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Artificial cell containing superoxide dismutase--selection of folding aids for stabilisation of SOD.

Superoxide dismutase (abbreviated as SOD) has been vigorously studied in the fields of radical chemistry and related life science. One of practical problems is how to keep its activity in certain adverse conditions causing denaturation. Artificial cell containing SOD can be prepared by polymer encapsulation or nanocapsulation which has been found to be effective to improve the stability of SOD. For construction of an ideal artificial cell system, some folding aids or aggregation inhibitors were utilised to enhance SOD stability. In this study, three groups of biopolymers are selected as folding aids or aggregation inhibitors for stabilisation of SOD, i.e. albumin, carbohydrates and glycoproteins. Results indicate that the thermostability of SOD is affected by different sort of albumin while some carbohydrates such as cyclodextrins are found to be able to enhance SOD stability. In addition, it is firstly found that selected glycoproteins such as alpha-macroglobulin and ovalbumin are several types of effective folding aids for stabilisation of SOD. They can protect SOD against denaturation even at very high temperature(over 100 degrees C). The stability was tested by the measurement of SOD activity loss using autooxidation method in different adverse conditions such as high temperature, extreme pH medium, proteolytic hydrolysis and long shelf life storage. The possible stabilisation mechanism of using cyclodextrins and glycoproteins as folding aids were discussed.

Albumins↗

Centromeric and noncentromeric ADE2-selectable fragmentation vectors for yeast artificial chromosomes in AB1380.

We have constructed a set of fragmentation vectors for the truncation of either the centromeric or the noncentromeric end of YACs containing a human DNA insert. These vectors carry ADE2 or HIS5 as the selectable marker, enabling direct use in AB1380, the host strain of most publicly available YAC libraries. Centromeric fragmentation vectors for AB1380 have not been reported previously; the noncentromeric vectors show high frequencies of fragmentation.

Carboxy-Lyases↗

Genetic improvement of mastitis through selection on somatic cell count.

Heredity influences both clinical mastitis and somatic cell score. Intramammary infection is the major cause of elevated somatic cell score. A nationwide program of genetic evaluation of dairy cattle for somatic cell score is being developed. Proper selection of artificial insemination sires, considering their genetic merit for both milk production and somatic cell score, will reduce the genetic increase in mastitis susceptibility that accompanies selection for high production.

Animals↗

An artificial cell-cycle inhibitor isolated from a combinatorial library.

Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members. Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity. In contrast to naturally occurring inhibitors, such as p21(Cip1), which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity. We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive. Expression of pep8 in human cells retards their progression through the G1 phase of the cell cycle. Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G1-to-S transition. This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution. The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems.

Binding, Competitive↗

Mechanical properties of artificial teeth.

In selecting the teeth for fabrication of complete or partial dentures, each patient's anatomic and physiologic requirements and the properties of the artificial teeth themselves should be taken into consideration. The purpose of this study was to evaluate the mechanical properties of the artificial teeth by the static compression test and the impact test. Specimens were the lower first molar porcelain and resin teeth (Livdent FB-20 teeth by G. C. Co., Tokyo, Japan). All were of the same shape. In the static compression test, the fracture load and deformation of the artificial teeth were measured with an Instron-type universal testing machine at a cross-head speed of 1.0 mm/min. Elastic modulus, ultimate strength and absorbed energy were calculated. In the impact test, the acceleration of a falling impactor was measured with a drop impact apparatus. The load applied to the specimen was equivalent to 300N. Absorbed energy and deformation were calculated. The resin teeth showed a lower elastic modulus, higher fracture toughness and shock-absorbing ability than the porcelain teeth. Resin teeth should be selected when the first requisite is high shock-absorbing ability, and porcelain teeth should be selected when the first requisite is high masticating efficiency.

Dental Porcelain↗

[Selection in the context of provocation in artificial populations of Drosophila melanogaster].

The experimental potentials of the method for selection against provocative background (autoselection) in artificial populations of Drosophila melanogaster were analysed. The dynamics of adaptation of Drosophila population under increasing NaCl concentration in food was studied. It appeared that selection was successful in case of feedback between the selection pressure, mediated by alteration of salt concentration, and the rate of flies adaptation determined by the intensity of their reproduction. Within a short period of time, as the result of autoselection, the flies' ability for development with NaCl of up to 8% concentration in food was registered. It was shown that this resistance of flies to the salt was inherited within twelve successive generations.

Adaptation, Physiological↗

Monolithic beds of artificial gel antibodies.

