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Novel artificial ion channels (1 and 2) based on CB[n] (n = 6 and 5, respectively) synthetic receptors with carbonyl-fringed portals (diameter 3.9 and 2.4 A, respectively) can transport proton and alkali metal ions across a lipid membrane with ion selectivity. Fluorometric experiments using large unilamellar vesicles showed that 1 mediates proton transport across the membranes, which can be blocked by a neurotransmitter, acetylcholine, reminiscent of the blocking of the K+ channels by polyamines. The alkali metal ion transport activity of 1 follows the order of Li+ > Cs+ approximately Rb+ > K+ > Na+, which is opposite to the binding affinity of CB[6] toward alkali metal ions. On the other hand, the transport activity of 2 follows the order of Li+ > Na+, which is also opposite to the binding affinity of 2 toward these metal ions, but virtually no transport was observed for K+, Rb+, and Cs+. It is presumably because the carbonyl-fringed portal size of 2 (diameter 2.4 A) is smaller than the diameters of these alkali metal ions. To determine the transport mechanism, voltage-clamp experiments on planar bilayer lipid membranes were carried out. The experiments showed that a single-channel current of 1 for Cs+ transport is approximately 5 pA, which corresponds to an ion flux of approximately 3 x 107 ions/s. These results are consistent with an ion channel mechanism. Not only the structural resemblance to the selectivity filter of K+ channels but also the remarkable ion selectivity makes this model system unique.
Enzymatic and homogeneous catalysis offer complementary means to produce enantiopure products. Incorporation of achiral, biotinylated aminodiphosphine-rhodium complexes in (strept)avidin affords enantioselective hydrogenation catalysts. A combined chemogenetic procedure allows the optimization of the activity and the selectivity of such artificial metalloenzymes: the reduction of acetamidoacrylate proceeds to produce N-acetamidoalanine in either 96 % ee (R) or 80 % ee (S). In addition to providing a chiral second coordination sphere and, thus, selectivity to the catalyst, the phenomenon of protein-accelerated catalysis (e.g., increased activity) was unraveled. Such artificial metalloenzymes based on the biotin-avidin technology display features that are reminiscent of both homogeneous and of enzymatic catalysis.
We compared two selected groups of patients with acute Guillain-Barré syndrome requiring artificial ventilation. Both groups were treated with conventional therapy, group A (n = 35) between 1970 and 1978, group B (n = 21) between 1979 and 1983. Neither plasmapheresis nor any other immunosuppressive treatment was applied. Significant differences were found favouring group B: There was a decline of mortality from about 60% to less than 10% (p less than 0.001) and a smaller number of severe complications due to improvement of supportive care.
Quantitative traits are shaped by networks of pleiotropic genes . To understand the mechanisms that maintain genetic variation for quantitative traits in natural populations and to predict responses to artificial and natural selection, we must evaluate pleiotropic effects of underlying quantitative trait genes and define functional allelic variation at the level of quantitative trait nucleotides (QTNs). Catecholamines up (Catsup), which encodes a negative regulator of tyrosine hydroxylase , the rate-limiting step in the synthesis of the neurotransmitter dopamine, is a pleiotropic quantitative trait gene in Drosophila melanogaster. We used association mapping to determine whether the same or different QTNs at Catsup are associated with naturally occurring variation in multiple quantitative traits. We sequenced 169 Catsup alleles from a single population and detected 33 polymorphisms with little linkage disequilibrium (LD). Different molecular polymorphisms in Catsup are independently associated with variation in longevity, locomotor behavior, and sensory bristle number. Most of these polymorphisms are potentially functional variants in protein coding regions, have large effects, and are not common. Thus, Catsup is a pleiotropic quantitative trait gene, but individual QTNs do not have pleiotropic effects. Molecular population genetic analyses of Catsup sequences are consistent with balancing selection maintaining multiple functional polymorphisms.
