Dihydrofolate Reductase Activity in Strains of Streptococcus faecium var. durans Resistant to Methasquin and Amethopterin

Resistance to the antifolates methasquin and amethopterin has been studied in new strains of Streptococcus faecium var. durans. Two methasquin-resistant strains (SF/MQ, SF/MQ(T)) and an amethopterin-resistant strain (SF/AM) were selected independently from the wild-type S. faecium var. durans (SF/O). SF/MQ(T) is a thymine auxotroph. Total dihydrofolate reductase activity was elevated in each of the resistant strains. The greatest increase (36-fold) was observed in extracts of SF/AM. The methasquin-resistant strains, SF/MQ and SF/MQ(T), had 29-fold and 8-fold, respectively, more dihydrofolate reductase activity than the parental strain. Total dihydrofolate reductase activity of SF/O was separable by gel filtration into two components: a folate reductase (11%) and a specific dihydrofolate reductase (89%). Folate reductase activity was associated with 88% of the total dihydrofolate reductase activity of SF/MQ(T), with specific dihydrofolate reductase activity accounting for the remaining 12%. In SF/MQ and SF/AM, folate reductase activity was associated with 97% of the total dihydrofolate reductase activity. Studies of the inhibition by methasquin and amethopterin of partially purified folate reductase and specific dihydrofolate reductase of the mutant strains suggested that resistance was not accompanied by changes in the affinities of these enzymes for either antifolate.     

Increased resistance to Listeria monocytogenes following subchronic cyclophosphamide exposure: Relationship to altered bone marrow function

Mice administered multiple doses of cyclophosphamide demonstrated a marked resistance to infection with the bacterium, Listeria monocytogenes. In contrast, acute exposure rendered mice more susceptible to infection than untreated controls. Resistance to infection with Listeria, a facultative intracellular organism, is thought to be dependent upon normal antimicrobial activity early after infection and subsequently through generation of primed T cells. Examination of various macrophage and immune functions, however, failed to demonstrate a significant difference between the two cyclophosphamide-treated groups although both groups were immunosuppressed when compared to untreated controls. Adoptive transfer studies into X-irradiated recipients revealed that repopulation with bone marrow cells from subchronic but not acute cyclophosphamide-treated mice, restored resistance. Furthermore, the numbers of granulocyte/macrophage progenitor cells in the bone marrow were elevated in subchronically treated mice but not acute or unexposed controls. These data suggest the selection of a granulocyte/macrophage progenitor cell possessing a high degree of antilisterial activity following subchronic cyclophosphamide treatment. The effects induced by this exposure regimen are probably related to the enrichment of this cell population resulting from the cell cycle stage specific activity of the drug.     

Three genetic variants of human plasma apolipoprotein A-IV. apoA-IV-1(Thr347----Ser), apoA-IV-0(Lys167----Glu,Gln360----His), and apoA-IV-3(Glu165----Lys).

Human apolipoprotein (apo) A-IV is a genetically polymorphic glyco-protein of 376 residues. We have recently reported the molecular basis for the two most common isoproteins, apoA-IV-1 and apoA-IV-2(Gln360----His), and for two rare variants, apoA-IV-0 (4-amino acid insertion) and apoA-IV-3(Glu230----Lys). In this report, we present the genetic basis for three additional isoproteins of apoA-IV. Sequence analysis of the apoA-IV-1 allele revealed a common nucleotide substitution which converts threonine (ACT), residue 347 of the mature protein, into serine (TCT). In one out of the five subjects with the apoA-IV-1/0 phenotype we identified two point mutations: 1) replacing the positively charged lysine (AAG), amino acid 167, with a negatively charged glutamic acid (GAG), and 2) converting the neutral residue 360, glutamine (CAG), to a positively charged histidine (CAT). We have also characterized four additional heterozygotes for the apoA-IV-3 allele. One individual was found to have the previously described substitution of lysine for glutamic acid at amino acid 230. The three other subjects had the identical mutation but at a different position in the polypeptide chain, residue 165. These results indicate that one predominant allele codes for the isoproteins apoA-IV-2 and apoA-IV-0 and that at least two major allelic variants for the isoproteins apoA-IV-1 and apoA-IV-3 are present in the population analyzed.     

Telemetered renal responses in dogs during detection of explosives.

