Antioxidant depletion in aortic crossclamping ischemia: increase of the plasma alpha-tocopheryl quinone/alpha-tocopherol ratio.

To assess whether oxidative stress contributes to the ischemia/reperfusion injury of aortic surgery, the contents of alpha-tocopherol, alpha-tocopheryl quinone, ascorbate, lipid-derived malondialdehyde, protein thiols, cholesterol, and lactate were analyzed in plasma samples from 24 patients subjected to aortic crossclamping. alpha-Tocopherol, ascorbate, and protein thiols decreased during ischemia, whereas alpha-tocopheryl quinone increased in all but two cases, doubling on average in proportion to alpha-tocopherol. Upon reperfusion alpha-tocopherol, ascorbate, and protein thiols remained low, whereas alpha-tocopheryl quinone returned to the preischemic level. Lipid-derived malondialdehyde (a measure of lipid hydroperoxides) increased significantly only during reperfusion. The results suggest that oxidative stress occurs simultaneously with ischemia/reperfusion during aortic surgery, and that measurement of the tocopheryl quinone/tocopherol ratio may shed new light on the underlying pathological events.     

Comparative studies of Babesia spp. from white-tailed and sika deer

Babesia odocoilei from white-tailed deer (Odocoileus virginianus) in Texas (USA) and B. capreoli isolated from sika deer (Cervus nippon) in Ireland were compared morphologically and antigenically. Babesia odocoilei and B. capreoli paired pyriforms resembled each other closely when in sika deer, but B. odocoilei pyriforms in white-tailed deer were slightly different. Babesia odocoilei in white-tailed deer also differed from B. odocoilei and B. capreoli in sika deer in the frequency of its developmental forms. Indirect immunofluorescence antibody test titres showed that there was some antigen cross-reactivity, but not as much as between B. capreoli and the bovine parasite, B. divergens. The Babesia spp. from deer that we studied appear to be distinct but related species. The low infectivity of B. odocoilei for a splenectomised sika deer suggests that sika deer in North America are probably not very susceptible to this parasite in the wild.     

A gibberellin-regulated gene from wheat with sequence homology to cathepsin B of mammalian cells

A previous report described several cDNAs corresponding to mRNAs which accumulated in wheat aleurone layers treated with gibberellic acid (GA) (Baulcombe and Buffard, 1983). The protein sequence deduced from one of these clones (2529) has extensive similarity to the thiol protease, cathepsin B from mammalian cells. Southern analysis of wheat DNA has shown that the 2529 mRNA is encoded by a small family of genes carried on the group 4 chromosome. The nucleotide sequence of a member of the gene family expressed at a low level in aleurone layers and the use of a primer extension assay to identify a clone of a member of the gene family producing an abundant mRNA are reported. The 2529 mRNA accumulates in the scutellum and the aleurone layer of germinating grains where its expression is regulated by GA. In the scutellum the expression was restricted to the parenchyma, suggesting that the 2529 product may have a role other than for mobilization of the endosperm.     

p lambda Zd39: a new type of cDNA expression vector for low background, high efficiency directional cloning.

We have developed a new type of bacteriophage lambda vector which provides a strong biological selection against non-recombinants that is independent of the sequences immediately surrounding the cloning site. This system, which we call 'selective substitution', is ideally suited for cDNA expression vectors where it is necessary to flank the cDNA insert with sequence elements (promoters etc.) required to produce a biologically active mRNA in vivo. Selective substitution is a general method, which may be applied to many types of vectors. In this report, we have specifically applied selective substitution to the construction of a new mammalian retrovirus expression vector. The level of background obtained with this vector (that is, the number of plaques obtained when the vector is ligated in the absence of insert DNA) is 0.02% when compared to ligation with restriction fragments and 0.1% to 0.4% when compared to ligation with newly synthesized cDNA. These features have allowed us to easily and efficiently generate several large cDNA libraries using total and size selected cDNA.     

Two smooth muscle myosin heavy chains differ in their light meromyosin fragment

Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment.     

Measurement of cytosolic free magnesium ion concentration by 19F NMR

Fluorinated derivatives of the chelator o-aminophenol-N,N,O-triacetic acid (APTRA) have been developed, synthesized, and analyzed for use as 19F NMR indicators of free cytosolic magnesium concentration. Magnesium dissociation constants for the 4-fluoro, 5-fluoro, and 4-methyl-5-fluoro species were determined to be 3.1, 0.9, and 0.6 mM, respectively, on the basis of UV absorption measurements at 37 degrees C in 115 mM KCl and 20 mM NaCl, pH 7.1, buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-tris-(hydroxymethyl)aminomethane. The corresponding pK values, which reflect protonation of the nitrogen atom, were determined by 19F NMR to be 4.15, 5.45, and 5.55, respectively, so that the chelators are insensitive to pH variations near the normal physiological range. The dissociation constants of these chelators for calcium ions are lower than those for magnesium but roughly 2-3 orders of magnitude above typical basal cytosolic free calcium levels, so that calcium ions will not interfere with the determinations of magnesium levels. 19F NMR studies carried out at 339.7 MHz indicate that magnesium ions are in slow exchange with the 5-fluoro and 4-methyl-5-fluoro APTRA derivatives and in fast exchange with the 4-fluoro APTRA derivative. In contrast, calcium ions were found to be in intermediate to fast exchange with all chelators. The apparent anomaly of higher thermodynamic stability of the APTRA complexes for calcium relative to magnesium but lower kinetic stability (higher k-1 values) for the calcium complexes reflects the very different association rates for the two ions. Thus, the magnesium association rates are 3 orders of magnitude slower than those for calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The distribution of oocyte 5S,somatic 5S and 18S + 28S rDNA sequences in the lampbrush chromosomes of Xenopus laevis

The nucleolus organizer locus of Xenopus laevis lampbrush chromosomes was identified by in situ hybridization of a ³H-labelled probe complementary to 18S + 28S rDNA. The nucleolus organizer is an axial granule on chromosome III that lies four-fifths the way down this chromosome reading from its larger (left) telomere, just within an exploded region that extends to its right end, where the lateral loops are exceptionally long. By in situ hybridization of ³H-labelled oocyte and somatic 5S spacer cRNA probes to similarly RNase-treated and denatured lampbrush chromosomes, the multiple sites of oocyte and somatic 5S gene families were identified. Oocyte 5S genes lie at the larger telomeres of the 15 chromosomes that possess these structures; that is, all but chromosomes X, XVII and XVIII. There are a further four sites, all peripheral, and in three of these, on chromosomes VII, X and XI, the sequences lie on lateral loops that are resolvable with the light microscope. By contrast all of the somatic 5S gene clusters occupy peripheral sites. There are two sites on chromosome III, one of which may be shared with oocyte 5S sequences; one on chromosome VII, which is very likely shared with oocyte 5S sequences; one terminal site on chromosome X; one site on chromosome XI that lies on a single pair of long loops which are inserted in a conspicuous and recognizable axial granule, loops which certainly carry oocyte 5S sequences too; two nearly terminal sites alongside the larger telomeres on chromosomes XII and XIV; and single interstitial sites on all three of the sphere-bearing chromosomes, VIII, IX and XVI. We suggest that 5S sequences on resolvable loops are transcribed by readthrough from upstream promoters, probably by polymerase II.