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W H Telfer

Publications and source records attributed to W H Telfer.

At least 19 recordsLinked to original sources
PubMed2001

Storage hexamer utilization in two lepidopterans: differences correlated with the timing of egg formation.

Most insects produce two or more storage hexamers whose constituents and developmental profiles are sufficiently different to suggest specialization in the ways that they support metamorphosis and reproduction. Hexamerin specializations are compared here in the Cecropia moth (Hyalophora cecropia), which produces eggs during the pupal-adult molt, and the Monarch butterfly (Danaus plexippus), which produces eggs under long-day conditions after adult eclosion. In both sexes of both species, reserves of arylphorin (ArH) were exhausted by the end of metamorphosis. In Cecropia, the same was true for the high-methionine hexamerins, V-MtH and M-MtH. But in short day Monarch females 20-30% of the pupal reserves of V-MtH and M-MtH survived metamorphosis, persisting until long-day conditions were imposed to stimulate egg formation. Differences in storage sites have been documented in other lepidopterans, with MtH reserves being found primarily in fat body protein granules and the ArH reserve being found primarily in the hemolymph. Similar differences could explain how a fraction of the MtH's, but not of ArH, escapes utilization during metamorphosis in a species with post-eclosion egg formation. No differences in utilization schedules were detected between V- and M-MtH, despite divergent compositions and antigenic reactivity.

Amino Acid Sequence↗
PubMed2000

Cyclic nucleotide-dependent protein phosphorylation in vitellogenic follicles of Hyalophora cecropia.

(1) In homogenates of vitellogenic follicles from Hyalophora cecropia, cyclic nucleotides promoted the transfer of label from [gamma32P]-ATP to at least four polypeptides. PKI (6-20) amide, an inhibitor of PKA (cAMP-dependent protein kinase), prevented all four reactions. Quantitative tests using kemptide as a substrate indicated that 80% of the total follicular PKA activity was localized in the follicle cells; labeling at 45, 32, and 27 kDa was particle-associated and also restricted to the follicle cells, while a 58 kDa substrate was labeled only in homogenates of the oocyte. (2) When intact follicles were incubated in [32P]-phosphate and okadaic acid, a protein phosphatase inhibitor, the 32 kDa substrate again exhibited cAMP-dependent labeling. There was thus a physiological relationship between PKA activation and 32 kDa protein phosphorylation, while exposure of at least two of the other three substrates to appropriate kinases required homogenization. The latter was illustrated by phosphorylation of the 42 kDa small subunit of vitellogenin, which occurred only when homogenization mixed the proteins of the yolk bodies with cytoplasmic kinases. (3) PKA activation is known to promote the termination of vitellogenesis, even in the absence of detectable labeling of the 32 kDa substrate. The possibility remains that phosphorylation at 32 kDa concerns later aspects of postvellogenic development that were not tested by the assay system used here.

Animals↗
PubMed1999

Equivalence of riboflavin-binding hexamerin and arylphorin as reserves for adult development in two saturniid moths.

The riboflavin-binding hexamerin (RbH) and arylphorin (ArH) were compared as storage reservoirs for adult development in Hyalophora cecropia. The two hexamerins were metabolically labeled with [3H]leucine and [35S]methionine, isolated by column chromatography, and separately injected into pupae whose diapause had been terminated by chilling. By the time of eclosion at least 98% of both hexamerins had been cleared from the hemolymph. Every reproductive and somatic tissue tested contained trichloroacetic acid-precipitable label; consistent differences between the two hexamerins were not detected in the distribution of their label to these tissues. While incorporation of intact hexamerins was not ruled out, hydrolysis and reincorporation of the liberated amino acids were indicated by label in vitellogenin and lipophorin, and by differences in 35S/3H ratios, which ranged from over 1.0 in chorions to 0.4 in wings, as compared with 0.75 in the injected hexamerins. Injection of [35S,3H]RbH from H. cecropia into A. luna, a species in the same subfamily whose pupae lack this hexamerin, resulted in a pattern of isotope incorporation similar to that yielded by RbH in the donor species. In neither species was there indication of a developing adult tissue that distinguished between RbH and ArH as precursor reservoirs for morphogenesis. This equivalence helps explain how many species of Lepidoptera are able to complete metamorphosis and reproduce without expressing an RbH gene. Evidence is also presented that ArH stored in the fat body protein granules during pupation may be utilized differently from that remaining in pupal hemolymph.

