Imaging the development of the zebrafish line

The lateral line system of the zebrafish offers the qualities of simplicity, accessibility and reproducibility which are most appropriate for the analysis of neural development and pattern formation in vertebrates. Furthermore, the transparency of the fish embryos allows the application of a variety of optical and molecular methods in the living animal, which is essential for a more complete analysis of the system.     

The role of deer in facilitating the spatial spread of the pathogen Borrelia burgdorferi

Borrelia burgdorferi is a vector-bourne zoonosis which propagates in wild populations of rodents and deer. The latter are incompetent for the pathogen but are required for the life cycle of hard-backed ticks which act as a vector for the pathogen. Increasing the diversity of hosts has previously suggested the presence of a ‘dilution effect’ in which such an increase reduces successful pathogen transmission as it increases the chance that a tick will encounter an incompetent host. This paper will produce a model which shows that whilst a dilution effect is possible for a system in which deer are the only incompetent host, this effect is not likely to be strong. Extending the population dynamics to include movement of deer into regions previously only inhabited by competent hosts, we find that, although ticks come in with the deer, there is a significant time lag before Borrelia appears.     

On the interpretation of the effect of area on the critical flicker frequency

The effects of area and intensity on the critical flicker frequency, threshold, and reaction time are considered in terms of neural net theory. An attempt is made to develop a mechanism which can account for the phenomena associated with the empirically observed laws of Ricco, Granit, Talbot, and Ferry-Porter as well as observations on reaction time and threshold. A simple model gives results which are substantially in agreement with observation except for a few apparent discrepancies. Experimental procedures are suggested which can determine whether these are apparent or real. This research was supported by the United States Air Force through the Air Force Office of Scientific Research of the Air Research and Development Command under contract No. AF 18(600)-1454. Reproduction in whole or in part is permitted for any purpose of the United States Government.     

On the origin of the genetic code and the stability of the translation apparatus

An “error theory” is developed which can be applied to determine the stability of a macromolecular translation machinery which reproduces itself. It is shown that the overall effects of a multitude of possible error versions of macromolecules can be treated statistically, and that such a statistical approach is of considerable usefulness in the theoretical treatment of complex macromolecular systems. The theory is developed within the context of a detailed treatment of the “frozen accident” hypothesis for the origin of the genetic code. A model is described which permits some thermodynamic characterization of the components involved in the code nucleation. The model also proves useful in resolving a stability “paradox” described by Orgel, which relates to the translation stability in present-day organisms and mechanisms of ageing. It indicates that any experimentally found decrease in translation accuracy with age is probably not due to an inherent instability in the translation apparatus. Relevant experiments are suggested.     

On the possibilities of diagnosis of the state of the phytoplankton photosynthetic apparatus by the method of nonlinear laser fluorimetry

The possibilities of diagnosis of the state of the phytoplankton photosynthetic apparatus by the method of nonlinear laser fluorimetry (saturation fluorimetry), which can be realized in remote sensing mode, were investigated. A procedure for the determination of the nonsaturated fluorescence parameter phi0, which is proportional to the concentration of chlorophyll a molecules, and the parameter A, which is a product of three photophysical parameters of chlorophyll a molecule in native chloroplast, was elaborated. Laboratory experiments with the axenic culture of eurihaline Thalassiosira weissflogii showed that the parameter A depends on the state of the photosynthetic apparatus of the alga, which was varied by either DCMU treatment or exposure to actinic light.     

A theoretical study of the acidification of the rhinovirus capsid

Electrostatic calculations for human rhinovirus 14 indicate that histidine-base residue pairs in the region of a beta-strand interaction between pentamers may be involved in a pH-induced process that leads to the release of viral RNA. Other picornavirus sequences are examined for these residue pairs, a subset of which is present in enteroviruses. Foot and mouth disease virus possesses one of the residue pairs, and cardioviruses, which undergo a separate pH and halide ion-induced capsid dissociation, possess none.     

The fidelity of the translation of the genetic code.

Aminoacyl-tRNA synthetases play a central role in maintaining accuracy during the translation of the genetic code. To achieve this challenging task they have to discriminate against amino acids that are very closely related not only in structure but also in chemical nature. A 'double-sieve' editing model was proposed in the late seventies to explain how two closely related amino acids may be discriminated. However, a clear understanding of this mechanism required structural information on synthetases that are faced with such a problem of amino acid discrimination. The first structural basis for the editing model came recently from the crystal structure of isoleucyl-tRNA synthetase, a class I synthetase, which has to discriminate against valine. The structure showed the presence of two catalytic sites in the same enzyme, one for activation, a coarse sieve which binds both isoleucine and valine, and another for editing, a fine sieve which binds only valine and rejects isoleucine. Another structure of the enzyme in complex with tRNA showed that the tRNA is responsible for the translocation of the misactivated amino-acid substrate from the catalytic site to the editing site. These studies were mainly focused on class I synthetases and the situation was not clear about how class II enzymes discriminate against similar amino acids. The recent structural and enzymatic studies on threonyl-tRNA synthetase, a class II enzyme, reveal how this challenging task is achieved by using a unique zinc ion in the active site as well as by employing a separate domain for specific editing activity. These studies led us to propose a model which emphasizes the mirror symmetrical approach of the two classes of enzymes and highlights that tRNA is the key player in the evolution of these class of enzymes.     

