Compartmentation of intracellular nucleotides in mammalian cells

The important role of nucleotides in cellular metabolism requires that serious consideration be given to the question of the homogeneity or inhomogeneity of nucleotide pools in cells. The purpose of this review is to summarize the existing evidence for compartmentation of nucleotide pools, discuss the limitations of this evidence, and to discuss the implications of compartmentation for the interpretation of nucleotide concentration measurements. Evidence for nucleotide compartmentation comes from the following types of evidence: compartmentation of RNA precursors; compartmentation of deoxynucleoside triphosphates; mitochondrial compartmentation; the existence of tightly bound nucleotides; pools derived from alternative synthetic routes; compartmentation in cyclic nucleotide metabolism; channeling in the synthesis of pyrimidine nucleotides; and others. The types of evidence adduced for compartmentation will be considered critically and in detail, and alternative explanations considered, as well. Implications of the data and hypotheses on nucleotide compartmentation for the interpretation of nucleotide pool measurements in various types of experiments will be discussed.     

Influenza vaccination in the elderly: 2. The economics of sending reminder letters

Reminder letters and follow-up telephone calls were used to increase influenza vaccination acceptance by 273 well elderly registered at an urban community health centre. The net effect of the reminder letters was to increase overall coverage to 43%, from 17% in the previous year. Follow-up telephone calls to patients who had not responded to the letters increased coverage to only 55%. Calculation of costs per additional vaccination given revealed that the use of reminder letters alone was much more cost-effective than follow-up telephone calls in increasing coverage. However, with the current fee-for-service reimbursement by medical care insurance in Ontario, neither means of improving vaccination coverage would result in net practice earnings. The implications for an effective and efficient annual influenza program in Canada are discussed.     

T-cell-mediated regression of "spontaneous" and of Epstein-Barr virus-induced B-cell transformation in vitro: studies with cyclosporin A

The regression of Epstein-Barr (EB) virus-transformed B-cell outgrowth which is seen in experimentally-infected cultures of blood mononuclear (UM) cells from healthy seropositive donors can be abolished in medium containing the T-cell-suppressive agent cyclosporin A (CSA) at concentrations of 0.05 microgram/ml and above. CSA mediates its effect within the first 4 days post-infection of the UM cells and this prevents subsequent in vitro generation of the EB virus-specific cytotoxic-T-cell response which normally brings about regression. Regression can be fully restored by supplementing the CSA-treated culture with interleukin 2 (IL-2)-containing culture supernatants or indeed with purified IL-2 itself, suggesting that CSA mediates its effect in this system through inhibiting the endogenous production of IL-2 which is required to amplify the virus-specific cytotoxic response. "Spontaneous transformation" to EB virus genome-positive lymphoblastoid cell lines in noninfected cultures of UM cells from healthy seropositive donors, though rare in normal medium, is enhanced to such a degree in the presence of CSA that, for many donors, the phenomenon becomes titratable against input cell dose across the 2.0 X 10(6)-2.5 X 10(5) cells/culture range. Cell mixing experiments suggest that the spontaneously transformed cell lines which arise with such efficiency under these conditions do so not by direct in vitro outgrowth of progenitor cells transformed by the virus in vivo, but by a two-step mechanism involving virus release and secondary infection in vitro.     