Animals↗
PubMed1998

Pertussis toxin-sensitive G protein that supports vitellogenin uptake by promoting patency.

Ovarian follicles of Hyalophora cecropia stopped accumulating [35S]vitellogenin when incubated in pertussis toxin, a Gi protein inactivator. At a cellular level, the responses to pertusis toxin resembled those described earlier to cell-permeant analogs of cyclic AMP. They included accelerated 36Cl-exchange, 86Rb+ uptake, and follicle cell swelling, which in turn resulted in a loss of epithelial patency. A 34% rise in follicular cAMP content accompanied these changes. In particulate fractions of follicle homogenates, pertussis toxin catalyzed the ADP-ribosylation of a polypeptide that resolved at 39 kDa in SDS-PAGE; rabbit antibodies to a C-terminal decapeptide common to 39 kDa mammalian Gi alpha-3 and G(o) alpha were bound in immunoblots at this same location. The findings suggest that a pertussis toxin-sensitive G alpha facilitates epithelial patency during vitellogenesis by suppressing cAMP levels. When follicles are released from this restraint, either experimentally with pertussis toxin or by progressing to the next phase in their normal program of development, cAMP levels rise and vitellogenesis terminates.

Adenosine Diphosphate Ribose↗
PubMed1998

Cyclic-AMP-induced water uptake in a moth ovary: inhibition by bafilomycin and anthracene-9-carboxylic acid.

The ion physiology of osmotic swelling and a consequent loss of epithelial patency was examined in the ovaries of the moth Hyalophora cecropia. After 30 min in the presence of an activator of cyclic-AMP-dependent protein kinase (PKA), the membrane potentials of both oocyte and follicle cells had hyperpolarized by approximately 30 %, cytoplasmic pH had dropped from 7.26 to 7.06, a normally low Cl- conductance had increased and the follicle cells had begun to swell. Since ion distribution studies have indicated that conductance increases should depolarize membranes in this system, it is proposed that hyperpolarization may be effected by an azide-inhibitable component of the membrane potential. Nanomolar levels of bafilomycin, an inhibitor of H+ V-ATPase, blocked the active component and prevented osmotic swelling in response to PKA activation. Under a variety of circumstances, correlations were seen between membrane potential and cytoplasmic pH, suggesting that substrate availability to the proton pump may contribute to hyperpolarization. H+ V-ATPases are known to energize ion and water transport across many epithelia, but in this case they generate water absorption by the epithelium. The increase in Cl- conductance was also required for the swelling response: the Cl- channel blocker anthracene-9-carboxylic acid prevented both swelling and hyperpolarization, as did Cl- substitution in the medium. Differences in isotope loading rates between 36Cl- and 86Rb+ suggested that, after PKA activation, Cl- functions other than as a counterion for K+ uptake.

Animals↗
PubMed1996

Methionine-rich hexamerin and arylphorin as precursor reservoirs for reproduction and metamorphosis in female luna moths.

The storage proteins of Lepidoptera include a pair of methionine-rich hexamerins (MtH) that are more abundant in female pupae than in males. Their inferred support of female reproduction could be achieved either by enhancing general pools of amino acids, or by hydrolyzing MtH at times and/or sites that direct its constituents to the synthesis of egg proteins. The two models were tested in Actias luna, a saturniid moth that makes its eggs during adult development. MtH and arylphorin (ArH), the third major storage protein of this species, were labeled metabolically with [35S]-methionine and [3H]-leucine, and injected individually into wandering stage caterpillars. Isotope distributions at eclosion indicated that both hexamerins supported egg formation as well as adult tissue protein synthesis. In the absence of evidence for targeting, MtH appears to support egg formation in A. luna by enhancing the amino acid pools derived from ArH. Analysis of 35S labeling and of 35S/3H ratios indicated, however, that ArH is consumed over a period that extends somewhat later in adult development than MtH. Differences in timing should prove to be much greater in Lepidoptera that delay egg formation until after eclosion.

