Historical review: another 50th anniversary--new periodicities in coiled coils

In 1953, Francis Crick and Linus Pauling both proposed models of supercoiled helices (‘coiled coils’) for the structure of keratin. These were the first attempts at modelling the tertiary structure of a protein. Crick emphasized the packing mode of the side-chains (‘knobs-into-holes’), which required a periodicity of seven residues over two helical turns (7/2) and a supercoil in the opposite sense of the constituent helices. By contrast, Pauling envisaged a broader set of periodicities (4/1, 7/2, 18/5, 15/4, 11/3) and supercoils of both senses. Crick's model became canonical and the ‘heptad repeat’ essentially synonymous with coiled coils, but 50 years later new crystal structures and protein sequences show that the less common periodicities envisaged by Pauling also occur in coiled coils, adding a variant packing mode (‘knobs-to-knobs’) to the standard model. Pauling's laboratory notebooks suggest that he searched unsuccessfully for this packing mode in 1953.     

Determination of the polymorphic composition of smooth pea starch

A method for estimating the proportions of ‘A’ and ‘B’ polymorphs comprising a sample of ‘C’ type starch is proposed which uses established experimental techniques with commercially available spreadsheet and X-ray analysis software. Waxy maize, potato and smooth pea starches were used to provide X-ray diffraction patterns characteristic of the ‘A’, ‘B’ and ‘C’ starch polymorphs. Samples of amorphous starches were also prepared. The method initially involved subtraction of the amorphous phase and instrumental background from the X-ray diffraction patterns of each starch sample using the spreadsheet program, Lotus 1-2-3. The remainder of the pattern, representing the crystalline portion of the starch sample, was then analysed by profile fitting to elucidate the positions and areas of individual diffraction peaks. The ratio of the total peak area to the areas under peaks characteristic of ‘A’ and ‘B’ type starches, respectively, were used to calculate the relative proportions of these polymorphs in smooth pea starch. These proportions were found to be 56±3% ‘A’ polymorph to 44±3% ‘B’ polymorph. A ‘C’ type pattern was constructed by using Lotus 1-2-3 to combine diffraction patterns from the crystalline portions of ‘A’ and ‘B’ type starches in the proportions given above. Polymorph patterns were obtained by manipulation of the diffraction patterns from the crystalline portions of starches using Lotus 1-2-3. An ‘A’ type pattern was obtained by subtraction of a ‘B’ type pattern from that of a ‘C’ type. Similarly, a ‘B’ type pattern was obtained by subtraction of an ‘A’ type pattern from that of a ‘C’ type.     

Comparative assessment of two algorithms for calibrating stereophotogrammetric systems

In this paper, a comparison is carried out between two photogrammetric algorithms aimed at camera calibration and three-dimensional target point reconstruction by ‘absolute’ distributions of control points; the first is Marzan and Karara's DLT, the second is CESNO, by the author, an algorithm quite close to Hatze's modified DLT (MDLT). The comparative assessment is especially aimed at testing the capability of the two methods to produce good results when calibration data are to be extrapolated beyond the space spanned by the control distribution. The assessment was carried out not by a real stereophotogrammetric system, but by the computer simulation of a two-camera set-up. Various combinations of internal camera parameters, such as the scaled principal distances, were tried out. As for the magnitude of the simulated non-linear lens distortion, three configurations were used which produced ‘low’, ‘medium’ and ‘strong’ distortion. The influence of decreasing the number of control (calibration) points on the accuracy performance of the two algorithms was also investigated. The results show the superiority of CESNO, especially with medium or strong lens distortion, or when the two camera principal distances sensibly differ.     

Intracellular signalling proteins as ‘smart’ agents in parallel distributed processes

In eucaryotic organisms, responses to external signals are mediated by a repertoire of intracellular signalling pathways that ultimately bring about the activation/inactivation of protein kinases and/or protein phosphatases. Until relatively recently, little thought had been given to the intracellular distribution of the components of these signalling pathways. However, experimental evidence from a diverse range of organisms indicates that rather than being freely distributed, many of the protein components of signalling cascades show a significant degree of spatial organisation. Here, we briefly review the roles of ‘anchor’, ‘scaffold’ and ‘adaptor’ proteins in the organisation and functioning of intracellular signalling pathways. We then consider some of the parallel distributed processing capacities of these adaptive systems. We focus on signalling proteins-both as individual ‘devices’ (agents) and as ‘networks’ (ecologies) of parallel processes. Signalling proteins are described as ‘smart thermodynamic machines’ which satisfy ‘gluing’ (functorial) roles in the information economy of the cell. This combines two information-processing views of signalling proteins. Individually, they show ‘cognitive’ capacities and collectively they integrate (cohere) cellular processes. We exploit these views by drawing comparisons between signalling proteins and verbs. This text/dialogical metaphor also helps refine our view of signalling proteins as context-sensitive information processing agents.     