The introductions of the continuous beds, now often called monoliths [S. Hjertén, J.-L. Liao, R. Zhang, J. Chromatogr. 473 (1989), 273-275] and the artificial, highly selective gel antibodies against antigens as large as proteins, viruses and cells [J.-L. Liao, Y. Wang, S. Hjertén, Chromatographia 42 (1996), 259-262] were breakthroughs in the design of chromatographic beds. This paper deals with a combination of these two methods, i.e., the artificial gel antibodies have been synthesized in the monolithic mode. As antigen we have used human hemoglobin. A comparison of the ion-exchange chromatograms of the eluates from the monolithic columns shows that the monolith prepared in the presence of hemoglobin adsorbed this protein, but not the other proteins in the sample (ribonuclease A and cytochrome c), i.e., this monolith was selective for hemoglobin, whereas the blank column (prepared in the absence of hemoglobin) had no selective properties, since none of the applied proteins were adsorbed. The diameter of the column was 6mm, but the same approach to synthesize a monolithic selective bed can very likely also be used for capillaries and microchips.

Antibodies↗

The effects of genetic selection for increased growth rate on mucosal and muscle weights in the different regions of the small intestine of the domestic fowl (Gallus domesticus).

1. The total organ weights and the constituent weights of mucosal and muscle tissues have been determined in the small intestines of three lines of domestic fowls subjected to different degrees of artificial genetic selection for rapid growth rates. 2. Effects of selection have been examined on the basis of wet and dry tissue weights in duodenum, jejunum and ileum from each line of birds. 3. Effects of selection have been compared on both absolute tissue weights and relative weights (i.e. expressed per kilogram body weight). 4. Selection for rapid growth rate is associated with increases in absolute weight and length of small intestine but marked reductions in relative weight and length. 5. In the fastest growing group of birds the major effect is a decrease in the relative mass of mucosa which is most pronounced in the more proximal regions of the small intestine. 6. These findings are discussed in relation to possible enhancement of intestinal digestive and absorptive efficiency in birds selected for improved growth rate and feed conversion. 7. The differences in tissue composition of the small intestine between lines and between regions of the intestine in the same line emphasise the difficulty in selecting an anatomical parameter upon which to base the in vitro and in vivo measurements of nutrient absorption in a number of established preparations.

Animals↗

Rearing management of rabbit males selected by high growth rate: the effect of diet and season on semen characteristics.

A total of sixty-six young males were used to evaluate the effect of low (L), medium (M) and high (H) concentrations of dietary digestible energy received during the rearing seasons (autumn and spring) on the performance and main semen characteristics of males for artificial insemination selected by a high growth rate. Males reared during the spring season presented a significantly higher weight at weaning than those reared during the autumn season (P < 0.001), and these differences were maintained until the end of the trial. The requirements of the males were easily covered as a general rule. In the autumn group, the males were unable to intake the digestible protein recommended only during their 3rd month of life, especially with low concentrate diets (P < 0.05). H males showed higher semen concentration and production during the autumn season, while L males showed a higher semen concentration and production than M males during the spring season, the H group showed intermediate values (P < 0.001). Males reared during the spring season showed significantly higher values of sperm concentration (P < 0.01) and production (P < 0.01). H males presented a lower percentage of spermatozoa with cytoplasmic droplets than the L group (P < 0.05) and the lowest values for sperm abnormalities during the autumn season, while the L group presented higher values for percentage abnormalities, especially during the last month controlled (P < 0.05). As a general rule, the main motility parameters controlled were not affected by the rearing diet received nor the season. These results seem to indicate that the management of rabbit males during the growing and rearing periods seem to significantly affect their subsequent performance and semen production.

Animal Feed↗

Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA.

The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.

Animals↗

Selection of molecular descriptors with artificial intelligence for the understanding of HIV-1 protease peptidomimetic inhibitors-activity.

Quantitative Structure Activity Relationship (QSAR) techniques are used routinely by computational chemists in drug discovery and development to analyze datasets of compounds. Quantitative numerical methods like Partial Least Squares (PLS) and Artificial Neural Networks (ANN) have been used on QSAR to establish correlations between molecular properties and bioactivity. However, ANN may be advantageous over PLS because it considers the interrelations of the modeled variables. This study focused on the HIV-1 Protease (HIV-1 Pr) inhibitors belonging to the peptidomimetic class of compounds. The main objective was to select molecular descriptors with the best predictive value for antiviral potency (Ki). PLS and ANN were used to predict Ki activity of HIV-1 Pr inhibitors and the results were compared. To address the issue of dimensionality reduction, Genetic Algorithms (GA) were used for variable selection and their performance was compared against that of ANN. Finally, the structure of the optimum ANN achieving the highest Pearson's-R coefficient was determined. On the basis of Pearson's-R, PLS and ANN were compared to determine which exhibits maximum performance. Training and validation of models was performed on 15 random split sets of the master dataset consisted of 231 compounds. For each compound 192 molecular descriptors were considered. The molecular structure and constant of inhibition (Ki) were selected from the NIAID database. Study findings suggested that non-covalent interactions such as hydrophobicity, shape and hydrogen bonding describe well the antiviral activity of the HIV-1 Pr compounds. The significance of lipophilicity and relationship to HIV-1 associated hyperlipidemia and lipodystrophy syndrome warrant further investigation.