The role of the medial septum in the acoustic trachea of the cricket Gryllus bimaculatus

The auditory organs of the cricket which are situated in the front legs are joined together by a large transverse trachea which decisively influences their directional characteristics. The transverse trachea is medially divided by a septum. The importance of this septum for the localization of a sound source was tested by means of behavioural experiments in which the phonotactic movements of intact Gryllus bimaculatus females were compared quantitatively with those of the same specimen after perforation of the septum. The septal perforation does not noticeably influence locomotion in the absence of acoustic stimuli but selectively changes essential characteristics of phono taxis: 1) The animals walk in less straight lines. The oscillations around the mean course, typical of phonotaxis, are increased in amplitude, while the frequency decreases. 2) Course deviations from the direction of the sound source become more pronounced. 3) The threshold for phonotaxis is raised by about 10 dB. 4) Both the speed at which the animals walk and the proportion of time during which they are mobile are reduced. The results are discussed in relation to the role of the septum in the mechanism of sound localization, and with regard to its possible importance for the recognition of acoustic patterns.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

Echinococcus granulosus equinus: the histochemistry of the laminated layer of the hydatid cyst

The laminated layer of the hydatid cyst of E. granulosus equinus grown in mice has a polysaccharide substrate, which is not glycogen, but which possesses 1,2 glycols. This periodate-reactive component is demonstrated at the ultrastructural level using the thiosemicarbazide-silver proteinate technique which is also positive in Golgi-derived vesicles in the cytons and within the superficial cytoplasm at the parasite tissue/laminated layer interface. A carboxyl-acid mucopolysaccharide, lacking uronic and sialic acid residues, is present and weak reactions for sulphated acid mucopolysaccharide are recorded. The equivocal evidence for the presence of a chitin-like substrate is discussed. The predominantly basic protein component of the laminated layer has histochemically identifiable SS groups which are only very weakly demonstrated at the cytochemical level. Arginine and tryptophan are recorded. Lipid levels are low, and divalent cations, possibly calcium, are present.     

Absorption of phosphorus from the spermatophore through the cuticle of the bursa copulatrix of the butterfly, Calpodes ethlius

In lepidoptera the spermatophore is deposited in a bursa copulatrix, which is lined by a cuticle through which solubilized components of the spermatophore must be absorbed by the underlying epithelium. This study follows the route taken by the phosphorus which, with calcium, forms a significant portion of the spermatophore of Calpodes ethlius in the form of homogeneous, amorphous concretions. Lead nitrate was used in the primary fixative to precipitate the phosphorus in order to follow the pathway taken by it through the cuticle and bursal epithelium. The epicuticle-free pits, which cover the bursal cuticle, are identified as the points of entry, and the epicuticular filaments as major pathways for transport of phosphorus and presumably soluble molecules. Lead phosphate deposits also occurred in the epithelium extracellularly between the apical microvilli, lateral membranes and basal folds, in calcium granules and in large compound structures which appear to be made up, at least in part, of calcium granules. The confinement of the phosphorus to extracellular compartments of the epithelium suggests that its function is to be found elsewhere.     

A study of the investigatory behavior of the dairy cow

A commission to investigate the possible ingestion by dairy cattle of air-dropped wildlife baits for vaccination against sylvatic rabies provided an excellent opportunity for a detailed study of bovine investigatory behavior. Twelve unarmed baits (half of which were placed in a bag with liver slurry serving as a fox attractant) were placed in each of two pastures. A group of 9 cows was then allowed to graze freely on each of the pastures from approximately 08.00 to 15.00 h for 5 consecutive days. Continuous direct observation revealed that the tendency of the cows to approach the baits varied with the type of bait encountered, and habituated over the 5-day study. However, once an investigation was initiated, the likelihood of contacting the bait and the manner in which the bait was contacted was not readily influenced by prior exposure to the baits. The type of bait being investigated did not influence the likelihood of a cow making contact, but did influence the manner in which the bait was contacted. Flehmen, which only occurred following head contact with a bait, most commonly followed mouth or tongue contact and was less likely to be employed as cows habituated to the objects being investigated.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat.

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

Operant determination of the cage and feeder size preferences of the laying hen

The design of an apparatus which allows hens to work for feeder space or for cage size is described. Using this apparatus, it is shown that groups of 4 hens will only rarely work to obtain more than the 40-cm length of feed trough which is usually provided in Europe for a group of 4 medium hybrid hens in a deep battery cage. This implies that there is no requirement for a larger feeder.Given the opportunity, the hens chose to spend up to 50% of the day in a cage larger than their usual battery cage, but this effect was highly variable both between groups and from day to day. A more detailed study of which birds in a group work, and of what the birds do in the enlarged cage, is needed before firm conclusions about the cage-size requirements of laying hens can be made.     