Protein turnover and cell-cycle initiation in yeast

The effects of short pulses of cycloheximide on the traversal of the G1 phase of the cell cycle of the yeast Saccharomyces cerevisiae were examined. Cells were released from a block at the regulatory stage of G1, termed ‘start’, and pulsed with cycloheximide. Delays in budding which were considerably longer than the length of the pulse were observed. During the delay the cells remained blocked at ‘start’. No delay in budding was observed after cycloheximide pulses, when cells were released from a cdc 24 block which arrests the budding process but not ‘start’. Overall protein synthesis did not show an additional delay after the pulse. The extra lag following cycloheximide pulses appears to reflect a unique feature of ‘start’. It may be accounted for by a requirement at ‘start’ for a labile protein with a half-life time of about 6 min.     

Rapid multichannel microfluorometry for metabolic and spectral studies in single living cells

A multichannel microspectrofluorometer has been developed for operation on two modes, a ‘morphological’ mode for the assay of intracellular fluorochromes (e.g. NAD(P)H) in correlation with topography, and a ‘spectral’ mode for wavelength analysis of natural cell fluorescence. This instrument is based on an electron bombardment silicon camera tube (EBS) operated in conjunction with a multiscaling computer. The total NAD(P)H emission from 2 × 30 μm cell strips can be analysed in real time (32.8 msec frame scan) with a signal-to-noise ratio over 100:1. The metabolic changes in cytoplasmic regions are compared with those in regions comprising cytoplasm + nucleus, where the major contribution may be nuclear (cf earlier studies). The observation of a ‘multilocalized’ and asynchronous metabolic response is facilitated with substrates such as glucose-1-phosphate, associated with a longer lag period before the initiation of fluorescence changes. The latter largely occur in the 440–480 nm region. Fluorescence spectra recorded from intracellular regions are nearly super-posable to the spectrum obtained from NAD(P)H crystals.     

3-D attitude representation of human joints: A standardization proposal

In view of the singularities, asymmetries and other adverse properties of existing, three-dimensional definitions for joint and segment angles, the present paper proposes a new convention for unambiguous and easily interpretable, 3-D joint angles, based on the concept of the attitude ‘vector’ as derived from Euler's theorem. The suggested standard can be easily explained to non-mathematically trained clinicians, is readily implemented in software, and can be simply related to classical Cardanic/Eulerian angles. For ‘planar’ rotations about a coordinate system's axes, the proposed convention coincides with the Cardanic convention.

The attitude vector dispenses with the ‘gimbal-lock’ and non-orthogonality disadvantages of Cardanic/Eulerian conventions; therefore, its components have better metrical properties, and they are less sensitive to measurement errors and to coordinate system uncertainties than Cardanic/Eulerian angles.

A sensitivity analysis and a physical interpretation of the proposed standard are given, and some experimental results that demonstrate its advantages.     

A characteristic pharmacological action of ‘Yang-invigorating’ Chinese tonifying herbs: Enhancement of myocardial ATP-generation capacity

In order to investigate the pharmacological basis of ‘Yang-invigorating’ action, the effect of oral treatment with the methanolic extract of ‘Yang-invigorating’ herbs on ATP-generation capacity was examined, using heart homogenates prepared from herb-pretreated mice. Tonifying (i.e., health-promoting) herbs of other functional categories were also included for comparison. The results indicated that ‘Yang-invigorating’ Chinese tonifying herbs could invariably enhance myocardial ATP-generation capacity, with the extent of stimulation varying among the herbs. In contrast, ‘Yin-nourishing’ herbs either did not stimulate or even decreased myocardial ATP-generation capacity. While ‘Qi-invigorating’ herbs produced variable effects on myocardial ATP-generation capacity, most of the ‘blood-enriching’ herbs did not cause any significant changes. The results obtained from studies using myocardial mitochondrial fractions isolated from herb-pretreated mice suggest that ‘Yang-invigorating’ herbs might speed up ATP generation by increasing mitochondrial electron transport. The ensemble of results has provided evidence for the first time to support the pharmacological basis of ‘Yang invigoration’ in Chinese medicine.     

