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[Recapitulatory significance of coeloblastulas].

The problem is discussed whether in Metasoa evolution there was a blastea stage, or else they were of parenchymal structure from the very beginning. The author favours the colonal theory in Metasoa origin and, as their progressive form, he considers spherical colonies where the cells are not united by intracellular substance but have immediate contact with each other. There are two ways of flagellar cells union: by their lateral surfaces or by basal ends. With the first way of union, a rather large colony acquires inevitably the form of blastea. With the second way of union, the number of cells increases, the colony becomes either a homogenous parenchimella or a blastea. The possibility of both ways of evolutionary development were studied experimentally. The results of artificial mutant selection of zootrophic colonial Euglena, Anthophysa vegetans are represented. The author comes to the following conclusions from the results obtained: 1. In Metosoa phylogenesis a blastea-like stage should be present. 2. Recapitulatory significance of the coeloblastulas is evident.

Animals↗

-Entrance of PCR for studying murine coronaviruses-.

This paper describes a number of applications of the Polymerase Chain Reaction (PCR) in the study of enterotropic murine coronaviruses [mouse hepatitis virus (MHV)]. A diagnostic PCR was developed which detected all of 11 different MHV strains. This fast and reliable method was also able to differentiate MHV from other non-murine coronaviruses. On the basis of this assay a quantitative PCR was designed using a mutant template containing a point mutation which competed for the PCR primers. The amplification and cloning of the structural protein genes of enterotropic MHV strains it plasmid vectors for subsequent sequencing is described. In addition an RT PCR was developed which was able to selectivity detect artificially generated recombinant coronavirus.

Animals↗

Modification of bacterial artificial chromosome clones using Cre recombinase: introduction of selectable markers for expression in eukaryotic cells.

Bacterial artificial chromosome clones (BACs) are widely used at present in human genome physical mapping projects. To extend the utility of these clones for functional genomic studies, we have devised a method to modify BACs using Cre recombinase to introduce a gene cassette into the loxP sequence, which is present in the vector portion of the BAC clone. Cre-mediated integration is site specific and thus maintains the integrity of the genomic insert sequences, while eliminating the steps that are involved in restriction digest-based DNA cloning strategies. The success of this method depends on the use of a DNA construct, RETRObac, which contains the reporter marker green fluorescent protein (GFP) and the selectable marker neomycin phosphotransferase (neo), but does not contain a bacterial origin of replication. BAC clones have been modified successfully using this method and the genomic insert shows no signs of deletions or rearrangements. Transfection efficiencies of the modified BACs into human or murine cell lines ranged from 1% to 6%. After culture in media containing G418 for 3 weeks, approximately 0. 1% of cells previously sorted for GFP expression acquired stable antibiotic resistance. Introduction of a human BAC clone that contains genomic p53 sequences into murine NIH3T3 cells led to expression of human p53 mRNA as determined by RT-PCR, demonstrating that sequences contained on the BAC are expressed. We believe that GFP-neo modified BAC clones will be a valuable resource in efforts to study biological effects of known genes as well as in efforts to clone and analyze new genes and regulatory regions.

Animals↗

Response of the Cu(II) ion selective electrode to Cu titration in artificial and natural shore seawater and in the measurement of the Cu complexation capacity.

The Orion 94-29 Cu(II) jalpaite ion selective electrode (Cu-ISE) was used to measure both the concentration of the aqueous free Cu(II) ion ([Cu(II)aq]) and its changes due to additions of Cu, in artificial seawater (ASW) and in seawater from San Diego Bay, CA. The range of free copper ion (i.e., pCu, -log [Cu(II)aq]) determined in seawater samples from the San Diego Bay area (11.3-12.6, 11.9 +/- 0.4, average +/- SD) is consistent with that previously reported for estuarine and coastal areas (10.9-14.1). The changes in [Cu(II)aq] as a result of the additions of Cu were used to determine the Cu complexation capacity (Cu-CC), which has a measured range (2.7 x 10(-8)-2.0 x 10(-7) M; 7.6 x 10(-8) +/- 4.8 x 10(-8) M) comparable to the range of values previously reported for estuarine and coastal zones (i.e., L1+L2, 1.1 x 10(-8)-2.0 x 10(-7) M). The narrow range of pCu at the Cu-CC (pCuCu-CC, 11.1-11.9, 11.5 +/- 0.2) indicates the predominant role of the Cu-CC in regulating the concentration of ambient Cu(II)aq to a level < or =1 x 10(-11) M Cu(II)aq. These results attest to the capability of the Cu-ISE to measure pCu and Cu-CC in aquatic coastal environments with relatively high total Cu concentrations and organic loads, such as those from heavily used coasts and bays.

