiolipin H_II phase has been suggested with Mg²⁺ and Mn²⁺, but not with Ca²⁺. (3) Models for the H_II to lamellar phase transition and for the H_II phase to the lipidic particles are presented. (4) Lipidic particles are exclusively found in lipid model systems, which contain H_II phase favouring lipids. Morphological evidence is presented which suggests these lipidic particles represent inverted micelles. These observations include: (${}_{\mathrm{<mtext>i</mtext>}}$) there is a strong topological and quantitative relation between H_II tubes and lipidic particles, (${}_{\mathrm{<mtext>ii</mtext>}}$) lipidic particles occur densely packed in conglomerates without the presence of a smooth layer.     

An Antigen in Human Breast Cancer Sera related to the Murine Mammary Tumour Virus

THE possibility of an oncogenic virus in human breast cancer has been increased by recent findings. Virus-like particles resembling the mammary tumour virus (MTV) were found in electron micrographs of human breast cancer tissue¹ and particles physically and morphologically similar to MTV particles have been seen in human milk². These particles were found more frequently in the milk of American women with a history of breast cancer in their immediate families and in the milk of Parsi women in Bombay than in the milk of nonselected American women³. Parsi women are three times more likely to have breast cancer than other women in Bombay³. The detection of RNA-dependent DNA-polymer-ase activity in such particles isolated from human milk emphasized the possibility that these particles represent an oncogenic RNA virus⁴. In addition to the physical and morphological resemblance of human milk particles and MTV an immunological relationship between these two kinds of particles seems probable; sera from breast cancer patients neutralize the biological activity of MTV whereas normal human sera did not do so⁵. We report data supporting the hypothesis of an immunological cross relationship between MTV and human breast cancer.     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

Utilization of enzyme markers to determine the location of plasma membrane from Pisum epicotyls on sucrose gradients

Using linear sucrose gradients, particulates derived from pea (Pisum sativum L. cv. Alaska) epicotyls have been fractionated and examined for marker enzyme activity. The coincidence of three reputed plasma-membrane markers [cellulase (EC 3.2.1.4), K⁺-stimulated Mg²⁺-ATPase, and glucan synthetase] at the same position on sucrose density gradients, in combination with electron microscopic evidence reported by G. Shore and G. Maclachlan (J. Cell Biol. 64, 557–571; 1975), indicates that plasma membrane of pea epicotyl has a buoyant density of about 1.13 g/cm³. This density disagrees with those usually reported for plant plasma membranes and also with recent reports for Pisum. It is, however, shown to be distinct from the equilibrium densities of enzymic markers for particulate components derived from Pisum endoplasmic reticulum (1.10–1.11 g/cm³), Golgi (1.12 g/cm³) and mitochondria (1.18 g/cm³). Furthermore, other recent literature indicates that the 1.13 g/cm³ buoyant density may be characteristic of the plasma membrane of many members of the Leguminosae. Our data indicate that the conditions of differential centrifugation (time, centrifugal force), coupled with the amount of protein utilized, affect the resolution and interpretation of profiles of marker enzymes on sucrose gradients (e.g. glucan synthetase and K⁺-stimulated Mg²⁺-ATPase were sometimes found to be associated not only with particles of 1.13 g/cm³ density, but with particles of higher densities as well). Particulate cellulase was found to be associated only with particles with equilibrium densities of about 1.13 g/cm³. Cellulase thus proved to be the most useful marker for establishing a differential centrifugation regime which would permit examination of the 1.13 g/cm³ particulate components with minimal contamination by particles of higher densities.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

Detection of Differences in Surface-charge-associated Properties of Cells by Partition in Two-polymer Aqueous Phase Systems

WHEN aqueous solutions of two polymers are mixed in certain proportions they may form two-phase systems^1,2 which can be buffered and used to partition and separate cells, particles and macromolecules by countercurrent distribution (CCD). Partition generally depends on polymer composition and concentration, the ionic composition and the charge sign of the material being partitioned. Such systems have been used to separate erythrocytes from white cells and erythrocytes on the basis of age. Changes in the surface properties of cells resulting from enzyme treatment or storage have also been demonstrated by this means³. Higher cell partition often accompanies increasing electrophoretic mobility which suggests that surface charge may be an important factor in partitioning^4–6. An apparent exception to this is the increased partition of stored human erythrocytes as compared with fresh⁷, as opposed to the mean electrophoretic mobility of both cell populations which remain identical⁸.     