Contamination effects by a ‘conventional’ and a ‘biodegradable’ lubricant oil on infaunal recruitment to Antarctic sediments: A field experiment

To investigate the impacts of synthetic lubricants on Antarctic infaunal communities, a field experiment was setup near Australia's Casey Station, East Antarctica. Two types of synthetic lubricants were tested: an ‘Unused’ and ‘Used’ conventional synthetic lubricant, and an alternative marketed as ‘biodegradable’. Clean defaunated sediment was contaminated with the lubricants, decanted into trays, and deployed by divers onto the seabed in a randomised block design. Sediments were sampled 5 and 56 weeks after deployment. After 5 weeks, benthic assemblages that had recruited to the lubricant contaminated sediments were significantly different to those in ‘Control’ sediments, and differences were more pronounced after 56 weeks. Total number of individuals did not significantly differ between treatments after 5 weeks. However, after 56 weeks total individuals in the ‘Control’ sediments were significantly greater than in the contaminated sediments. Nototanais antarcticus (tanaid) and to a lesser extent Monoculodes sp. (gammarid), Tanaid sp. IV and Eudorella sp. (cumacean) had significantly higher abundances in the control sediments after 56 weeks compared to the contaminated sediments. Copepods numerically dominated the benthic assemblages at both sampling times; however, their abundance did not significantly differ across treatments. The community recruiting to the contaminated sediments remained different from that in the ‘Control’ sediments for the duration of the experiment (1 year). The ‘biodegradable’ lubricant was just as environmentally harmful to the Antarctic infauna as the ‘conventional’ lubricant currently used at Australia's Antarctic stations. Our results demonstrate that changes to recruitment are one of the potential environmental consequences of a lubricant spill to Antarctic benthic communities, and reinforce the importance of preventative oil spill management and effective clean-up procedures. Further monitoring of this field experiment will provide much needed information about the long-term impacts by synthetic lubricants in the Antarctic marine environment.     

Photoacoustic detection of photosynthetic activities in isolated broken chloroplasts

Methodology and demonstration how to utilize the photoacoustic technique in photosynthesis research are presented. Photoacoustic signals were obtained from suspensions of isolated broken chloroplasts. In the presence of strong, continuous (non-modulated) background light the signals were normally larger than without the background light. The effect of the background light was saturable and was absent when non-active (e.g. heat-treated) samples were used, showing that the normal smaller signal in the absence of background light is a genuine reflection of the loss of heat due to the competing photochemistry. The effect of the background light is to close the reaction-centers and hence to inhibit the photochemical process. The percent difference of the photoacoustic signal (± background light) is taken as a measure of the photochemical activity (‘photochemical loss’).

Initial results demonstrate the wavelength dependence of the ‘photochemical loss’. As expected there was a ‘red-drop’ decrease of the ‘photochemical loss’ for λ > 690 nm, when the cofactor methyl viologen was present. Surprisingly, however, there was a ‘red-rise’ increase for λ > 690 nm when no cofactor was present. These findings indicate that under the last conditions there is an unsuspected photoactivity of PS I which was not detected hitherto by the conventional techniques. The dependence on the background light intensity confirms this result. This photoactivity can be explained tentatively as a cyclic electron flow around PS I, present without any added cofactor.

Initial results on the modulation frequency dependence in the presence of electron acceptors are also demonstrated.     

Orthology,paralogy and proposed classification for paralog subtypes

The paper clears up confusions about the concepts of orthology and paralogy, particularly in cases involving gene family expansions. The terms ‘inparalog’ and ‘outparalog’ are defined to distinguish ancient paralogs from lineage-specific ones.     

Male sterility and restored fertility in annual mercuries, relations with sex differentiation