Animals↗

An artificial six-zinc finger peptide with polyarginine linker: selective binding to the discontinuous DNA sequences.

Artificial DNA binding peptides recognizing separated sequences would expand varieties of the target genes for desirable transcriptional control. Here we demonstrated that polyarginine linker between two 3-zinc finger domains gives DNA binding selectivity to the separated target sequences. We created a six-zinc finger peptide, Sp1ZF6(Arg)8, by connecting two DNA binding domains of transcription factor Sp1 with a bulky and cationic polyarginine linker. The DNA binding properties to continuous and discontinuous target sequences were examined and compared to those of Sp1ZF6(Gly)10 containing a flexible and neutral polyglycine linker. The dissociation constants indicate that Sp1ZF6(Arg)8 has an obvious DNA binding preference to discontinuous target sequences but not Sp1ZF6(Gly)10. Footprinting analyses also showed that Sp1ZF6(Arg)8 binds properly only to the discontinuous target sites, while Sp1ZF6(Gly)10 does not distinguish them. The results provide helpful information for linker design of future zinc finger peptides to various states of DNA as gene expression regulators.

Amino Acid Sequence↗

Selection of high fertility donors for artificial insemination programmes.

One hundred and seventy-seven semen donors were used in an artificial insemination service using frozen semen over a 4 year period. Although the donors were selected for good semen quality, pregnancy rates varied markedly from less than five to greater than 20 pregnancies per 100 treatment cycles. When the characteristics of the 25 most fertile and 25 least fertile donors were compared, only sperm morphology was significantly different. Sperm concentration and motility before and after thawing of semen were similar in the two groups as were the proportions of adverse prognostic factors in the recipients. It is concluded that using more stringent criteria for selection of donors on the basis of sperm morphology and discarding donors who do not produce pregnancies within 12 cycles should improve overall pregnancy rates.

Adolescent↗

Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases.

Animals↗

Selective adhesion of endothelial cells to artificial membranes with a synthetic RGD-lipopeptide.

A constrained cyclic ArgGly-Asp-D-Phe-Lys, abbreviated as cyclo(-RGDfK-), lipopeptide has been synthesized and incorporated into artificial membranes such as giant vesicles with DOPC and solid-supported lipid bilayers. The selective adhesion and spreading of endothelial cells of the human umbilical cord on solids functionalized by membranes with this RGD-lipopeptide have been observed. Furthermore, we have demonstrated strong selective adhesion of giant vesicles to endothelial cells through local adhesion domains by combined application of hydrodynamic flow field and reflection interference contrast microscopy (RICM). The adhesion can be inhibited by competition with a water-soluble RGD peptide. We suggest that this strategy could improve the efficiency of liposomes targeting used as vectors or as drug carriers to cells.

Cell Adhesion↗

[Teeth arrangement of anterior artificial teeth. 1. Age and teeth exposure].

In selecting and arranging anterior artificial teeth for edentulous patients, it is necessary to consider their sex, personality, age and other factors. The relation of upper and lower lip lines to incisal edge has been one of the standards for the selection and the arrangement of anterior artificial teeth. The purpose of this report was to establish and to evaluate decision bases for the selection and arrangement. We recorded and measured the exposure rate of anterior natural teeth (48 men, 46 women) during function, such as smile, pronunciation, by means of the image analysis. Following results were obtained. 1. As the age of patients increased, upper anterior teeth appeared less than lower's. 2. Differences in exposure ratio between upper and lower teeth were found more remarkable in smiling rather than in pronunciation.

Adult↗

Tuning and dissecting electronic and steric effects in ammonium receptors: nonactin vs artificial receptors.

Ion selective electrodes (ISE) based on three different tripodal receptors (5, 6, and 7) have been investigated for sensing ammonium ion. Each receptor is based on three pyrazole groups that can accept three H-bonds from the bound ammonium ion. The receptor based on 4-bromo-3,5-dimethylpyrazole (6) is the most sensitive with a detection limit for ammonium ion of 2.5 x 10(-5) M at pH 8. The detection limits for the receptors based on 2,3-dimethylpyrazole (5) and unsubstitued pyrazole (7) are 1.0 x 10(-4) and 2.0 x 10(-4) M, respectively. The selectivities of the receptors 5, 6, and 7 for sensing ammonium ion over potassium ion (logK(NH)4+(/K)+) are -2.8, -2.3, and -1.7, respectively. In contrast, the detection limit and the selectivity of a nonactin-based ISE are 2.2 x 10(-5) M and -1.3, respectively. Crystallographic studies reveal that 6 accepts three H-bonds from the bound ammonium and singly protonated receptor 5 forms three H-bonds with the bound water molecule.

