PCR amplification of murine immunoglobulin germline V genes: strategies for minimization of recombination artefacts

Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the 'plateau phase' of the reaction was reached. Recombination events were located throughout the approximately 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.     

Structural studies of ribosomal proteins

Crystal and solution structures of fourteen ribosomal proteins from thermophilic bacteria have been determined during the last decade. This paper reviews structural studies of ribosomal proteins from Thermus thermophilus carried out at the Institute of Protein Research (Pushchino, Russia) in collaboration with the University of Lund (Lund, Sweden) and the Center of Structural Biochemistry (Karolinska Institute, Huddinge, Sweden). New experimental data on the crystal structure of the ribosomal protein L30 from T. thermophilus are also included.     

A dual thrombin receptor system for platelet activation

Platelet-dependent arterial thrombosis triggers most heart attacks and strokes. Because the coagulation protease thrombin is the most potent activator of platelets, identification of the platelet receptors for thrombin is critical for understanding thrombosis and haemostasis. Protease-activated receptor-1 (PAR1) is important for activation of human platelets by thrombin, but plays no apparent role in mouse platelet activation. PAR3 is a thrombin receptor that is expressed in mouse megakaryocytes. Here we report that thrombin responses in platelets from PAR3-deficient mice were markedly delayed and diminished but not absent. We have also identified PAR4, a new thrombin-activated receptor. PAR4 messenger RNA was detected in mouse megakaryocytes and a PAR4-activating peptide caused secretion and aggregation of PAR3-deficient mouse platelets. Thus PAR3 is necessary for normal thrombin responses in mouse platelets, but a second PAR4-mediated mechanism for thrombin signalling exists. Studies with PAR-activating peptides suggest that PAR4 also functions in human platelets, which implies that an analogous dual-receptor system also operates in humans. The identification of a two-receptor system for platelet activation by thrombin has important implications for the development of antithrombotic therapies.     

Nitric oxide prevents IL-1beta and IFN-gamma-inducing factor (IL-18) release from macrophages by inhibiting caspase-1 (IL-1beta-converting enzyme)

Procytokine processing by caspase-1 is required for the maturation and release of IL-1beta and IFN-gamma-inducing factor (IGIF) (or IL-18) from activated macrophages (Mphi). Nitric oxide (NO) has emerged as a potent inhibitor of cysteine proteases. Here, we tested the hypothesis that NO regulates cytokine release by inhibiting IL-1beta-converting enzyme (ICE) or caspase-1 activity. Activated RAW264.7 cells released four to five times more IL-1beta, but not TNF-alpha, in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine. Stimulated peritoneal Mphi from wild-type mice (inducible NO synthase (iNOS)+/+) also released more IL-1beta if exposed to N(G)-monomethyl-L-arginine, whereas Mphi from iNOS knockout mice (iNOS-/-) did not. Inhibition of NO synthesis in stimulated RAW264.7 cells also resulted in a threefold increase in intracellular caspase-1 activity. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited caspase-1 activity in cells as well as the activity of purified recombinant caspase-1 and also prevented the cleavage of pro-IL-1beta and pro-IGIF by recombinant caspase-1. The inhibition of caspase-1 by NO was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. An in vivo role for the regulation of caspase-1 by NO was established in iNOS knockout animals, which exhibited significantly higher plasma levels of IL-1beta and IFN-gamma than their wild-type counterparts at 10 h following LPS injection. Taken together, these data indicate that NO suppresses IL-1beta and IGIF processing by inhibiting caspase-1 activity, providing evidence for a unique role for induced NO in regulating IL-1beta and IGIF release.     

New multidimensional editing experiments for measurement of amide deuterium isotope effects on Cbeta chemical shifts in 13C, 15N-labeled proteins

Novel multidimensional NMR pulse sequences for measurement of the three- and four-bond amide deuterium isotope effect on the chemical shifts of 13Cbeta in proteins are presented. The sequences result in editing into two subspectra of a heteronuclear triple resonance spectrum ?omega(N), omega(Cbeta), omega(Halpha)? according to there being a deuterium or a proton attached to 15N for the pertinent correlations. The new experiments are demonstrated by an application to the first module of the 13C,15N-labeled protein RAP 18-112 (N-terminal module of alpha2-macroglobulin receptor associated protein).     