The diets of Hispaniolan colubrid snakes

Summary Approximately 1590 Hispaniolan colubrid snakes representing six genera and eight species were examined for prey remains (Alsophis cantherigerus, Antillophis parvifrons, Darlingtonia haetiana, Hypsirhynchus ferox, Ialtris dorsalis, Uromacer catesbyi, U. frenatus, and U. oxyrhynchus). The snakes were collected at many localities over a span of 80 years.Of 426 prey items, 77.9% were lizards (of which 69.6% were anoles), 19% frogs, 2.6% birds and mammals, and 0.5% other snakes. Darlingtonia was the only snake that did not exploit lizards; it fed exclusively on Eleutherodactylus frogs, including egg clutches. Disregarding Darlingtonia, there is no size class of Hispaniolan colubrids between 20–90 cm SVL that does not prey primarily on Anolis. Certain prey genera are added to, or deleted from, diets depending on snake size, but the data suggest that snake SVL alone does little to dictate what prey genera (or groups) are eaten. Shannon-Wiener values (H') indicate that Darlingtonia has the narrowest trophic niche, while Alsophis and Ialtris have the widest. Values of H' are not correlated with snake SVL, but highly significant (P<0.001) correlations exist between H' and mid-body circumference, head width, and snout width, and these characters may be indicators of trophic generalists and specialists. Anolis lizards are the most ubiquitous and conspicuous vertebrates on Hispaniola, and it is not surprising that they are widely exploited as a food source. Although as some snake species grow larger, anoles play a decreasingly important role in their diets, there is no evidence to suggest that they are ever abandoned as a food source by any Hispaniolan colubrid of any size.Secretive lizards of low vagility are eaten almost exclusively by wide ranging foragers (Alsophis, Antillophis); very active prey (Ameiva) is taken by sit-and-wait strategists (Hysirhynchus, U. frenatus). Those snakes which exploit the most prey groups are active foragers. Uromacer catesbyi exhibits both foraging modes, and predictably, eats diurnally active (anoles) and diurnally quiescent (hylid frogs) prey with almost equal frequency.Within Maglio's cantherigerus species assemblage, in which an Alsophis cantherigerus-like snake was ancestral to the other species, and in which longsnouted Uromacer are the most morphologically derived, there is an obvious trend toward trophic specialization on Hispaniola. The West Indies have provided an ideal natural laboratory for the investigation of many aspects of vertebrate ecology, and an arena in which to test theories of island biogeography. The most extensively studied West Indian vertebrates have been the lizards of the iguanid genus Anolis. Conversely, the ecology of West Indian snakes has been largely ignored. This is surprising in light of the fact that much has been written about Anolis predation, but little has been written about predators of Anolis; snakes may be important, frequent consumers of anoles.Hispaniola is physiographically and ecologically the most diverse of the Greater Antilles and, concomitantly, it has the most diverse snake fauna, including six colubrid genera containing 11 described species. It has rich frog and lizard faunas, but only two endemic mammals. Study of the diets of Hispaniola's colubrid snakes was undertaken to gain initial insights into the ecology of the snakes and to determine 1) what the snakes eat; 2) what relationships exist between snake diet and snake size as well as head and body proportions; 3) what relationships exist between snake foraging mode and prey type and size; 4) if anoles, as the most ubiquitous and conspicuous vertebrates on Hispaniola, comprise an important source of food; 5) if significant geographical differences in diet exist, expecially on satellite islands; 6) if north island and south island (sensu Williams 1961) Anolis ecomorphs are preyed upon by the same snake species in similar proportions; 7) if snakes are selective or opportunistic predators.This paper, the first in a series that will address all of the above topics, will briefly describe methods, snake species and prey genera. Prey genera are analyzed in terms of what snake taxa prey upon them, what size classes of snakes prey upon them, and prey genera diversity versus snake size and proportions.     

Location of a structural gene for xylose-H+ symport at 91 min on the linkage map of Escherichia coli K12

Mutations in the xylose-H+ transport activity of Escherichia coli K12 were isolated using Mud(ApRlac). The initial selection was for simultaneous acquisition of ampicillin and xylose resistance in an fda background. Colonies were then screened for xylose-inducible beta-galactosidase and for growth on xylose of their fda+ derivatives. Two of the xylose-positive derivatives were shown to be impaired in xylose-H+ symport in whole cells and in xylose transport into subcellular vesicles. Their xylose transport in whole cells showed increased sensitivity to arsenate. The site of prophage insertion was mapped to 91.4 min on the E. coli genome between pgi and malB. It is proposed that the gene for the xylose-H+ symport system be called xylE.     

LH concentrations in two cattle with XY gonadal dysgenesis

Two animals with XY gonadal dysgenesis both had a reproductive tract similar in size to that found in sexually immature heifers, but neither had normal testicular or ovarian tissue. All cells examined in both animals contained XY chromosomes and spinal cord neurones were sex chromatin negative. Basal LH concentrations averaged 3.1 ng/ml in Animal 1 and 2.4 ng/ml in Animal 2 but increased within 12 h of injecting oestradiol to peak concentrations of 125 and 11 ng/ml respectively. Animal 1 displayed a distinct pulsatile LH release pattern with a highly repeatable decline phase at each pulse. A GnRH injection produced a rapid rise in plasma LH in both animals, sustained in Animal 1 at greater than 500 ng/ml for more than 2 h. Each animal displayed behavioural symptoms of oestrus within 12 h of being injected with 3 mg oestradiol benzoate and was repeatedly served by a bull. These studies indicated that both animals differed from freemartins and had some hypothalamic and pituitary response patterns resembling those reported for female cattle.     