The paper summarizes the researches conducted on male sterility in Mercurialis annua. Totally sterile individuals are very scarce in the dioecious species showing as the other Mercuries, unisexual flowers devoid of rudiments of the opposite sex. From one sterile male mutant, a ‘sterile series’ was conducted and genetics was studied. Sterile, semisterile, restored fertile male lines were constructed as well as female lines containing the inducer gene of male sterility, both fertility restorers and the sensitive cytoplasm. Morphology and ontogeny of these isogenic lines were presented. Male sterile anthers (empty) present a splitted tapetum and an abnormal meiotic end. Restored fertile male lines were normal. The relative abundance of auxin and cytokinins was studied. A specific cytokinin pathway measured as a background in fertile lines, the cis-oxidized pathway characterised the ‘sterile series’. Restoration of normal meiosis and tapetum appeared for the highest quantities of cis-zeatin (669 ng instead of 192 ng/100 g fresh weight in totally sterile). Auxin quantities were abundant compared with the normal males. Gene expression in the ‘sterile series’ was also compared with the fertile lines. t-RNAs specific for normal females were expressed in the male ‘sterile series’. Hybridization kinetics and in vitro translations pf poly(A)⁺RNAs demonstrate specific sequences for each line. Comparisons between identical organs (normal fertile male/restored fertile male or normal female/female of the ‘sterile series’) exhibited nearly 10% differences. The results suggest that for stamen development, a cascade of regulators probably exists: sex genes acting on the induction of stamen or pistil, then genes for sterility/restoration of fertility acting in anthers. Fertility-sterility regulators control the synthesis of a specific cytokinin pathway. The new hormonal signals are linked to several specific genes expressed in the floral morphology characterizing each line of the ‘sterile series’.     

Effect of epiphytes on the extent of necrotic injuries of resistant and susceptible poplar clones infected with Dothichiza populea.

Poplar cuttings of a resistant clone, Populus ‘Grandis’, and susceptible clones, Populus nigra ‘Italica’ and Populus ‘Robusta’, were infected with the pathogenic fungus Dothichiza populea alone, or with the pathogen and one of five strains of epiphytes antagonistic towards it (in vitro), isolated from poplar bark. The extent of injury was examined for 28 days after infection by determining the length of necrotic patches and their area as expressed in per cent of the total area of a cutting or the area of necrotic injuries caused by the pathogen alone.

All the poplar cuttings of both the resistant and susceptible clones became diseased when infected with the pathogen alone. Surprisingly enough, however, the least affected clone was the susceptible P. ‘Robusta’, in which necrotic injuries covered 28% of the total area, as against 40% and 70% in the resistant P. ‘Grandis’ and the susceptible P. nigra ‘Italica’, respectively.

When the cuttings were infected simultaneously with Dothichiza populea and its antagonistic epiphytes, the diseased area in the resistant clone diminished by as much as two-thirds, and in the susceptible P. nigra ‘Italica’, by one-third in comparison with the area affected by the pathogen alone. In turn, in the susceptible P. ‘Robusta’ the introduction of three out of five epiphytes stimulated the growth of the pathogenic fungus producing on average a double increase in the necrotic area. The differences in the response of the pathogen to the presence of epiphytes recorded in the susceptible clones indicate a marked influence of the plant on the nature of interactions between its epiphytic microflora and the pathogen.     

Sequence dependent hydration of DNA

The transitions between the different helical conformations of DNA depend on the base sequence and the ambient conditions such as humidity and counter-ion concentration. In this study energy minimization techniques have been used to locate water molecule sites around nucleotides especially those which form hydrogen bonds between two or more nucleotide atoms and thus form solvent mediated bridges. We have studied several sequences and find that those which are known not to exist in the low hydration ‘A’ form have very similar number of bridging sites in both ‘A’ and ‘B’ conformations. Those sequences which are found in the ‘A’ conformation have considerably more bridging sites in this low hydration form than in the ‘B’ conformation. Sequence related solvent effects for a given conformation have also been analysed.     

The significance of the free energy of hydrolysis of GTP for signal-transducing and regulatory GTPases

A large number of GTP/GDP binding proteins, which in general have intrinsic and/or stimulatable GTPase activity, have been identified in recent years and are involved in a wide range of cellular regulatory and signal transducing processes. A common property of these proteins is that they exist in what is generally described as an active form when GTP is bound and an inactive (resting) form when GDP is present. Thus, the intrinsic or stimulated GTPase activity of these ‘enzymes’ serves to turn off a signal or to terminate a regulated process. It has been suggested that these proteins, together with ATPases whose prime function is to convert the free energy of ATP hydrolysis into another form of energy or into energy-requiring chemical reactions should be grouped together under the heading of ‘energyases’. In this article, this suggestion is examined from the point of view of identifying the role of the free-energy of hydrolysis of GTP in the signal-transducing or regulatory process of the GTPases. It is concluded that there is a qualitative difference between ATPases and classical GTPases, in the sense that a quantitative relationship between the free-energy of GTP hydrolysis and the appearance of this energy in a different form cannot be directly defined. The significance of the high free energy of hydrolysis is that it allows efficient transition from the active to the inactive state of GTPases in spite of the tendency of the strong interaction of the GTP-bound form with a partner molecule (‘effector’), an essential feature of their mode of action, to stabilize the GTP-bound form.     