Anti-Bacterial Agents↗

Electronmicroscopic changes in selected vital organs after long-term total artificial heart pumping in animal experiments.

Electronmicroscopic (EM) evaluation of selected vital organs (liver, kidney, lung) informs us about otherwise hardly detectable changes during total artificial heart (TAH) pumping. In our experiments, we compared 2 groups of long-surviving animals in which the TAH TNS-BRNO pneumatic device was implanted (TNS-BRNO-II and VII in 45 experiments, TNS-BRNO-III in 1 experiment, and TNS-BRNO-VIII in 1 experiment). In 4 experiments, the Rostock TAH (NABEL, TAH Research Center, Rostock, Germany) was implanted. One group of 22 animals with an average survival of 162 days (the longest, 293 days) was submitted to an antihypertensive treatment; another 1 of 29 calves with an average survival of 98 days (the longest, 173 days) was untreated. The evaluation was performed using a scale (0 to 3) based on very precisely fixed criteria. EM pathologic changes documented various stages of ischemic damage. Except for the liver, no significant difference was found between both groups, despite the substantially prolonged survival in the treated group. Very important was the general state of mitochondria, endoplasmic reticulum, and nucleus. Further, the state of glomerular podocytes in the kidney and the state of interalveolar septa and of pneumocytes constituting the air-blood barrier for gas exchange in the lungs are especially important. In some animals of both groups, the EM findings were completely normal, especially in the lung.

Animals↗

The heritability theory of heterosis and its meaning for global agriculture.

This paper begins with the overthrow of the concept of combining ability in crossbreeding by the concept of heritability. The reason is that general combining ability changes with the number and kind of pure strains in the foundation stock and hence special combining ability changes also, so that work with different kinds of pure strains in the foundation stock cannot be compared. Hence combining ability is useless as a parameter to predict the amount of heterosis expected in the next generation. On the other hand, since each cross has a separate heritability, it can be applied to a cross population just as successfully as in purebreeding. Since the same concept holds in both cases, resort to any other concept would be superfluous. That's why combining ability must be rejected. Another reason (not given in the full text) is, an infinite number of pure strains would be required in the foundation stock for its results to be comparable with those of the heritability theory, which disposes of its utility altogether. The main content of the thesis is then the centennial enigma of heterosis can be resolved by Descarte's theoretic method of deduction. Accordingly we start from the definition of heterosis. [formula: see text] where H is heterosis, F1 is the first generation offspring, MP is the mean of the parents or midparent, and from the use of a binomial random variable and its extension to the multinomial case derive the basic relations of heterosis with its components. Starting with second degree statistics, we obtain [formula: see text] where V and cov stand for variance and covariance. The equations of heterosis are [formula: see text] where N is number of genes controlling a trait, a = (Pi - Pj)/2, d is deviation from midparent, while the variance components are all indicated by their names under the respective terms. It turns out that all these can be easily computed from the data so that the problem becomes a simple one which any college student may solve. In other words, the right answers are found when the right questions are asked. Who had ever shown that the heritability principle is inapplicable in crossbreeding, e.g., in a crossing of two pure strains? From this cue arose the realization that the F1 of a cross of two pure strains must also be a Mendelian population, with p and q both equal to 1/2 which simplifies the algebra outright. This Heritability Theory of Heterosis, or HTH in capital letters, rests on 2 initial arguments: 1) Since 0.5 + 0.5 = 1, crossing two pure strains gives a population which is only a special case of pure-breeding, therefore a heritability coefficient must exist for the F1; 2) Our problem reduces to that of finding that coefficient; the answer is given by the additive component divided by VF1, i.e., (1/2) Na2/VF1. which is readily found from the solution of the heterosis equations. Thus the eternal enigma of heterosis is resolved! This happened at the end of the 20th century. We now come to the second point of the discovery, the new genetic parameter crossheritability which will rise in size with the increase of the number of times it's used and form the link between breeding and evolution. The advent of the Age of Evolution Engineering in the 21st century marks a totally new era, showing that artificial will ultimately supercede natural selection, with the long span of time element eliminated. For agriculture at least, it means there is no limit to the increase of food supply by the new method, with the concentration of desirable genes by hybridization in place of the old theory of their fixation. Genetic gain is achieved through artificial selection, with an 80% saving of time, labor and cost by adoption of the new method. Applied to a further increase in all kinds of agricultural products including hybrid rice, it means that a huge escalation, in fact a New Green Revolution, on a much larger scale than that of any such before, is in view, provided it is adopted in our research and educational institutions as early as possible, ere its spread elsewhere. The possibilities from the evolution point of view can only be pictured by science fiction.

