Parameters affecting the yield of DNA from human blood

An examination of variables affecting the yield of DNA from blood was undertaken in order to improve sample processing and to evaluate alternative methods of mailing blood samples for DNA analysis. A rapid, high-yield method was developed for the isolation of high-molecular-weight DNA from fresh and frozen blood. In addition, the following observations were made: (1) Of the anticoagulants examined, acid citrate dextrose (ACD) solution B was found to be superior to EDTA and heparin for preserving yields of DNA during incubation at room temperature. If DNA is isolated from frozen blood, high yields of undegraded DNA are achieved after incubation at 23 degrees C for 5 days with ACD solution B. (2) High yields of undegraded DNA are obtained from blood stored with ACD solution B for at least 1 day at 42 degrees C, 5 days at 0 degrees C, or 1 month at -20 degrees C. (3) Three cycles of freezing and thawing may have little if any affect on the yield of DNA. The results indicate that blood for DNA extraction may be mailed in an ambient temperature container and, in many cases, sent by first-class mail rather than by overnight delivery services.     

Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development

Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16-cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa expression in this embryo is restricted early by translational regulation to the small micromere lineage.     

Aluminum stress and protein synthesis in near isogenic lines of Triticum aestivum differing in aluminum tolerance

Aluminum (Al) stress was examined in three lines of wheat ( Triticum aestivum L.) by measuring root lengths, protein synthesis and protein accumulation in seedling root tips grown in a hydroponic system. An Al-sensitive, recurrent wheat parent (cv. Katepwa) showed very little root growth in low Al concentrations. In contrast, an Al-tolerant near isogenic line (Alikat) and Al-tolerant donor (cv. Maringa) had much greater root growth. Segregation data from an F₂ population (Katepwa × Alikat) showed that one major gene controlled Al tolerance based on root growth ( X ²= 0.651). All three lines showed an approximately 2-fold increase in [³⁵S]-Met incorporation in root tips after 3 days in Al and a comparable increase in root-tip dry weight. Maringa and Alikat root tips showed an increased total protein content while Katepwa root tips showed no increase in total protein content during the Al stress. Based on higher specific activities, insoluble proteins were preferentially translated in all three lines during Al stress. Proteinase activity in Katepwa root tips was 1.7-fold higher during Al stress, with Maringa and Alikat showing no change in proteinase activity. The Al-induced, increased proteinase activity in Katepwa appeared to inhibit soluble protein accumulation.     

Relationship of food intake to the induction of plasma sex steroid-binding protein and testicular activity in immature male little brown bats (Myotis lucifugus lucifugus)

Immature male little brown bats were aroused prematurely from their first hibernation and fed ad libitum (Group AL) or given a restricted diet (Group FR). All animals were weighed daily and the food intake of Group FR males was restricted to maintain their body weights at or near initial post-arousal values (5.3-6.8 g). The average body weights of Group AL males increased during the first week after arousal and then stabilized at a level which was 20% higher than those of Group FR males. The post-arousal induction of plasma SBP was similar in Groups FR and AL: plasma SBP activity was significantly increased 1 week after arousal and by 3 weeks had reached levels which were more than 10-fold higher than those of immature hibernating males which served as controls. Although food restriction had no effect on plasma SBP levels, it did inhibit reproductive development. Arousal-induced increases in testicular and epididymal (head/body) weights in Group FR males were less than 50% of those in Group AL males. However, histological examination of the testes revealed similar degrees of spermatogenic activation in both groups. Plasma testosterone concentrations were increased markedly in Groups FR and AL; values were generally lower in Group FR but wide individual variations were observed. Despite these elevated peripheral testosterone values, the accessory sex glands in both groups remained unstimulated.     

The importance of belowground mineral element stores in cattails (Typha latifolia L.)

We measured the amount of N, P, K, Ca, Mg, Fe, B, Mn, Na, Sr, Cu and Zn in above- and belowground parts of cattails (Typha latifolia L.) every 2 weeks during the growing season (April–October) in plants growing in a marsh on the shore of Lake Mendota, Wisconsin. Elements differed considerably in their distribution between above- and belowground parts and the amount of apparent exchange between parts. The ratio of the amount of an element in aboveground plant parts to that belowground (A:B) was between 1:1 and 2:1 for most elements, as compared with the 2.2:1 ratio of biomass. The maximum amounts of Fe and Zn belowground exceeded their aboveground maxima, while K, Ca and Mn had A:B ratios greater than 2:1. N, P and K in belowground plant parts decreased considerably during the spring, and belowground decreases were large enough to be potentially important sources of these elements for shoot growth. Belowground stores of Ca, Mg, Mn, Na and Sr decreased little in the spring and do not function as reserves.     

Plasma sex steroid binding in Chiroptera

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Large-scale production and characterization of Bacillus thuringiensis subsp. tenebrionis insecticidal protein from Escherichia coli

Bacillus thuringiensis subsp. tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration of transgenic potato plants. The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp. tenebrionis by translation initiation at an internal initiation site in the native DNA sequence. The gene sequence specific for this truncated protein was expressed in E. coli strain JM 101 and fermented at the 1000-l scale. The protein accumulated as insoluble inclusion bodies, and was purified by extraction at pH␣10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation. No chromatography steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95 % and demonstrating full insecticidal activity against Colorado potato beetle larvae. The protein was further characterized to assure identity and suitability for use in safety-assessment studies. Received: 31 May 1996 / Received revision: 11 September 1996 / Accepted: 13 October 1996     

Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.

Some bisphosphonates used for the treatment of bone disorders are also potent inhibitors of squalene synthase, a critical enzyme for sterol biosynthesis. Among seven drugs tested, YM 175 (cycloheptylaminomethylene-1,1-bisphosphonic acid) was the most potent inhibitor of rat liver microsomal squalene synthase (Ki = 57 nM) and sterol biosynthesis from [14C]mevalonate in rat liver homogenate (IC50 = 17 nM). EB 1053 (3-(1-pyrolidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) and PHPBP (3-(1-piperidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) were less potent inhibitors in both these assays. Pamidronate and alendronate were poor inhibitors of squalene synthase (IC50 > 10 microM) but were potent inhibitors of sterol biosynthesis from mevalonate (IC50 = 420 and 168 nM, respectively), suggesting that the latter two agents may have inhibited other enzymes involved in the synthesis of farnesyl pyrophosphate from mevalonate. Etidronate and clodronate were inactive in both these assays. YM 175 also inhibited sterol biosynthesis in mouse macrophage J774 cells (IC50 = 64 microM) and in rats, when administered acutely, it inhibited cholesterol biosynthesis in the liver (ED50 = 30 mg/kg, s.c.). Structural modifications on YM 175 to enhance cell permeability may result in a new class of cholesterol-lowering agents.