The use of coated paramagnetic particles as a physical label in a magneto-immunoassay

An ideal label for use in an immunoassay would require no further chemical or electromagnetic stimulation prior to its detection and would be free from interference from the sample matrix. Micron sized paramagnetic particles are able to perturb magnetic fields. This perturbation can be directly detected using a suitable electronic device and is independent of the sample matrix. In this study coated paramagnetic particles were used as a physical label in a non-competitive solid phase ‘sandwich’ assay for the detection of human transferrin. The transferrin acted as a ‘biological bridge’ allowing a dose dependant immobilization of the paramagnetic particles to a polyethylene terephthalate solid phase. Quantitation of the paramagnetic label was achieved using an electronic detection system allowing a linear dose response with a femtomolar detection limit (260 fmol).     

PATHWAYS OF INCORPORATION OF FATTY ACID INTO GLYCEROLIPIDS OF THE MURINE PERIPHERAL NERVOUS SYSTEM IN VIVO: ALTERATIONS IN THE DYSMYELINATING MUTANT TREMBLER MOUSE

In vivo glycerolipid metabolism was studied in sciatic nerves of normal and Trembler mice. The results showed that two kinetically independent pathways were implicated in the labeling of diacylglycerophospholipids from [³H]palmitate: the Kennedy pathway and a ‘direct acylation’ pathway. In normal nerves, 45% of the glycerophospholipids were labeled, with a rate constant k₃ = 3.9 × 10⁻³ min⁻¹, from phosphatidic acid and diacylglycerol intermediates, themselves formed with a rate constant of k₁ = 0.24 min⁻¹ from a free ³H-fatty acid pool, FFA₁, that represents 45% of the total injected label. The remaining 55% of the glycerophospholipids were labeled from a kinetically distinct free ³H-fatty acid pool, FFA₂, with a rate constant of k₄ = 9.8 × 10⁻², via a process that does not implicate a detectably labeled metabolic intermediate (‘direct acylation’). Glycerophospholipid labeling via the Kennedy pathway in the Trembler mouse sciatic nerves was reduced to 75% of the normal level, while labeling via the ‘direct acylation’ pathway was increased 1.4-fold. The values of the rate constants for free ³H-fatty acid utilisation (k₁ and k₄) were both increased about 2.5-fold, while that of glycerophospholipid formation from diacylglycerol (k₃) was close to normal. Copyright © 1996 Elsevier Science Ltd     

Binding affinities and protein ligand complex geometries of nucleotides at the F(1) part of the mitochondrial ATP synthase obtained by ligand docking calculations

F₀F₁ ATP synthases utilize a transmembrane electrochemical potential difference to synthesize ATP from ADP and phosphate. In this work, the binding modes of ADP, ATP and ATP analogues to the catalytic sites of the F₁ part of the mitochondrial ATP synthase were investigated with ligand docking calculations. Binding geometries of ATP and ADP at the three catalytic sites agree with X-ray crystal data; their binding free energies suggest an assignment to the ‘tight’, ‘open’ and ‘loose’ states. The rates of multi-site hydrolysis for two fluorescent ATP derivatives were measured using a fluorescence assay. Reduced hydrolysis rates compared to ATP can be explained by the ligand docking calculations.     

Suppression of the transformed phenotype with retention of the viral ‘src’ gene in cell hybrids between rous sarcoma virus-transformed rat cells and untransformed mouse cells

Hybrid cell lines between untransformed mouse 3T3TK-cells and normal rat kidney (NRK) cells transformed by the B77 strain of Rous Sarcoma Virus (RSV) express a non-transformed phenotype, as determined by anchorage-dependent growth and organization of microfilament bundles. Virus rescue experiments and genetic experiments using an RSV mutant temperature-sensitive for maintenance of the transformed phenotype demonstrate that RSV is retained in the non-transformed hybrids. The action of the viral transformation gene ‘src’ therefore appears to be ‘suppressed’ in these hybrids. The suppressed hybrids generate variants in which the expression of the transformed phenotype and the ‘src’ gene is regained. This system should prove to be of value in identifying cellular genes involved in the expression of virally induced transformation.