Algorithms↗

Development and transmission of psoroptic mange of cattle in feed lots in endemic and non-endemic regions.

A test was begun in November 1982 to evaluate the development of Psoroptes ovis, the severity of psoroptic mange of cattle, and the incidence of transfer of P. ovis from artificially infested cattle to non-infested cattle held in feed lots in either an endemic (Bushland, TX) or non-endemic region (Kerrville, TX). The 2 herds each consisted of 24 Hereford steers. The 24 steers were randomly assigned to 3 pens of 8 steers per pen. Two steers from each pen that were randomly selected were artificially exposed to ca. 1000 P. ovis (AINF). The development of mange on these 6 AINF steers and the incidence of transfer to the other steers (INF) were monitored approximately every 3 weeks for 17 weeks. The 6 AINF steers at Bushland (endemic region) developed significantly more lesions than the 6 AINF steers at Kerrville. Three AINF steers in Bushland died from hypothermia and mange while no deaths were recorded at Kerrville. The numbers of mites sampled from affected skin areas of AINF steers from both regions were not significantly different. All 18 of the naturally exposed steers (INF) in Bushland contracted mange; whereas, only 9 INF steers in Kerrville did. The severity (% lesions) and number of mites per sample from affected skin were significantly greater on the INF steers in Bushland. These differences among INF steers in Bushland and Kerrville may be attributed to the higher level of parasitism attained in Bushland on the AINF steers. We propose that the extremely cold weather in Bushland contributed to the greater level of parasitism attained on AINF steers in Bushland by stressing the host steers.

Animals↗

Recombinant pICln forms highly cation-selective channels when reconstituted into artificial and biological membranes.

pICln has been proposed to be the swelling-activated anion channel responsible for ICl, swell, or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pICln into artificial and biological membranes. Single channels were observed when pICln was reconstituted into planar lipid bilayers. In the presence of symmetrical 300 mM KCl, the channels had a high open probability and a slope conductance of 48 pS, and were outwardly rectifying. Reduction of trans KCl to 50 mM shifted the reversal potential by -31.2 +/- 0.06 mV, demonstrating that the channel is at least seven times more selective for cations than for anions. Consistent with this finding, channel conductance was unaffected by substitution of Cl- with glutamate, but was undetectable when K+ was replaced by N-methyl-D-glucamine. Reconstitution of pICln into liposomes increased 86Rb+ uptake by three- to fourfold, but had no effect on 36Cl- uptake. Phosphorylation of pICln with casein kinase II or mutation of G54, G56, and G58 to alanine decreased channel open probability and 86Rb+ uptake. When added to the external medium bathing Sf9 cells, pICln inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pICln and not minor contaminant proteins. However, these findings do not support the hypothesis that pICln is the anion-selective ICl, swell channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pICln, or may be a consequence of in vitro reconstitution of purified, recombinant protein.

Animals↗

TOPAAS, a tomato and potato assembly assistance system for selection and finishing of bacterial artificial chromosomes.

We have developed the software package Tomato and Potato Assembly Assistance System (TOPAAS), which automates the assembly and scaffolding of contig sequences for low-coverage sequencing projects. The order of contigs predicted by TOPAAS is based on read pair information; alignments between genomic, expressed sequence tags, and bacterial artificial chromosome (BAC) end sequences; and annotated genes. The contig scaffold is used by TOPAAS for automated design of nonredundant sequence gap-flanking PCR primers. We show that TOPAAS builds reliable scaffolds for tomato (Solanum lycopersicum) and potato (Solanum tuberosum) BAC contigs that were assembled from shotgun sequences covering the target at 6- to 8-fold coverage. More than 90% of the gaps are closed by sequence PCR, based on the predicted ordering information. TOPAAS also assists the selection of large genomic insert clones from BAC libraries for walking. For this, tomato BACs are screened by automated BLAST analysis and in parallel, high-density nonselective amplified fragment length polymorphism fingerprinting is used for constructing a high-resolution BAC physical map. BLAST and amplified fragment length polymorphism analysis are then used together to determine the precise overlap. Assembly onto the seed BAC consensus confirms the BACs are properly selected for having an extremely short overlap and largest extending insert. This method will be particularly applicable where related or syntenic genomes are sequenced, as shown here for the Solanaceae, and potentially useful for the monocots Brassicaceae and Leguminosea.

Chromosomes, Artificial, Bacterial↗

The influence of plasticizer loss on the viscoelasticity of temporary soft liners.

