Contribution of 3-O- and 6-O-sulfated glucosamine residues in the heparin-induced conformational change in antithrombin III

The role of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide critical for biological activity, iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, was determined by comparing its ability to bind antithrombin, induce a conformational change in this protease inhibitor as monitored by the enhancement of intrinsic fluorescence, and accelerate (at saturation) the interaction of this protein with human factor Xa. The octasaccharide produced a maximum 48% increase in intrinsic fluorescence at 37 degrees C and a rate of factor Xa inhibition of 6 X 10(5) M-1 s-1 as measured by stopped-flow fluorometry at 25 degrees C. The basal rate of the antithrombin-factor Xa interaction observed in the absence of oligosaccharide was 2 X 10(3) M-1 s-1. The synthetic pentasaccharide, consisting of residues 2-6, produced fluorescence enhancement and rate of inhibition equal to those of the octasaccharide. However, a similar pentasaccharide, identical in all respects except that it lacked the 3-O-sulfate on residue 4, produced less than a 5% fluorescence enhancement and a rate of factor Xa inhibition of 8 X 10(3) M-1 s-1. The tetrasaccharide consisting of residues 2-5 produced a 35% fluorescence enhancement and a rate of factor Xa inhibition of 3 X 10(5) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP as an alternative inhibitor of bacterial and endogenous nucleases and its effect on native chromatin compaction

The studies reported here demonstrate that ATP may be used in lieu of EDTA to inhibit nuclease digestion of DNA and chromatin. Because ATP is a milder chelator than EDTA and is a biochemical common to the cellular microenvironment in vivo, critical studies of cellular processes that require native structure to be maintained are more feasible without the presence of strong chelators. During the digestion of chromatin into its components by nuclease treatment, ATP assures the retention of nucleoprotein compaction, particularly for large to intermediate-sized oligosomes (2400bp–1000bp in length). ATP used at a concentration of 3.3 mM appears to be somewhat better than EDTA, 1.0 mM, for minimizing degradation of nuclease-treated chromatin. However, termination of nuclease digestion of chromatin and minimization of further degradation by the addition of ATP to a concentration of 1.0 mM was almost equivalent to the addition of EDTA to a concentration of 1.0 mM. Slightly more degradation was observed for the latter condition. In addition, ATP can be used to inhibit endogenous nuclease activity when specific restriction enzymes are needed. Standard low ionic strength DNP, deoxyribonucleoprotein, and DNA electrophoresis of proteinized and deproteinized chromatin oligomers, respectively, indicated that ATP effectively inhibits staphylococcal nuclease. Low ionic strength nucleoprotein electrophoresis to resolve staphylococcal nuclease-digested chromatin indicates that as little as 10^–4 M EDTA can promote structural unfolding resulting in changes in apparent mobilities for chromatin oligomers 250 and 600 by in length. Comparative digestion of chromatin with staphlococcal nuclease followed by reaction termination by ATP or EDTA showed that this observation was not merely the result of degradation due to inefficiency of ATP enzyme inhibition.     

Ammonium Triggers Uptake of NO-3 by Chenopodium rubrum Suspension Culture Cells and Remobilization of their Vacuolar Nitrate Pool

Photoautotrophic cell suspension cultures of Chenopodium rubrumrequire high concentrations of nitrate and ammonium. Duringthe growth phase total NH₄⁺ and the greater portion of NH₃^–were consumed. During the stationary phase nitrate uptake continuedbut at a substantially smaller rate than during the growth phase.During growth the bulk of the absorbed N was incorporated intoprotein, the amount of which was then maintained constant untilsenescence. NH₃^– was accumulated upon transition betweenthe growth and the stationary phase. NH₃^–, like the freeamino acids, was deposited in the vacuole but, unlike thesecompounds, could not be remobilized upon transfer of the cellsinto N-free medium. Readdition of NH₄⁺ to the medium, however,resulted in a mobilization of the vacuolar NH₃^–-pool.Reutilization of both vacuolar N-storage pools must have beenaccomplished by recycling from the vacuole to the cytoplasmbecause N-metabolizing enzymes could not be detected in isolatedvacuoles. Transfer of the cells of the stationary phase intomedium containing NH₃^– and NH₄⁺ resulted in an inductionof nitrate uptake by the cells, but only after a lag phase of4–5 days. It is conceivable that NH₄⁺ induces NH₃^–-translocatingsystems in the plasmalemma and in the tonoplast. (Received December 19, 1988; Accepted March 2, 1989)     