Cabergoline, a potent dopamine agonist and inhibitor of prolactin secretion, was investigated as a potential fertility control agent in the red fox ( Vulpes vulpes). Sixty silver fox vixens were selected randomly and artificially inseminated. Cabergoline was fed to groups of 12 vixens in a minced beef ration either as a single dose of 25, 50 or 100 microg kg(-1), or a dose of 50 microg kg(-1) that was repeated 2 days later (2 x 50 microg kg(-1)). Four foxes from each group of 12 were given cabergoline at day 28, day 35 or day 48 after artificial insemination, and a control group of four foxes was used as a comparison for each dose day. In a separate trial, two groups of five foxes were selected randomly from the farm population and fed 100 microg kg(-1) of either cabergoline or a placebo each day from day 42 to day 46 of pregnancy. Foxes that received single doses of cabergoline of 100 microg kg(-1) or 2 x 50 microg kg(-1) aborted at day 28, but the same doses did not result in abortions when administered on days 35 and 48. Although lactation was not terminated in groups that received a single or double dose of cabergoline, increased post-natal cub mortality was associated with cabergoline administration. Growth of cubs between 4 and 8 weeks of age was not inhibited in vixens that received cabergoline. Doses of 100 microg cabergoline kg(-1) administered each day from day 42 to day 46 resulted in abortions and terminated lactation. The capacity of single doses of cabergoline to cause abortions in the red fox during mid- rather than late pregnancy is contrary to reported observations for the domestic dog. This finding indicates that luteotrophic support of the corpus luteum by prolactin may be more important at mid-pregnancy in the red fox. The results of this study support previous field observations that cabergoline delivered in bait affects the reproductive success of vixens and may be a practical adjunct to the lethal control of wild red foxes in Australia.
The effects of interaction of artificial directional and stabilizing selection were studied using a recessive mutation of Drosophila melanogaster radius incompletus which causes interruption of the second longitudinal wing vein (L2). The character under directional selection was the length of the proximal segment of L2; stabilizing selection was conducted for a complex of morphometric wing characters. Three experimental designs in three replications were used: 1) directional and stabilizing selection; 2) directional selection; 3) unselected controls. The dynamics and variation of the character under directional selection was shown to be different in the stabilized and unstabilized lines. Coefficient of variation which increased in the unstabilized lines remained at the same level in the stabilized lines and in the controls. The results are in agreement with those of the previous experiment, where directional selection was conducted for a behavioral trait, which suggests general character of the relationships observed.
Selection of spermatozoa on Percoll's medium has enabled to obtain 33 pregnancies in 217 couples. This simple and inexpensive technique, which is most successful during the first nine months, deserves to be attempted, in case of deficient sperm, before contemplating more complex techniques.
We report on the manipulation of intracellular filaments using a nanosurgery system based on a subnanosecond pulsed UV laser optimized for the localized severing of biological polymers. By inducing artificial catastrophe of selected microtubules (MTs), we perform shrinkage-rate measurements in interphase Ptk-2 cells throughout the entire cell. We quantify the impact of two labeling methods and three fluorescent markers, showing a 25% faster depolymerization with Alexa-488 tubulin compared with Rhodamine and yellow fluorescent protein (YFP) tubulins and a 20% higher variability induced by microinjection compared with stable transfection. Using EB3-GFP as a tip marker, we establish a new protocol to measure shrinkage rate, growth rate and rescue frequency simultaneously with high temporal and spatial specificity in live cells. As our analysis shows, laser-induced MT dynamics are physiologically relevant. The high statistical efficiency that the method offers in terms of numbers of measured events and therefore reduced standard deviations represents an important quantitative improvement in the measurement of dynamic instability parameters in vivo. We extend the application of the method by demonstrating induced dynamic behavior of actin-stress fibers after severing. This new method enables the quantitative investigation of cytoskeleton dynamics in a local confinement.