PURPOSE: This study examined the changes in mechanical properties of temporary soft liners introduced by the differential loss of alcohol and plasticizer in different storage media. MATERIALS AND METHODS: Four brands of temporary soft liners were used to prepare 128 specimens on Lucite LS substrates. Each group of specimens was stored in a different medium (air, 10% ethanol, water, and artificial saliva), selected to affect the levels of plasticizer and alcohol over time. Data on weight changes, plasticizer leaching, and recovery rate were recorded at the following intervals: baseline, 2, 7, 14, 28, 56, and 176 days. The storage media were periodically renewed except during the drying stage between day 56 and 176. Two-way analysis of variance and Duncan multiple-range tests were performed. RESULTS: Plasticizer leaching occurs at higher levels in artificial saliva than in other storage media (P <.0001). The mean recovery rates exhibited a statistically significant increase (indicating reduced viscoelasticity) in more of the artificial saliva specimens than in all other test groups (P <.0001). Comparatively, the rates of recovery were lower in the 10% ethanol storage medium (except Lynal) with a statistically significant difference attained with the higher air-stored specimens in Tempo and Coe-Comfort (P <.0001). During the drying phase, weight changes in the 10% ethanol group were higher than those in the other storage media. CONCLUSIONS: The study supports the contention that alcohol may have been retained in the 10% ethanol group in a manner that extended the viscoelasticity of the material. The roles of alcohol and plasticizer content in the mechanical properties of temporary soft liners depend on the type of temporary soft liners and on the nature/duration of service desired. Storing dentures lined with tissue conditioners under dry conditions and those lined with true soft-liners in an ethanol humidor may be justified to extend serviceability by minimizing the loss of the component most relevant to viscoelasticity.

Air↗

[A new zoonosis--investigation of Gardnerella vaginalis disease of fox. I. Causative agent isolation and artificial infection].

It is the first time that this paper reports on using improved rabbit blood agar culture medium to isolate 145 strains Gardnerella vaginalis from the foxes and their abortion fortus organs of main fox farms. Among the strains, 26 strains were isolated from the abortion foetus organs, isolation rate was 92.86%; 118 strains were isolated from the vaginal exeretion of the abortion and empty foxes, isolation rate was 34.01%; 1 strain was isolated from inguinal lymph node of the pelted positive fox, isolation rate was 2%. None was isolated from blood. By causative agent isolation, we revealed the bacterium survival position in vivo, and the best isolation route isolation opportunity and isolation methods were selected. By artificial infection test, we have proved pathogenicity of the bacterium.

Abortion, Veterinary↗

Receptor selectivity of enkephalin analogs carrying artificial address peptides.

The synthetic [Leu]enkephalin analogs YGGFLGP(KS'S'S')2-OMe (S' represents Sar residue, ENK-S'8), YGGFLGP(KPPP)2-OMe (ENK-P8), and YGGFLGP(KA'LA')2-OMe (A' represents 2-aminoisobutyric acid (Aib), ENK-A'8) were designed to investigate the role of membrane affinity in opioid receptor binding. CD measurement in ethanol revealed that ENK-S'8 took a random coil conformation, while ENK-P8 and ENK-A'8 partly adopted a 3(1)- and alpha-helical conformation, respectively. ENK-A'8 was shown to be distributed to lipid bilayer membrane due to the amphiphilic alpha-helical structure, while ENK-S'8 and ENK-P8 stayed in aqueous phase. Opioid receptor affinities ENK-S'8 and ENK-P8 were similar to [Leu]enkephalinamide, indicating that the connection of hydrophilic peptide segments to the C-terminal of [Leu]enkephalinamide did not affect the receptor affinity. On the other hand, delta- and mu-receptor affinities of ENK-A'8 were about 1/20 and 1/6 times, respectively, those of [Leu]enkephalinamide. Therefore, ENK-A'8 has higher selectivity for mu-receptor than [Leu]enkephalinamide. These results are explained in terms of the membrane compartment concept proposed by Schwyzer.

Amino Acid Sequence↗

Towards an embryocentric world: the current and potential uses of embryo technologies in dairy production.

Structural features of the dairy industry make it well situated to use embryo technologies as tools for enhancing the genetic merit of dairy cattle and improving fertility. Technologies dependent upon embryo transfer have the potential to increase the efficiency of quantitative genetic selection as well as marker-assisted selection, simplify cross-breeding and germplasm conservation procedures and allow incorporation of transgenes into dairy cattle. In addition, embryo technologies may prove useful in improving fertility in infertile populations of lactating cows. The realisation of the promise of embryo technologies has been constrained by suboptimal efficiency in the production of embryos, alterations in embryonic and fetal survival and development associated with in vitro embryo production and cloning, as well as other technical and societal concerns. Solutions to many of these constraints are possible and the use of embryo technologies in both nucleus and commercial herds is likely to increase. Eventually, embryo transfer may compete with artificial insemination as a dominant method for establishing pregnancies in dairy cattle.

Animals↗