Fluorometric analysis of an attempt to reclaim ischemic flaps in rats with Fluosol

An experiment was designed to answer two questions as they apply to random skin-flap survival: Is there a therapy that can improve random skin-flap survival when given postoperatively? And if so, when does one start such a therapy? Fluosol-DA 20% (Fluosol) has increased random skin-flap survival when given preoperatively in our laboratory. An experiment was devised to see if it could rescue failing flaps. One-hundred Sprague-Dawley rats were divided into a control (N = 25) and five experimental groups (N = 15). All had 10 X 13 cm reverse McFarlane random flaps raised and reinset. The experimental groups underwent hemodilution with either Ringer's lactate or Fluosol at 4, 8, and 12 hours after flap elevation. All were kept in 50% oxygen for 72 hours postoperatively. The flaps and their corresponding necrotic areas were measured on day 7. As to when to institute a therapy, we simultaneously evaluated the use of a microfluorometer as a monitor of flap survival. Analysis of flap survival showed little difference between control and experimental Ringer's lactate or Fluosol groups. Analysis of the microfluorometric data led to the following points. First, as a monitor of flap viability, it is limited by a lack of specificity and sensitivity. Second, comparison of the data from portions of the flap destined to live with those destined to die suggests that it may not be failure of circulatory inflow that leads to flap death.     

Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Benthic algal biomass and productivity in high subarctic streams,Alaska

Year-round measurements of the standing crop of epilithic algae (as chlorophyll a concentration) in two streams — one second and one fourth order (map scale 1:63 360) — in interior Alaska (64°–65° N) were only about one tenth that reported from streams of temperate North America. Cell densities in these streams, however, were similar to those in comparable temperate streams. Year-round domination of the benthic flora by very tiny diatoms (Achnanthes spp.) may explain the apparent disparity between low chlorophyll a content and nearly average cell densities. Chlorophyll a standing crop in a more alkaline groundwater-fed stream, however, was higher and within the range of similarly sized temperate streams. Maximum chlorophyll a standing crop varied positively with alkalinity in 5 clear-water streams where standing crop was measured on natural or artificial substrates. Seasonal mean concentrations of sestonic chlorophyll a (used as estimates of benthic algal chlorophyll a standing crop) varied directly and significantly with alkalinity among ten clear-water streams; and, with total phosphorus among 8 of 10 clear-water and 5 brown-water streams studied. During the summer, when there is little darkness, gross primary productivity (as estimated by the diurnal dissolved-oxygen method) was similar to that of northern temperate streams. Gross primary productivity was also seen to vary directly with alkalinity in 5 clear-water streams of this region.U.S. Fish and Wildlife Service     

Early detection and treatment of hyperlipidemia: physician practices in Canada.

We surveyed primary care physicians in Canada to determine their current practices regarding the detection and treatment of hyperlipidemia in asymptomatic adults 20 years of age or more and to determine the role of selected patient characteristics (age, sex and the presence of coronary heart disease [CHD] risk factors) in their management decisions. The self-administered questionnaire was completed by 428 of 804 family physicians and general practitioners. The proportion of physicians who reported having tested at least 50% of their adult patients varied from 29% to 85% and was related to the number of CHD risk factors present and the patient''s age. The proportion of respondents who reported starting dietary or drug therapy among patients with a cholesterol level of 6.2 mmol/L or less increased as the number of CHD risk factors increased and was not related to patient age or sex. According to the factors examined our results suggest that primary care physicians in Canada select patients for screening and treatment mainly on the basis of CHD risk factors present and that their approach is more conservative than that recommended by the Canadian and US consensus conferences.