PDZ domains are a recently characterized protein-recognition module. In most cases, PDZ domains bind to the C-terminal end of target proteins and are thought thereby to link these target proteins into functional signaling networks. We report the isolation of artificial PDZ domains selected via a mutagenesis screen in vivo, each recognizing a different C-terminal peptide. We demonstrate that the PDZ domains isolated can bind selectively to their target peptides in vitro and in vivo. Two of the target peptides chosen are the C-terminal ends of two cellular transmembrane proteins with which no known PDZ domains have been reported to interact. By targeting these artificial PDZ domains to the nucleus, interacting target peptides were efficiently transported to the same subcellular localization. One of the isolated PDZ domains was tested and shown to be efficiently directed to the plasma membrane when cotransfected with the full-length transmembrane protein in mammalian cells. Thus, artificial PDZ domains can be engineered and used to target intracellular proteins to different subcellular compartments.
Transgenic green fluorescent protein (GFP) reporter embryonic stem (ES) cells are powerful tools for studying gene regulation and lineage choice during development. Here we present a rapid method for the generation of ES cells expressing GFP under the control of selected genes. Bacterial artificial chromosomes (BACs) from a previously constructed GFP transcriptional fusion library (Gene Expression Nervous System Atlas [GENSAT]) were modified for use in ES cells, and multiple BAC transgenic ES cell lines were generated. Specific GFP expression in transgenic cell lines was confirmed during neural differentiation marking neural stem cells, neuronal precursors, and glial progeny by Hes5, Dll1, and GFAP, respectively. GFP was dynamically regulated in ES cell progeny in response to soluble factors that inhibit Notch signaling and a factor that directs astroglial fate choice. Our protocols provide a simple and efficient strategy to utilize the whole GENSAT BAC library to create hundreds of novel fluorescent cell lines for use in ES cell biology.
In this study we present novel evidence that strengthens the paradigm of selective transfer of energy mediated by a random gating of ion channels. Specifically, we investigated the spectral response of a noisy artificial biomembrane whose electrical properties were largely dictated by embedded alamethicin oligomers. In this respect, we first evaluated experimentally the linear transfer function of the system via the white-noise analysis method. We prove that such a system displays specific ranges of frequency over which input signals pass preferentially, depending on their spectral content and the holding potential across the artificial bilayer which contains alamethicin. By employing voltage-driven periodic stimulation of alamethicin oligomers, we demonstrate that overall response of the system obeys qualitatively the predictions inferred from the transfer function analysis of it. These results emphasize the exquisite ability of excitable membranes to behave as band-limited filters and allow for maximal transfer of energy from an external stimulus over well-defined frequency ranges.
Plasma branched-chain amino acids and urinary C-peptide-creatinine excretion was determined at 3, 4 1/2 and 6 months of age in a group of 50 infants who were either breast-fed or artificially fed and selected at random. The average concentrations of valine in plasma and C-peptide in urine as well as the ratio between C-peptide and creatinine in urine were 2-3 times higher (p less than 0.01) in artificially fed as compared to breast-fed infants at all the ages studied. Plasma valine values correlated significantly with the urinary C-peptide/creatinine ratio (r = 0.76, p less than 0.01), which suggests that the enhanced insulin response induced by the artificial formula is related to its protein content.
The benzene-based tripodal tris(oxazolines) have been developed as the most selective and strong receptors toward linear alkylammonium ions reported to date. Among six tris(oxazolines) based on 2,4,6-trimethylbenzene framework, the phenylglycinol-derived receptor 4 exhibits the largest association constant toward nBuNH3+ (logK(ass) = 6.65 +/- 0.02), while a similar value toward tBuNH3+, (logK(ass) = 3.80 +/- 0.01) compared with others, which corresponds to the selectivity ratio of nBuNH3+/tBuNH3+ as high as approximately equals 700. The tris(oxazoline) 6 that has bare oxazoline ring exhibits still a large association constant toward sterically hindered tBuNH3+ (logK(ass) = 5.26 +/- 0.02). Both receptors 4 and 6 extract beta-phenethylammonium ion from water into chloroform almost completely. When the benzene frame is changed from 2,4,6-trimethylbenzene to 2,4,6-triethylbenzene, dramatic changes in the affinity as well as in the selectivity are observed. The association constant observed by tris(oxazoline) 8 toward nBuNH3+ approaches 10(8)M(-1) and the selectivity ratio of nBuNH3+/tBuNH3+ is increased to 2,700. This selectivity is even more enhanced to 4,000 with tris(oxazoline) 9. The enhanced binding affinity and high selectivity observed with receptors 4 and related derivatives 7-9 compared with others can be explained by an optimized steric and electronic environment provided by the phenyl substituents, which has been unambiguously demonstrated by X-ray crystallographic and 1H NMR spectroscopic studies on the host-guest complexes. The new receptor system has several unique features such as ready availability, structural simplicity, and in particular, versatility in derivatization. By virtue of these advantages, it can be readily tailored as selective receptors toward biologically important amines.