Messenger RNA Nuclear Particles and their Attachment to Cytoplasmic Membranes in Krebs Tumour Cells

THE observation that some nuclear particles contain DNA-like RNA (D-RNA) supports the idea that messenger RNA (mRNA) species bind to specific proteins to form ribonucleoprotein complexes^1–4. The mechanism by which the nuclear particles are transported and eventually bind to ribosomes in a translation complex is still uncertain^5–9. We now present evidence that D-RNA nuclear particles of about 40–50S contain mRNA species which are transported into the cytoplasm and assemble on the network of the endoplasmic reticulum (ER). Ribosomes then bind to this membrane-bound mRNA to form a translation complex. The concept of membrane-bound mRNA has been suggested before¹⁰ but has not been conclusively demonstrated.     

High-frequency transduction of pBR322 by cytosine-substituted T4 bacteriophage: evidence for encapsulation and transfer of head-to-tail plasmid concatemers

Transduction of plasmid pBR322 by cytosine-substituted T4 phages has been studied. Three T4 phage mutants which substitute cytosine for all of hydroxymethylcytosine residues in the DNA, were shown to transduce pBR322 at frequencies of 2 × 10^?2 to 4 × 10^?3 transductants per singly infected cell. Also, three T4 phage strains which partially substitute cytosine for hydroxymethylcytosine, transduced pBR322 at frequencies of 2 × 10^?3 to 2 × 10^?4. The transduction frequencies of pBR322 we attained are at least 10-fold higher than those reported by G. G. Wilson, K. Young, and G. J. Edlin (1979, Nature (London)280, 80–82). We found that multiplicity of infection in preparation of the transducing phage is the most important factor affecting the frequency of pBR322 transduction. When a lysate made at a multiplicity of infection ranging from 0.5 to 0.05 was used as the donor phage, transduction frequency of pBR322 was 10- to 40-fold higher than that of high-m.o.i. lysate. The transduction frequency was not affected by either restriction systems or amber suppressors of the recipient cells. However, no pBR322-containing transductant was obtained when either recA or polA mutants were used as the recipients. DNA from T4dC phage containing pBR322-transducing particles was analyzed on agarose gel electrophoresis after cleavage with restriction endonucleases. It was suggested that the pBR322 DNA in the T4dC phage particles exists as head-to-tail concatemers.     

Seed dispersal of the hemiparasitic plant Thesium chinense by Tetramorium tsushimae and Pristomyrmex punctatus

Heterotrophic plants normally produce a vast number of dust‐like seeds containing minimal energy reserves, which are usually wind dispersed. However, some heterotrophic species have evolved adaptive strategies to use zoochorous seed dispersal. Seed dispersal by ants, known as myrmecochory, is one of the most widespread animal dispersal systems and has been reported in a diverse range of plant taxa. However, the combination of myrmecochory and heterotrophy seems to be very rare. Here I report the discovery of myrmecochory in the hemiparasitic plant Thesium chinense by Tetramorium tsushimae and Pristomyrmex punctatus. Myrmecochory would be an advantageous dispersal system for T. chinense because its fruits are quickly transferred to the ants' nests, which provide a refuge from the seed predator Canthophorus niveimarginatus. Myrmecochory is also potentially beneficial for T. chinense, as the nests of these ants are frequently located close to poaceous plants, which are the preferred hosts.     