The primary objectove of this work was to develop and test a method of measuring and characterizing renal hemodynamic responses in unrestrained dogs. Recordings of left kidney blood flow and abdominal aortic pressure were obtained from unrestrained dogs through the use of a two-channel implanted telemetry system during episodes of search and detection of simulated explosives. In each of a number of sequences, the dog was first given a start signal, and after locating the hidden device, was rewarded with food. Data were assessed at the start, find, and recovery segments. The dynamic flow and pressure, together with a hydraulic renal model, were used to derive the total preglomerular and postglomerular vascular resistances and the mean level of glomerular hydrostatic pressure. Data from three dogs obtained by telemetry and analyzed through the use of the model have shown that compared to the start of the test, the location of hidden explosives results in a decrease in both the level of mean aortic blood pressure and the preglomerular resistance; whereas, the reward results in an elevation of mean blood pressure and preglomerular resistance. The postglomerular resistance varied less than the preglomerular resistance, and mean flow did not vary significantly. This work has shown that the stimulation of a reward and the successful performance of a task lead to significant renal responses in dogs. It has further shown that telemetry, when employed with improved data analysis techniques, permits renal hemodynamics to be assessed in unrestrained animal subjects.     

Rapid separation of mouse T and B lymphocytes using wheat germ agglutinin.

A separation procedure has been developed for mouse splenic T and B lymphocytes which is based on their differential agglutination by wheat germ agglutinin (WGA). In the presence of 50-100 micrograms/ml of WGA, multicellular aggregates are formed which are enriched in B cells. These aggregates can be separated from monodisperse T cells by gravity sedimentation and subsequently dissociated into single cells by treatment with N-acetylglucosamine (NAG). Immunocytochemical analyses and mitogenic assays indicate approximately 10-15% cross contamination of the resultant B and T cell fractions. The separation procedure is not only convenient and rapid but also allows the simultaneous recovery of viable T and B cells from the same spleen preparation.     

Mayfly production in a Colorado mountain stream: an assessment of methods for synchronous and non-synchronous species

Year-round collections of mayflies (Ephemeroptera) from a Colorado mountain stream allowed critical examination of several methods of calculating production for species with different life cycles. Five of the six numerically dominant species exhibited slow seasonal, univoltine life cycles. Baetis tricaudatus was bivoltine. Two species demonstrated well synchronized development, three species were poorly synchronized and a sixth was intermediate. Mean density and biomass data from each sampling date were used to ascertain the goodness-of-fit of each species to the Allen curve. It is proposed that such information can provide quantitative criteria for identifying species with well synchronized development and thereby determine when it is appropriate to directly apply cohort methods while avoiding time intensive body size (e.g. head width) measurements necessary for size-frequency analyses. In addition, these data demonstrate that species specific production varies with gross changes in elevation.     

Hepatic proprotein convertases modulate HDL metabolism

The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.     

ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

ApoE plays an important role in lipoprotein metabolism. This study investigated the effects of adenovirus-mediated human apoE overexpression (AdhApoE3) on sterol metabolism and in vivo reverse cholesterol transport (RCT). In wild-type mice, AdhApoE3 resulted in decreased HDL cholesterol levels and a shift toward larger HDL in plasma, whereas hepatic cholesterol content increased (P < 0.05). These effects were dependent on scavenger receptor class B type I (SR-BI) as confirmed using SR-BI-deficient mice. Kinetic studies demonstrated increased plasma HDL cholesteryl ester catabolic rates (P < 0.05) and higher hepatic selective uptake of HDL cholesteryl esters in AdhApoE3-injected wild-type mice (P < 0.01). However, biliary and fecal sterol output as well as in vivo macrophage-to-feces RCT studied with (3)H-cholesterol-loaded mouse macrophage foam cells remained unchanged upon human apoE overexpression. Similar results were obtained using hApoE3 overexpression in human CETP transgenic mice. However, blocking ABCA1-mediated cholesterol efflux from hepatocytes in AdhApoE3-injected mice using probucol increased biliary cholesterol secretion (P < 0.05), fecal neutral sterol excretion (P < 0.05), and in vivo RCT (P < 0.01), specifically within neutral sterols. These combined data demonstrate that systemic apoE overexpression increases i) SR-BI-mediated selective uptake into the liver and ii) ABCA1-mediated efflux of RCT-relevant cholesterol from hepatocytes back to the plasma compartment, thereby resulting in unchanged fecal mass sterol excretion and overall in vivo RCT.