Analysis of the biological properties of antibodies raised against intact and deglycosylated porcine zonae pellucidae

Zonae pellucidae (ZP) were isolated from 1,500 porcine ovaries and heat solubilized to generate approximately 15 mg ZP glycoprotein. Analysis of this material by isoelectric focusing, one-dimensional electrophoresis, and gas chromatography indicated the presence of a major glycoprotein species that exhibited considerable microheterogeneity with respect to its charge (pI 7.5–3.5) and molecular mass (45–85 kDa) and that contained 39.6% carbohydrate, predominantly N-acetylglucosamine. Chemical deglycosylation of porcine ZP using trifluoromethanesulphonic acid (TFMS) resulted in the production of five discrete protein bands on one-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) with molecular masses of 66, 52, 36, 32, and 16 kDa. Antisera raised in rabbits and marmosets to ZP and/or deglycosylated ZP (DGZP) were used in immunoblotting experiments to demonstrate the retention of immunogenicity by DGZP and the cross-reactivity of the antisera with their heterologous antigen. These studies indicated that antisera that were capable of inhibiting the fertility of primates in vivo and the penetration of the human ZP in vitro reacted preferentially with 3 of the 5 products of deglycosylation, with molecular masses of 66, 52, and 36 kDa. Anti-DGZP antibodies were also shown to interact with intact porcine and human ZP and, with the latter, to block the ability of human spermatozoa to both bind to and penetrate this structure.     

Structural requirements for anion substrates of the methotrexate transport system in L1210 cells

A broad spectrum of structurally diverse anions reversibly inhibits the influx of methotrexate in L1210 cells. Several of the more effective anions and their respective inhibition constants (K_i values) were: 5-methyltetrahydrofolate (0.3 μm), bromosulfophthalein (2 μm), thiamine pyrophosphate (3 μm), 8-anilino-1-naphthalene sulfonate (7 μm), phthalate (20 μm), and AMP (50 μm). Moderate inhibition was observed with P_i (K_i = 400 μm) and other divalent inorganic anions, while small monovalent anions such as Cl^? (K_i = 30 mm) were the least effective. When these same anions were tested for an effect on methotrexate efflux, stimulation was observed with some anions, while others had no effect. Enhancement was produced by folate compounds and p-aminobenzoylglutamate, small monovalent (e.g., Cl^?, acetate, and lactate) and divalent (e.g., phosphate and succinate) anions, a few nucleotides (e.g., AMP), and thiamine pyrophosphate, while little or no effect was associated with trivalent anions (e.g., citrate), most nucleotides, and large organic anions (e.g., bromosulfophthalein, NAD, and NADP). Anions with the ability to promote methotrexate efflux in control cells lost this capacity upon exposure of the cells to an irreversible inhibitor of methotrexate influx. These results support the hypothesis that methotrexate transport proceeds via an anion-exchange mechanism and moreover provide evidence that anion substrates for this system can be identified by their ability to promote methotrexate efflux. Anions which appear most likely to participate in this exchange cycle in vivo are P_i and AMP.     

A kinetic analysis of the effects of inhibitor-1 and inhibitor-2 on the activity of protein phosphatase-1

The steady-state interaction between protein phosphatase-1 and its two inhibitor proteins was studied in vitro at low enzyme concentrations where the assumptions of the Michaelis-Menten equation appeared to be valid. Under these conditions, and in the absence of divalent cations, inhibitor-1 behaved as a mixed inhibitor using phosphorylase alpha as a substrate, whereas inhibitor-2 was a competitive inhibitor. The results demonstrate that inhibitor-1 and inhibitor-2 do not interact with protein phosphatase-1 in an identical manner. Inhibitor-1 was only a substrate for protein phosphatase-1 in the presence of Mn2+, and its dephosphorylation was inhibited competitively by inhibitor-2 (Kis = 8 nM). Inhibitor-1 did not inhibit its own dephosphorylation in the presence of Mn2+. Its Km as a substrate (190 nM) was very much higher than its Ki as an inhibitor (1.5-7.5 nM). The results are consistent with a model in which a single binding site for inhibitor-1 is present on protein phosphatase-1, distinct from the binding site for phosphorylase alpha. It is envisaged that the binding of inhibitor-1 to this site not only inhibits the dephosphorylation of other substrates but permits access of its phosphothreonine to the same catalytic group(s) responsible for the dephosphorylation of other substrates. G-substrate, a protein phosphorylated exclusively on threonine residues, did not inhibit the dephosphorylation of phosphorylase alpha and its dephosphorylation was potently inhibited by inhibitor-1 or inhibitor-2. The role of the phosphothreonine residue in inhibitor-1 is discussed in the light of these results.