Strongly correlated s-wave superconductivity in the N-type infinite-layer cuprate.

Quasiparticle tunneling spectra of the electron-doped ( n-type) infinite-layer cuprate Sr0.9La0.1CuO2 reveal characteristics that counter a number of common phenomena in the hole-doped ( p-type) cuprates. The optimally doped Sr0.9La0.1CuO2 with T(c) = 43 K exhibits a momentum-independent superconducting gap Delta = 13.0+/-1.0 meV that substantially exceeds the BCS value, and the spectral characteristics indicate insignificant quasiparticle damping by spin fluctuations and the absence of pseudogap. The response to quantum impurities in the Cu sites also differs fundamentally from that of the p-type cuprates with d(x(2)-y(2))-wave pairing symmetry.

Isolation and characterization of a 30 kDa protein with antifungal activity from leaves of Engelmannia pinnatifida.

During the course of screening plants for novel antifungal activity, we found that a high-molecular-mass fraction of an extract from leaves of Engelmannia pinnatifida exhibited potent and broad-spectrum antifungal activity. In this study a 30 kDa protein from E. pinnatifida leaves was purified to homogeneity by ammonium sulphate precipitation, gel filtration, Mono-Q and C18 reverse-phase column chromatographies. The purified protein showed potent antifungal activity against various plant pathogens with as little as 50 ng. The N-terminal amino acid sequence of the purified protein was determined as XXTKFDFFTLALQXPAXF, where X indicates an unidentified residue. This sequence showed 35-50% sequence identity with purified style glycoproteins associated with self-incompatibility from wild tomato, tobacco and petunia, a phosphate-starvation-induced ribonuclease from cultured tomato cells and the SIR 63.4 kDa protein from yeast.

5'-secondary structure formation, in contrast to a short string of non-preferred codons, inhibits the translation of the pyruvate kinase mRNA in yeast.

The effects of poor codon bias and secondary structure formation upon the translation of the pyruvate kinase (PYK1) mRNA have been investigated in Saccharomyces cerevisiae. Following insertion mutagenesis at the 5'-end of the PYK1 coding region, the gene was transformed into yeast, and translation assessed directly in vivo by determining the distribution of the modified PYK1 mRNAs across polysomes fractionated by sucrose density gradient centrifugation. The chromosomally-encoded (wild-type) PYK1 mRNA, and the actin, ribosomal protein L3 and glyceraldehyde-3-phosphate dehydrogenase mRNAs were used to control for minor differences between polysome preparations. An insertion containing 13 non-preferred codons at the 5'-end of the coding region was found to have no significant effect upon PYK1 mRNA translation. In contrast, translation was inhibited by an insertion which increased the formation of secondary structures at the 5'-end of the mRNA (overall delta G = -36.6 kcal/mol). Control insertions were also analysed to exclude the possibility that alterations to the amino acid sequence of pyruvate kinase affect the translation of its mRNA. These insertions, which introduced preferred codons or restored wild-type levels of secondary structure formation, did not significantly influence PYK1 mRNA translation.

Chemical synthesis, cloning and expression in mammalian cells of a gene coding for human tissue-type plasminogen activator.

A 1610-bp DNA duplex coding for human tissue-type plasminogen activator has been chemically synthesized using the phosphoramidite procedure, adapted for a custom-built gene synthesizer. The synthesizer, which was designed for both simplicity and speed, permits the rapid construction of relatively large genes and compares favorably in speed with alternative cDNA isolation procedures. The plasminogen activator gene has been expressed in mammalian cells and shown to produce authentic protein by an immuno-activity assay.

Novel modified beta-interferons: gene cloning, expression, and biological activity in bacterial extracts.

A series of novel, modified interferons based on the structure of human beta-interferon have been expressed in Escherichia coli. Modified interferon genes were constructed from sequences derived from the natural beta-interferon gene, a synthetic beta-interferon gene, or a specific combination of the two. A total of 23 out of the 25 novel interferons exhibited antiviral (AV) and antiproliferative (AP) activity which varied from 3 to 230% and 8 to 490% of the values for beta-interferon, respectively. None of the novel interferons had only AV or AP activity, although one had a much reduced ratio of AV/AP activity compared with beta-interferon. Substitution of beta-interferon amino acids 2-7 or 28-46 resulted in interferons with significantly increased AP activity on Daudi lymphoblastoid cells (four- to fivefold). All the novel interferons except two with modifications in the 82-105 region reacted with a neutralizing beta-interferon monoclonal antibody.

Hemodynamics after autogenous, interpositional grafting in small arteries.

This investigation initiates the quantitative, hemodynamic assessment of interpositional grafts in small rat arteries. The left femoral arteries in 16 Sprague-Dawley rats were transected at two levels, and were then repaired using interrupted suturing technique. This effectively provided an ideally sized and histologically matched interposition graft. The 20-MHz PUDVM method was used to measure blood velocities, and derive values for vessel lumen area, temporal mean of the spatial mean velocity (Vsm), and volumetric flows. Measurements were completed distal to the interposed graft. Variables were quantitated in the preoperative and at the 5-, 15-, and 30-minute postoperative intervals. Statistical analysis of data indicated that the interpositional grafting procedure resulted in increased vessel lumen area and decreased Vsm, but, importantly, volumetric flow (about 8.20 ml/min) remained unchanged. This study demonstrated that, although hemodynamic characteristics are altered, volumetric flow can be restored after an experimental, interpositional grafting procedure in small arteries.