Continuously tunable 1.55-/spl mu/m VCSEL implemented by precisely curved dielectric top DBR involving tailored stress

We present an optically pumped and continuously tunable 1.55-/spl mu/m vertical-cavity surface-emitting laser (VCSEL). The device shows 26-nm spectral tuning range, 400-/spl mu/W maximum output power, and 57-dBm side-mode suppression ratio. The VCSEL is implemented using a two-chip concept. The movable top mirror membrane is precisely designed to obtain a tailored air-gap length (L'=16 /spl mu/m) and a radius of curvature (ROC=4.5mm) in order to efficiently support the fundamental optical mode of the plane-concave resonator. It consists of a distributed Bragg reflector (DBR) with periodic, differently stressed silicon nitride and silicon dioxide multilayers implemented by plasma-enhanced chemical vapor deposition. The lower InP-based part, comprising the InP-InGaAsP bottom DBR and the active region, is grown monolithically using metal-organic vapor phase epitaxy.     

Theory of bound rubber

A theory of bound rubber formation has been developed which treats the effect as random adsorption of structural units of polymer on reactive sites which are assumed to exist on the surface of filler particles. Equations are derived for the fraction of bound rubber and for the molecular weight distribution of free (unbound) rubber. The theory contains only one adjustable parameter, the filler surface area per reactive site, A₀. It is shown for the case of the Schulz distribution that the amount of bound rubber depends but slightly on the dispersion parameter of the polymer and is determined essentially by the adsorption index M?_w cP/A₀N_A, where c is the filler concentration, P is its specific surface area, and N_A is the Avogadro number. All of the experimentally observed features of the bound rubber effect, including preferential adsorption of large molecules, are correctly predicted, the quantitative agreement of the theoretical equations with available experimental data being satisfatory. This supports the underlying assumption that the processes involved in the polymer–filler interaction may be approximated by a random-process model.     

Calculation of coupling of two grooves in a parallel-plate guide

An analysis of the coupling properties of uniformly coupled groove guides is presented using an exact method for the determination of the phase constants of fundamental TE even and odd modes.     

Effects of bis(2-ethylhexyl) phthalate on chromosomes of human leukocytes and human fetal lung cells

Blood from two male and two female donors was exposed at 37degrees for 4 hr to concentrations of 60.0, 6.0, 0.6, and 0.06 mug of a widely used plasticizer, bis (2-ethylhexyl) phthalate, per milliliter of blood. The bis(2-ethylhexyl) phthalate was solubilized with polysorbate 80. Appropriate polysorbate and nonpolysorbate controls also were established. Following the 4 hr of incubation, phytohemagglutinin was added and tissue cultures were established. In addition, human fetal lung cells were exposed in tissue culture to a medium containing 6.0 mug/ml of bis(2-ethylhexyl) phthalate in polysorbate 80 for 5 days. Similar controls also were established for these experiments. Analysis of chromosome preparations from all cultures obtained failed to show any increased evidence of isochromatid and chromatid breaks or gaps or abnormal forms at any studied concentration when compared to the control cultures. In addition, analysis of fetal lung cell preparations for aneuploidy failed to reveal any differences between cells from study and control cultures. This study involved a short-term exposure to bis(2-ethylhexyl) phthalate in various concentrations which did not cause damage in leukocytes or fetal lung cells.     

Paraganglioma of the cauda equina: a case report and review of the literature

The authors describe the technique of RARE sequences (Hennig, 1986) and their hydrographic application to urinary imaging, RARE urography. Heavily T2-weighted non-tomographic images of the urinary tract are obtained in less than 30 seconds, without contrast medium or ionizing radiation. The whole urinary tract is visualised on one image with a spatial resolution superior to that of sonography. The semiology of RARE urography is similar to that of the intravenous pyelogram. Silent kidneys are also visualised.