Selective knockdown of gene expression by short interference RNAs (siRNAs) has allowed rapid validation of gene functions and made possible a high throughput, genome scale approach to interrogate gene function. However, randomly designed siRNAs display different knockdown efficiencies of target genes. Hence, various prediction algorithms based on siRNA functionality have recently been constructed to increase the likelihood of selecting effective siRNAs, thereby reducing the experimental cost. Toward this end, we have trained three Back-propagation and Bayesian neural network models, previously not used in this context, to predict the knockdown efficiencies of 180 experimentally verified siRNAs on their corresponding target genes. Using our input coding based primarily on RNA structure thermodynamic parameters and cross-validation method, we showed that our neural network models outperformed most other methods and are comparable to the best predicting algorithm thus far published. Furthermore, our neural network models correctly classified 74% of all siRNAs into different efficiency categories; with a correlation coefficient of 0.43 and receiver operating characteristic curve score of 0.78, thus highlighting the potential utility of this method to complement other existing siRNA classification and prediction schemes.
Between April 1986 and July 1987, 21 patients underwent orthotopic implantation of a total artificial heart (Jarvik 7) at La Pitié Hospital. There were 18 men and 3 women with a mean age of 37.3 +/- 11.4 years. The device implanted was the 70 ml version in 10 patients and the 100 ml version in 11 patients. In the results, three variables were analysed: age, acute myocardial decompensation or chronic myocardial failure, and aetiology of the 21 patients treated. 10 (47.6%) had adequate support and were successfully transplanted. Eleven patients (52.4%) died during circulatory support. The main causes of death were sepsis and multiple organ failure. In only one patient was a mismatch between the heart and chest cavity present. There was no clinical evidence of thromboembolic complications. Patients of 40 years of age and less have an 80% chance of being successfully transplanted in comparison with a 25% success rate in older patients. Patients that developed sudden cardiac decompensation have a 75% success rate for transplantation in comparison with 44.4% success rate in patients with a chronic illness. Early implantation of the device, before the development of irreversible damage in other organs, is recommended in younger patients with acute or chronic disease and in older patients with acute myocardial failure. The use of this device is contraindicated in immunosuppressed patients due to the high risk of infection.
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A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was produced to assess the contribution of the Ser190 side-chain to the affinity and selectivity of lead uPA inhibitors in the absence of other differences present in comparisons of natural proteases. Crystallography and enzymology involving WT and Ala190 uPA were used to calculate free energy binding contributions of hydrogen bonds involving the Ser190 hydroxyl group (O(gamma)(Ser190)) responsible for the remarkable selectivity of 6-halo-5-amidinoindole and 6-halo-5-amidinobenzimidazole inhibitors toward uPA and against natural Ala190 protease anti-targets. Crystal structures of uPA complexes of novel, active site-directed arylguanidine and 2-aminobenzimidazole inhibitors of WT uPA, together with associated K(i) values for WT and Ala190 uPA, also indicate a significant role of Ser190 in the binding of these classes of uPA inhibitors. Structures and associated K(i) values for a lead inhibitor (CA-11) bound to uPA and to five other proteases, as well as for other leads bound to multiple proteases, help reveal the features responsible for the potency (K(i)=11nM) and selectivity of the remarkably small inhibitor, CA-11. The 6-fluoro-5-amidinobenzimidzole, CA-11, is more than 1000-fold selective against natural Ala190 protease anti-targets, and more than 100-fold selective against other Ser190 anti-targets.