Agriculture↗

Totally implantable artificial hearts and left ventricular assist devices: selecting impermeable polycarbonate urethane to manufacture ventricles.

In the development of a new generation of totally implantable artificial hearts and left ventricular assist devices (VADs) for long-term use, the selection of an acceptable material for the fabrication of the ventricles probably represents one of the greatest challenges. Segmented polyether urethanes used to be the material of choice due to their superior flexural performance, acceptable blood compatibility, and ease of processing. However, because they are known to degrade and to be readily permeable to water, they cannot meet the rigorous requirements needed for a new generation of implantable artificial hearts and VADs. Therefore, the objective of the present study was to identify alternative polymeric materials that would be satisfactory for fabricating the ventricles, and in particular, to determine the water permeability through membranes made from four commercial polycarbonate urethanes (Carbothane PC3570A, Chronoflex AR, Corethane 80A, and Corethane 55D) in comparison to those made from two traditional polyether urethanes (Tecoflex EG80A and Tecothane TT-1074A). In addition to determining the rate of water transmission through the six membranes by exposing them to deionized water, saline, and albumin-Krebs solution under pressure and measuring the displacement of liquid by means of a recently developed capillary method, the inherent surface and chemical properties of the six membranes were characterized by SEM, contact angle measurements, FTIR, DSC, and GPC techniques. The results of the study demonstrated that the rate of water transmission through the four polycarbonate urethane membranes was significantly lower than through the two polyether urethanes. In fact the lowest values were recorded with the two Corethane membranes, and the harder type 55D polymer had a lower value (2.7 x 10(-7) g/s cm2) than the softer 80A version (3.3 x 10(-7) g/s cm2). This level of water vapor permeability, which appears to be controlled primarily by a Fickian diffusion mechanism, is between 2 and 4 times lower than that obtained with traditional polyether urethane membranes of equivalent thickness. The superior performance of the polycarbonate urethanes is likely due to the inherently lower chain mobility of the carbonate structure in the soft segment phase. In addition, the study shows that additional impermeability to water vapor can be achieved by selecting a polyurethane polymer with a high hard segment content, an aromatic rather than aliphatic diisocyanate comonomer, and a more hydrophobic surface. The use of a higher molecular weight polyurethane is not necessarily efficacious if the above requirements are not met. As expected by Raoult's Law, the study found that the use of physiological media instead of deionized water further decreases the rate of water vapor transmission. Because none of today's commercial polyurethanes are totally impervious to water vapor transmission, additional work is needed to develop permeable polymers or to apply additional treatments to existing candidates to achieve an acceptable impermeable ventricle material.

Biocompatible Materials↗

Isolation of cDNA clones mapping around DXS178: a search for human X-linked agammaglobulinaemia gene using yeast artificial chromosomes, cosmids and direct cDNA selection.

X-linked agammaglobulinaemia (XLA) was previously mapped using genetic linkage analysis to Xq22. No recombinants have been found so far between the XLA locus and DXS178 in over 30 informative meioses. Two overlapping yeast artificial chromosomes (YACs), which cover a region of approximately 1000 kb around DXS178 and form part of a larger YAC contig, were hybridised to an ordered cosmid library constructed from a human fibroblast cell line with karyotype 49,XXXXX. Positive cosmids were gridded in high density arrays onto nylon filters and rescreened with a series of YACs, YAC end probes and additional markers from the region to assign cosmids into intervals. A 640 kb-YAC hybridising to p212 (DXS178) was used for direct selection of PCR-amplified cDNA from two cDNA libraries. To identify the cosmids containing transcribed sequences the enriched cDNA from each library was radiolabelled and hybridised back to positive clones which mapped to the region of interest. These clones were then hybridised to the subcloned enriched cDNA sublibraries. Candidate cDNA clones were isolated, grouped within the region of interest and their position was confirmed by mapping back to the cosmids.