Perspectives of the control of Opisthorchis invasion by the method of treating the molluskan population]

Sanitation of the mollusk population with highly effective cercaricide preparations can be used for the control of opisthorchosous infection. Low concentrations of molluskocides which are sublethal for non-infected mollusks have turned to be lethal for hyperinfected individuals. All tested molluskocides display a cercaricide effect in concentrations which are considerably lower than molluskocide ones. Low concentrations of cercaricides are toxic both for cercariae and parthenites from the liver of mollusks and for freely swimming cercariae. Disastrously affecting the freely swimming cercariae microdoses of cercaricides prevent the infection of the second intermediate hosts. Microdoses of cercaricides which are lethal for cercariae are not toxic for the majority of hydrobionts including fishes of all age groups. As far as the dynamics of the infection level of Bithynia with O. felineus is characterized by a one-peak curve with a distinct peak in the middle-end of July the treatment of water bodies with microdoses of cercaricides should be carried out just in this period.     

Repression of the Histidine Operon: Effect of the First Enzyme on the Kinetics of Repression

Kinetic studies on repression of the enzymes for histidine biosynthesis in Salmonella typhimurium showed that, upon addition of histidine to a derepressed culture, the enzymes became repressed in a temporal sequence which corresponds with the positional sequence of the genes in the histidine operon. This serial pattern of repression occurred under conditions in which the feedback site of the first enzyme for histidine biosynthesis is intact. When this site was rendered nonfunctional the pattern of repression was changed so that all of the enzymes became repressed concomitantly. These results suggest that the first enzyme for histidine biosynthesis plays a hitherto unrecognized role in control of the histidine system.     

The p127 subunit (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5

The V protein of simian virus 5 (SV5) blocks interferon signaling by targeting STAT1 for proteasome-mediated degradation. Here we present three main pieces of evidence which demonstrate that the p127 subunit (DDB1) of the UV damage-specific DNA binding protein (DDB) plays a central role in this degradation process. First, the V protein of an SV5 mutant which fails to target STAT1 for degradation does not bind DDB1. Second, mutations in the N and C termini of V which abolish the binding of V to DDB1 also prevent V from blocking interferon (IFN) signaling. Third, treatment of HeLa/SV5-V cells, which constitutively express the V protein of SV5 and thus lack STAT1, with short interfering RNAs specific for DDB1 resulted in a reduction in DDB1 levels with a concomitant increase in STAT1 levels and a restoration of IFN signaling. Furthermore, STAT1 is degraded in GM02415 (2RO) cells, which have a mutation in DDB2 (the p48 subunit of DDB) which abolishes its ability to interact with DDB1, thereby demonstrating that the role of DDB1 in STAT1 degradation is independent of its association with DDB2. Evidence is also presented which demonstrates that STAT2 is required for the degradation of STAT1 by SV5. These results suggest that DDB1, STAT1, STAT2, and V may form part of a large multiprotein complex which leads to the targeted degradation of STAT1 by the proteasome.     

Evaluating the stability of the classification of community data

We propose a method for a posteriori evaluation of classification stability which compares the classification of sites in the original data set (a matrix of species by sites) with classifications of subsets of its sites created by without‐replacement bootstrap resampling. Site assignments to clusters of the original classification and to clusters of the classification of each subset are compared using Goodman‐Kruskal's lambda index. Many resampled subsets are classified and the mean of lambda values calculated for the classifications of these subsets is used as an estimation of classification stability. Furthermore, the mean of the lambda values based on different resampled subsets, calculated for each site of the data set separately, can be used as a measure of the influence of particular sites on classification stability. This method was tested on several artificial data sets classified by commonly used clustering methods and on a real data set of forest vegetation plots. Its strength lies in the ability to distinguish classifications which reflect robust patterns of community differentiation from unstable classifications of more continuous patterns. In addition, it can identify sites within each cluster which have a transitional species composition with respect to other clusters.     

The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters

We constructed a library of synthetic promoters for Lactococcus lactis in which the known consensus sequences were kept constant while the sequences of the separating spacers were randomized. The library consists of 38 promoters which differ in strength from 0.3 up to more than 2,000 relative units, the latter among the strongest promoters known for this organism. The ranking of the promoter activities was somewhat different when assayed in Escherichia coli, but the promoters are efficient for modulating gene expression in this bacterium as well. DNA sequencing revealed that the weaker promoters (which had activities below 5 relative units) all had changes either in the consensus sequences or in the length of the spacer between the −35 and −10 sequences. The promoters in which those features were conserved had activities from 5 to 2,050 U, which shows that by randomizing the spacers, at least a 400-fold change in activity can be obtained. Interestingly, the entire range of promoter activities is covered in small steps of activity increase, which makes these promoters very suitable for quantitative physiological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories.