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Computer simulation of initial events in the biochemical mechanisms of DNA damage.

Understanding the systematic and quantitative correlation between the physical events of energy deposition by ionizing radiation and the ensuing chemical and biochemical processes leading to DNA damage is one of the goals in radiation research. Significant progress has been made toward achieving the stated goal by using theoretical modeling techniques. These techniques are strongly dependent on computer simulation procedures. A review of such techniques with details of various stages of simulation development, including a comparison with available experimental data, is presented in this article.

Base Sequence↗

A deficiency of the homeotic complex of the beetle Tribolium.

In Drosophila, the establishment of regional commitments along most of the anterior/posterior axis of the developing embryo depends on two clusters of homeotic genes: the Antennapedia complex (ANT-C) and the bithorax complex (BX-C). The red flour beetle has a single complex (HOM-C) representing the homologues of the ANT-C and BX-C in juxtaposition. Beetles trans-heterozygous for two particular HOM-C mutations spontaneously generate a large deficiency, presumably by an exchange within the common region of two overlapping inversions. Genetic and molecular results indicate that this deficiency spans at least the interval between the Deformed and abdominal-A homologues. In deficiency homozygous embryos, all gnathal, thoracic and abdominal segments develop antennal appendages, suggesting that a gene(s) has been deleted that acts to distinguish trunk from head. There is no evidence that beetles have a homologue of the segmentation gene fushi tarazu of similar genomic location and function. On the basis of the genetic tractability, convenient genome size and organization of Tribolium, and its relatively long phylogenetic divergence from Drosophila (>300 million years), we have integrated developmental genetic and molecular analyses of the HOM-C. We isolated about 70 mutations in the complex representing at least six complementation groups. The homeotic phenotypes of adults and lethal embryos lead us to believe that these beetle genes are homologous with the Drosophila genes indicated in Fig. 1 (see text).

Animals↗

A possible prebiotic synthesis of pantetheine, a precursor to coenzyme A.

The involvement of coenzyme A in many enzyme reactions suggests that it acted in this capacity very early in the development of life on Earth. Particularly relevant in this regard is its role in the activation of amino acids and hydroxy acids in the biosynthesis of some peptide antibiotics--a mechanism of peptide synthesis that forms the basis for the proposal that a thioester world could have preceded the RNA world. The components of coenzyme A have been shown to be probable prebiotic compounds: beta-alanine, pantoyl lactone and cysteamine and possibly adenosine. We show here that the pantetheine moiety of coenzyme A (which also occurs in a number of enzymes) can be synthesized in yields of several per cent by heating pantoyl lactone, beta-alanine and cysteamine at temperatures as low as 40 degrees C. These components are extremely soluble and so would have been preferentially concentrated in evaporating bodies of water, for example on beaches and at lagoon margins. Our results show that amide bonds can be formed at temperatures as low as 40 degrees C, and provide circumstantial support for the suggestion that pantetheine and coenzyme A were important in the earliest metabolic systems.

4-Butyrolactone↗

Synthesis of long prebiotic oligomers on mineral surfaces.

Most theories of the origin of biological organization assume that polymers with lengths in the range of 30-60 monomers are needed to make a genetic system viable. But it has not proved possible to synthesize plausibly prebiotic polymers this long by condensation in aqueous solution, because hydrolysis competes with polymerization. The potential of mineral surfaces to facilitate prebiotic polymerization was pointed out long ago. Here we describe a system that models prebiotic polymerization by the oligomerization of activated monomers--both nucleotides and amino acids. We find that whereas the reactions in solution produce only short oligomers (the longest typically being a 10-mer), the presence of mineral surfaces (montmorillonite for nucleotides, illite and hydroxylapatite for amino acids) induces the formation of oligomers up to 55 monomers long. These are formed by successive 'feedings' with the monomers; polymerization takes place on the mineral surfaces in a manner akin to solid-phase synthesis of biopolymers.

Amino Acids↗

Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection.

We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.

Base Sequence↗

A simple procedure for constructing 5'-amino-terminated oligodeoxynucleotides in aqueous solution.

A rapid method for the synthesis of oligodeoxynucleotides (ODNs) terminated by 5'-amino-5'-deoxythymidine is described. A 3'-phosphorylated ODN (the donor) is incubated in aqueous solution with 5'-amino- 5'-deoxythymidine in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), extending the donor by one residue via a phosphoramidate bond. Template- directed ligation of the extended donor and an acceptor ODN, followed by acid hydrolysis, yields the acceptor ODN extended by a single 5'-amino-5'-deoxythymidine residue at its 5'terminus.

Base Sequence↗

Diminished forearm vasomotor response to central hypervolemic loading in aerobically fit individuals.

The aim of this study was to test the hypothesis that cardiopulmonary baroreflex control of forearm vascular resistance (FVR) during central hypervolemic loading was less sensitive in exercise trained high fit individuals (HF) compared to untrained average fit individuals (AF). Eight AF (age: 24 +/- 1 yr and weight: 78.9 +/- 1.7 kg) and eight HF (22 +/- 1 yr 79.5 +/- 2.4 kg) voluntarily participated in the investigation. Maximal aerobic power (determined on a treadmill), plasma volume and blood volume (Evans blue dilution method) were significantly greater in the HF than AF (60.8 +/- 0.7 vs. 41.2 +/- 1.9 ml.kg-1.min-1, 3.96 +/- 0.17 vs 3.36 +/- 0.08 1, and 6.33 +/- 0.23 vs 5.28 +/- 0.13 1). Baseline heart rate (HR), central venous pressure (CVP), mean arterial pressure (MAP, measured by an intraradial catheter or a Finapres finger cuff), forearm blood flow (FBF, plethysmography), and FVR, calculated from the ratio (MAP-CVP)/FBF, were not different between the HF and the AF. Lower body negative pressure (LBNP, -5, -10, -15, and -20 torr) and passive leg elevation (LE, 50 cm) combined with lower body positive pressure (LBPP, +5, +10, and +20 torr) were utilized to elicit central hypovolemia and hypervolemia, respectively. Range of CVP (from LBNP to LE+LBPP) was similar in the AF (from -3.9 to +1.9 mm Hg) and HF (from -4.0 to +2.2 mm Hg). However, FVR/CVP was significantly less in the HF (-1.8 +/- 0.1 unit.mm Hg-1) than AF (-34 +/- 0.1 unit.mm Hg-1). The FVR decrease in response to increase in CVP was significantly diminished in the HF (-1.46 +/- 0.45 unit.mm Hg-1) compared to the AF (-4.40 +/- 0.97 unit.mm Hg-1), and during LBNP induced unloading the FVR/CVP of the HF (-2.01 +/- 0.49 unit.mm Hg-1) was less (P < 0.08) than the AF (-3.28 +/- 0.69 unit.mm Hg-1). We concluded that the cardiopulmonary baroreceptor mediated FVR reflex response was significantly less sensitive to changes in CVP in individuals who practice exercise training.

Adult↗

Binding of isolated plant lectin by rhizobia during episodes of reduced gravity obtained by parabolic flight.

Development of a legume root nodule is a complex process culminating in a plant/bacterial symbiosis possessing the capacity for biological dinitrogen fixation. Formation of root nodules is initiated by the binding and stabilization of rhizobia to plant root hairs, mediated in part by a receptor/ligand recognition system composed of lectins on the plant root surface and lectin-binding sites on the rhizobial cell surface. The dinitrogen fixation activity of these root nodules may be an important feature of enclosed, space-based life support systems, and may provide an ecological method to recycle nitrogen for amino acid production. However, the effects on nodule development of varied gravitational fields, or of root nutrient delivery hardware, remain unknown. We have investigated the effects of microgravity on root nodule formation, with preliminary experiments focused upon the receptor/ligand component. Microgravity, obtained during parabolic flight aboard NASA 930, has no apparent effect on the binding of purified lectin to rhizobia, a result that will facilitate forthcoming experiments using intact root tissues.

Fabaceae↗

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.).

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

Amino Acid Sequence↗

Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane.

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.

Animals↗

Extraterrestrial helium trapped in fullerenes in the Sudbury impact structure.

Fullerenes (C60 and C70) in the Sudbury impact structure contain trapped helium with a 3He/4He ratio of 5.5 x 10(-4) to 5.9 x 10(-4). The 3He/4He ratio exceeds the accepted solar wind value by 20 to 30 percent and is higher by an order of magnitude than the maximum reported mantle value. Terrestrial nuclear reactions or cosmic-ray bombardment are not sufficient to generate such a high ratio. The 3He/4He ratios in the Sudbury fullerenes are similar to those found in meteorites and in some interplanetary dust particles. The implication is that the helium within the C60 molecules at Sudbury is of extraterrestrial origin.

Carbon↗

Continuous in vitro evolution of catalytic function.

A population of RNA molecules that catalyze the template-directed ligation of RNA substrates was made to evolve in a continuous manner in the test tube. A simple serial transfer procedure was used to achieve approximately 300 successive rounds of catalysis and selective amplification in 52 hours. During this time, the population size was maintained against an overall dilution of 3 x 10(298). Both the catalytic rate and amplification rate of the RNAs improved substantially as a consequence of mutations that accumulated during the evolution process. Continuous in vitro evolution makes it possible to maintain laboratory "cultures" of catalytic molecules that can be perpetuated indefinitely.

Base Sequence↗

Prebiotic synthesis of 5-substituted uracils: a bridge between the RNA world and the DNA-protein world.

Under prebiotic conditions, formaldehyde adds to uracil at the C-5 position to produce 5-hydroxymethyluracil with favorable rates and equilibria. Hydroxymethyluracil adds a variety of nucleophiles, such as ammonia, glycine, guanidine, hydrogen sulfide, hydrogen cyanide, imidazole, indole, and phenol, to give 5-substituted uracils with the side chains of most of the 20 amino acids in proteins. These reactions are sufficiently robust that, if uracil had been present on the primitive Earth, then these substituted uracils would also have been present. The ribozymes of the RNA world would have included many of the functional groups found in proteins today, and their catalytic activities may have been considerably greater than presently assumed.

Amino Acids↗

In vitro chick pre-cardiac explant tissue differentiation during spaceflight on SpaceHab-02.

Chick precardiac tissue explants were cultured on the 8-day mission of STS-60, space shuttle Discovery. Development of in vitro cultures of precardiac chick tissue from embryo stages 5 though 8 (H-H) were initiated during orbit and were terminated after approximately fifteen hours of 37 degree C culture. Transmission electron microscopy and tritiated thymidine studies were performed postflight. No significant differences in cell proliferation were observed between flight and ground controls. Electron-microscopic studies revealed stage 8 explants were capable of differentiation during flight in a pattern which matched ground control tissues. As anticipated, stage 7 explant tissues had differentiated to a lesser extent compared to stage 8 tissues. Interestingly, stage 7 precardiac explant flight tissue differentiation was less than ground control tissue. This difference in differentiation between flight and ground cultures was enhanced in stage 6 tissues, as high levels of myofibril organization were only seen in ground controls. Other cellular components such as Golgi apparatus, junctional complexes, and mitochondria were present and appeared normal and healthy.

Animals↗