Dissociation and Reassociation of Infectious Poliovirus Particles

THE first reconstitution of an infectious virion was achieved when Fraenkel-Conrat and Williams¹ obtained the typical rods of tobacco mosaic virus (TMV) from its components, RNA and protein. Later, the conditions for the “self-assembly” of TMV were improved so that up to 50% of the viral RNA could be coated with the protein. The reconstituted TMV was shown to be infectious and indistinguishable from native virions by several criteria². Recent studies revealed that the reconstitution of TMV is a highly specific multi-step procedure, beginning at the 5′-end of the TMV-RNA^3–5. In the past five years a number of spherical plant viruses have also been reconstituted⁶. In experiments with small RNA-bacteriophages very low efficiencies of reconstitution in the range of 10^–8 to 10^–7 p.f.u. (plaque forming units) per input molecule of RNA were obtained^7,8. Reconstitution was improved by the addition of a minor viral protein, the A-protein, to the mixture of RNA and the structural protein. Even so the efficiency of conversion of RNA into infectious particles was in the range of 2×10^–6 (refs. 9 and 10). We have reported the first successful restoration of poliovirus infectivity lost on dissociation of the virion by urea-mercapto-ethanol treatment¹¹. Here we present evidence for reconstitution of infectious poliovirus particles from a mixture of poliovirus RNA and polypeptides.     

The margination propensity of spherical particles for vascular targeting in the microcirculation

The propensity of circulating particles to drift laterally towards the vessel walls (margination) in the microcirculation has been experimentally studied using a parallel plate flow chamber. Fluorescent polystyrene particles, with a relative density to water of just 50 g/cm ³comparable with that of liposomal or polymeric nanoparticles used in drug delivery and bio-imaging, have been used with a diameter spanning over three order of magnitudes from 50 nm up to 10 μm. The number of particles marginating per unit surface have been measured through confocal fluorescent microscopy for a horizontal chamber, and the corresponding total volume of particles has been calculated. Scaling laws have been derived as a function of the particle diameter d. In horizontal capillaries, margination is mainly due to the gravitational force for particles with d > 200 nm and increases with d ⁴; whereas for smaller particles increases with d ³. In vertical capillaries, since the particles are heavier than the fluid they would tend to marginate towards the walls in downward flows and towards the center in upward flows, with increasing with d ^9/2. However, the margination in vertical capillaries is predicted to be much smaller than in horizontal capillaries. These results suggest that, for particles circulating in an external field of volume forces (gravitation or magnetic), the strategy of using larger particles designed to marginate and adhere firmly to the vascular walls under flow could be more effective than that of using particles sufficiently small (d < 200 nm) to hopefully cross a discontinuous endothelium.     

Polymorphism in Human Casein

MANY human proteins exist in more than one genetically determined variety¹, but no polymorphism has been reported in human milk proteins, although some components have been sufficiently characterized from the biochemical standpoint^2,3 and extensive polymorphism has been detected in cow's milk. We therefore decided to investigate the possible occurrence of polymorphic variants of human casein, since electrophoresis has now given a clear picture of bovine casein genetics, which can be used as a model for study of the situation in man^4,5. Such variants would obviously be more easily used as genetic markers at the population than at the family level. Moreover, data reported by Grosclaude et al.⁶ suggest the existence of a supergene controlling bovine α_s1, β and k-caseins. For this reason, the possibility of a similar genetic control of human polymorphism (if any) was also tested.     

Campestroside,a new tetrahydroxanthone glucoside from Gentiana campestris

Campestroside has been isolated from the aerial parts of Gentiana campestris. From UV, MS, ¹H-NMR and ¹³C-NMR data its structure has been established as 1,3,5-trihydroxy-8-β-d-glucopyranosyl-5,6,7,8-tetrahydroxanthone. Campestroside which has also been detected in G. ramosa and G. germanica is the first reported tetrahydroxanthone glycoside.     

Perspectives in S-100 protein biology: Review article

The S-100 protein family constitutes a subgroup of Ca²⁺-binding proteins of the EF-hand type comprising three dimeric isoforms, S-100a₀, S-100a and S-100b, plus a number of structurally related proteins displaying 28–55% homology with S-100 subunits. S-100 protein was discovered in 1965; yet, its biological functions have not been fully elucidated. The present report will review the putative biological roles of S-100 protein. Both intracellular and extracellular roles have been proposed for S-100 protein. Within cells, S-100 protein has been reported to regulate protein phosphorylation, ATPase, adenylate cyclase, and aldolase activities and Ca²⁺-induced Ca²⁺ release. Also, cytoskeletal systems, namely microtubules and microfilaments have been reported to be regulated by the protein in the presence of Ca²⁺. Some molecular targets of S-100 protein within cells, have been identified. This is the case with microtubule proteins, caldesmon, and a brain aldolase. S-100 protein has been reported to be secreted; extracellular S-100 protein can stimulate neuronal differentiation, glial proliferation, and prolactin secretion. However, the mechanisms by which S-100 is secreted and stimulates the above processes are largely unknown. Future research should characterize these latter aspects of S-100 biology and find out the linkage between its intracellular effects and its extracellular activities.     

Conformational change of core particles studied by quasielastic light scattering

The diffusion translational coefficient D_T of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10⁻⁴ to 2M Na⁺ or K⁺. The observed values of D_T are independent of particle concentration in the range 0.1–2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40°–120°. When the salinity is progressively raised an increase of D_T from 1.9.10⁻⁷ cm²s⁻¹ to 3.2.10⁻⁷ cm²s⁻¹ was observed at about 2.10⁻³ M NaCl followed by a decrease of D_T beyond 0.6 M NaCl.The various possible causes of the changes of D_T such as interactions between particles or between particles and salt ions are discussed. We show that the single low ionic strength change is due to a conformational transition of the core particles, while the second variation of D_T accompanies the disorganization of the core particles.     

A yeast arginine specific tRNA is a remnant aspartate acceptor

High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNA^Asp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA₄^Arg. This tRNA has a sequence strikingly similar to that of tRNA^Asp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA₄^Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA₄^Arg into an aspartate acceptor, as efficient as tRNA^Asp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA₄^Arg. The latent aspartate acceptor capacity in a contemporary tRNA^Arg leads to the proposal of an evolutionary link between tRNA₄^Arg and tRNA^Asp genes.     

RNA Dependent DNA Polymerase in C Type Particles from Normal Rat Thymus Cultures

THE isolation and characterization of C type particles released from normal rat thymus cell cultures has been described^{1, 2}. The detection of RNA dependent DNA polymerase activity in these C type particles is reported here. Some properties of the enzymatic activities found in C type particles isolated from normal rat thymus cultures are described and compared with DNA polymerase found in isolated Moloney leukaemia virus (MLV) particles purified from the growth media of rat thymus cultures infected chronically with MLV². The sensitivity to actinomycin D is different in the two kinds of C type particles, suggesting that the DNA polymerases are not identical.     

Conductance studies on the interaction of sucrose with symmetrical tetraalkylammonium halides in formamide

The interaction of sucrose with symmetrical tetraalkylammoniurn halides (R₄NX) in formamide has been studied by employing conductance measurements. Conductance data of these salts in formamide saturated by sucrose at 50.0 ±0.05° are reported in the temperature range 25 to 70°. Plots of —log (specific conductance) against 1/T showed a break at the saturation temperature, where two straight lines intersect one another. Divergence of the pairs of straight lines in these homogeneous, ternary systems was found to be highly influenced by the structure-making or -breaking properties of the electrolytes. The results are interpreted in terms of the structural properties of the electrolytes and the hydrogen-bonding capabilities of the formamide and sucrose molecules. A similarity in the conductance behavior of R₄N⁺ ions in both aqueous and formamide solutions containing sucrose at saturation concentration has been observed. The transitional effect is more pronounced for R₄N⁺ ions in formamide than in aqueous systems, and this is attributed to the less-structured nature and higher dielectric constant of formamide.     

Hormone-activated Expression of the C-type RNA Tumour Virus Genome

THE concept of vertical transmission of specific viral information, particularly that possibly associated with the induction of malignancy in mice has been postulated. Moreover, it has been hypothesized that this genetic information may be expressed either in the form of whole virus in certain selected laboratory animal strains or as the operon involved in regulating cellular replication (oncogene)¹. To detect this proposed genetically transmitted message, one uses a group specific antisera against the C-type RNA tumour viruses having as one of its components, gs-3, first described by Gerring et al.². A similar group specific antigen has subsequently been reported by Schafer³, Gilden⁴ and Sarma et al.⁵ and designated “interspec”, meaning that the antigen is common to the internal components of the C-type RNA virion of several mammalian species.