A three-dimensional computational simulation of some sources of measurement artifact in microvascular pulsed ultrasound Doppler velocimetry.

Recent applications of 20 MHz pulsed ultrasound Doppler velocimetry (PUDVM) in microsurgical research have necessarily employed piezoelectric crystals whose diameter is not negligible compared to the lumen size (1-2 mm) of many vessels of interest. A three-dimensional numerical model was developed to explore relationships between actual and detected flow field parameters, for (steady) Poiseuille flow, when appreciable velocity gradients exist within the PUDVM sample volume. Validation studies showed that highly accurate velocity profiles could be obtained in the limiting case of a very small sample volume (0.1 mm radius), but that for currently employed crystals (approximately equal to 0.5 mm radius) there was appreciable underestimation of the centersteam velocity, and appreciable overestimation of the flow stream diameter. Errors in perceived velocity and flow rate were found to be relatively insensitive to perturbations in the sample volume thickness, in the size of the sampling range increment, or in the angle of insonation beam divergence. By contrast, these apparent flow parameters were found to be very sensitive to perturbations of sample volume diameter or of the Doppler angle. Small variations in the degree of partial sample volume overlap of the flowstream periphery were shown to be capable of causing large fluctuations in apparent flow stream diameter.

Some characteristics of laminar flow velocity spectra detected by a 20 MHz pulsed ultrasound Doppler.

For the purpose of improving accuracy of noninvasive flow measurements in small (1-2 mm diameter) blood vessels, an existing 20 MHz pulsed ultrasound Doppler velocimeter (PUDVM) has been augmented to allow fast Fourier transformation (FFT) of its Doppler shift signal. The modified instrument was used to collect velocity spectra for a benchtop test section delivering precise Poiseuille flows at velocities in the range of physiological interest. The velocity spectra demonstrated a substantial degree of broadening, much of which was attributable to the geometry of the finite sample volume size. Several spectral indices were studied as a function of flow field variables. Results showed that the intensity-weighted mean Doppler shift frequency, when converted to its corresponding velocity vM, agreed very closely with the theoretically predicted local fluid velocity. Measurement linearity and repeatability were evaluated for a number of system variables, indicating that FFT performance was essentially unaffected by several parameters capable of causing major degradation of (phasic) Doppler shift signals produced by conventional zero-crossing-counter circuitry. As presently configured, the augmented PUDVM instrument is fully capable of detailed flow field mapping in small subcutaneous vessels such as human digital arteries.

A hemodynamic evaluation of cuffed microarteriorrhaphy.

A cuffing technique and a standard interrupted suturing technique of microarteriorrhaphy were compared in the rat femoral artery. The comparison was based on hemodynamic variables, which were measured by 20-MHz pulsed ultrasound Doppler velocity meter (PUDVM) method. The hemodynamic variables included measured blood velocity and vessel lumen diameters, and calculated volumetric flows. Measurements were completed at 5-, 10-, and 20-minute intervals after vessel repair. Data from 26 experimental animals were suitable for analysis, with 15 animals in the standard repair group and 11 animals in the cuffed group. All variables were statistically similar prior to the surgical procedure, indicating that the study groups were appropriate for comparison. The measured lumen diameters were not statistically different at any time interval (P = .06). However, the velocities and the volumetric flow were statistically different (P less than .05) between groups at all time intervals. These values returned toward normal preoperative values over time. The cuffing technique used in this study was not as efficacious as the standard technique of microarteriorrhaphy, when evaluated by 20-MHz PUDVM-derived hemodynamic criteria.

Chemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification.

A 43-bp DNA duplex coding for poly(arginine) [poly(arg)] has been synthesised by modified phosphotriester procedures. It has been inserted into the Bg/II and BamHI restriction sites of a cloned synthetic beta-urogastrone (Uro) gene, under the control of the trp promoter. Subsequent induction with 3 beta-indole acrylic acid produces beta-Uro with a C-terminal poly(arg) fusion. The raised isoelectric point of this polypeptide fusion facilitates rapid purification by cation exchange chromatography. The C-terminal poly(arg) tail can be readily removed by treatment with carboxypeptidase B.

Chemical synthesis and cloning of a gene for human beta-urogastrone.

A DNA duplex coding for the 53 amino acids of human beta-urogastrone has been synthesised. Computer assisted design of the gene included restriction endonuclease sites for plasmid insertion, a termination codon and two triplets coding for lysine at the 5'-end of the structural gene. The synthesis involved preparation of 23 oligodeoxyribonucleotides by phosphotriester procedures coupled to rapid HPLC techniques. The gene was constructed in two halves by enzymatic ligation of the oligonucleotides and cloned into a specially constructed chimeric plasmid vector. Escherichia coli K12 MRC8 was transformed by the plasmid and clones containing the full gene sequence were isolated and characterised.