Agammaglobulinemia↗

Comparison of pregnancy outcome in mares among methods used to evaluate and select spermatozoa for insemination.

An artificial insemination dose for mares consisting of 500 million progressively motile spermatozoa is considered "standard" by most clinicians. However, little information is available directly comparing pregnancy outcome among methods of evaluating and selecting spermatozoa for insemination. The objective of this study was to determine if the method of spermatozoal evaluation and selection influences fertility as measured by pregnancy outcome. Mares were inseminated with 100 or 500 million spermatozoa that were selected for progressive motility, normal morphology, hypoosmotic swelling or absolute number regardless for evaluation method or quality. Thirty-two breeding cycles were tested for each treatment group and at each spermatozoal dose. Pregnancy outcomes were 44 and 41%, 55 and 41%, 39 and 31%, and 45 and 41%, for the 100 and 500 million progressively motile, morphologically normal, hypoosmotic swelling positive and absolute number treatment groups, respectively. Pregnancy outcome did not differ among methods of spermatozoal evaluation and selection for artificial insemination in the 100 (P=0.52) or 500 (P=0.78) million spermatozoa groups. Also the total number of spermatozoa and the absolute number of progressively motile, morphologically normal or hypoosmotic swelling positive spermatozoa inseminated, were not closely associated with pregnancy outcome in the 100 (P=0.24, 0.29, 0.33 and 0.38, respectively) or 500 (P=0.20, 0.84, 0.50 and 0.74, respectively) million spermatozoa groups. In this study, we found that the method of spermatozoal evaluation did not offer an advantage for pregnancy when used to select spermatozoa for insemination at the doses tested. These results were surprising, as we expected there would be differences among the evaluation methods. Instead, we found that evaluating spermatozoa offered no advantage for pregnancy over simply inseminating with a specified number of spermatozoa not selected for any particular characteristic under the conditions of our experiment.

Animals↗

[Direct measurement of nitric oxide levels in aqueous humor with selective iso-NO electrode under physiologic conditions and after extracapsular cataract extraction and artificial lens implantation with PMMA in experiments with rabbits].

PURPOSE: To estimate the level of the nitric oxide (NO) within the aqueous humor of the rabbit eye after ECCE and PMMA artificial lens implantation using selective, nitric oxide sensitive electrode Iso-NO. We analysed the NO level during early postoperative period between the 1st and the 5th day. MATERIAL AND METHODS: We examined 30 gray rabbits (weighing 3.0-3.5 kg). Just before surgery, samples of aqueous humor were aspirated to control the NO level in physiological condition. Lens was extracted with ECCE (envelope) technique. In 15 eyes PMMA IOL were implanted and 15 eyes were left aphacic. The aqueous samples were also collected 1, 3, 5 days after surgery. Nitric oxide in each sample was estimated directly with selective, nitric oxide sensitive electrode Iso-NO. Changing values of the NO concentration during the examination were expressed using evaluation of field under curve in the absolute values. Such results were compared with control group. RESULTS: During physiological conditions the mean value of the field under curve of the NO concentration was estimated as 4987.29 +/- 895.89. After ECCE with or without PMMA artificial lens implantation the levels of the NO were higher as compared to the control group. After ECCE and PMMA artificial lens implantation the highest level of NO was estimated on the 3rd postoperative day (7978.98 +/- 949.77). During the examinations the fields under curve of NO concentration were estimated as 6626.30 +/- 1176.53 and 6288.19 +/- 604.90 (on the 1st and the 5th days, respectively). On all postoperative days, values of the field under curve of NO were significantly higher as compared to the control group. In contrast, after ECCE without lens implantation the highest value of NO field under curve, was estimated on the 1st postoperative day as 11727.83 +/- 1032.44. During the examinations, the fields under curve of NO concentration were estimated as 9841.52 +/- 698.06 and 5446.96 +/- 568.47 (on the 3rd and the 5th postoperative days, respectively). Moreover, on all days of examination the value of NO fields under curve were significantly higher as compared to the control group. CONCLUSION: The level of nitric oxide in aqueous humor after ECCE with or without artificial lens implantation was higher than in the control group. The highest value of the field under curve was estimated on the 3rd postoperative day after ECCE and PMMA artificial lens implantation and on the 1st postoperative day after ECCE without lens implantation. The use of direct selective electrode Iso-NO is a simple method of nitric oxide estimation in aqueous humor, but the interpretation of results seems to be very difficult. This method may be suitable for comparative estimations of NO during the experimental works.

Animals↗