Animals↗
PubMed1996

Cyclic nucleotide-induced termination of vitellogenin uptake by Hyalophora cecropia follicles.

Endocytosis of vitellogenin by isolated follicles of Hyalophora cecropia terminated after membrane-permeable analogs of cAMP or cGMP were added to the culture medium. Depending on the concentration of the analog, a lag period of 30 min to 3 h preceded termination. Forskolin and IBMX both stimulated a rise in endogenous cAMP, and this also induced termination, as did pharmacological activation of the cyclic nucleotide-dependent protein kinases PKA and PKG. Inhibitors of PKA or PKG protected follicles from the corresponding cyclic nucleotide effect. When cAMP or cGMP was added to homogenates of vitellogenic follicles, a 32 kDa polypeptide was phosphorylated; inhibition of PKA, prevented phosphorylation of this protein. The rate of vitellogenin uptake did not accelerate significantly when PKA or PKG was inhibited in culture, which suggests that these kinases are normally inactive or operating below threshold during the several days of vitellogenesis. They seem thus not to be involved in the steady-state modulation of protein uptake. A more likely function of this control pathway in follicle development would be to trigger the termination of vitellogenesis, which normally occurs spontaneously in follicles of this species as they reach a length of 2 mm.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Steady-state gradient in calcium ion activity across the intercellular bridges connecting oocytes and nurse cells in Hyalophora cecropia.

Intracellular activities of K+, H+, Mg2+, Ca2+, and Cl-, measured with ion selective microelectrodes in the oocyte and the nurse cells in ovarian follicles of Hyalophora cecropia, indicated that a Ca2+ current is a key component of the electrical potential that is maintained across the intercellular bridges connecting these two cells. In vitellogenic follicles, Ca2+ activity averaged 650 nM in the oocyte and 190 nM in the nurse cells, whereas activities of the other ions studied differed between these cells by no more than 6%. Incubation in 200 microM ammonium vanadate caused a reversal of electrical potential from 8.3 mV, nurse cell negative, to 3.0 mV, oocyte negative, and at the same time the Ca2+ gradient was reversed: activities rose to an average 3.0 microM in the nurse cells and 1.6 microM in the oocyte, whereas transbridge ratios of the other cations remained at 0-3%. In immature follicles that had not yet initiated their transbridge potentials, Ca2+ activities averaged approximately 2 microM in both oocyte and nurse cells. The results suggest that vitellogenic follicles possess a vanadate-sensitive Ca2+ extrusion mechanism that is more powerful in the nurse cells than in the oocyte.

Animals↗
PubMed1994

Properties and significance of a riboflavin-binding hexamerin in the hemolymph of Hyalophora cecropia.

A riboflavin-binding hexamerin isolated from pupal hemolymph of Hyalophora cecropia has a native M(r) of 510,000, subunit M(r) of 85,000, and a 5% carbohydrate content. An intrachain cross-link was confirmed in protease limit digests. Ellman titration confirmed the presence of a sulfhydryl group, which is needed for this linkage. Though Cu2+ is known to promote the linkage, heavy metals were not detected in the isolate. Heat denaturation released ligand with the absorbency, fluorescence spectra, and chromatographic behavior of riboflavin. Binding resulted in substantial quenching of the fluorescence of both the isoalloxazine in riboflavin and of aromatic groups in the apoprotein. Kinetic analysis indicated a KD of 2.5 x 10(-7) M for riboflavin, 1.3 x 10(-7) M for lumiflavin, and greater than 1 x 10(-6) M for FMN and FAD. Over four moles of flavin were bound per mole of hexamerin. The amount of riboflavin in pupal hemolymph is sufficient to occupy only 2-3 of these sites. Riboflavin is also associated with lipophorin and vitellogenin, but the molar ratios after protein isolation were low. On a standard laboratory diet, riboflavin is in great excess, but most of it is apparently excreted before the apoprotein first appears in the hemolymph, just before wandering. The concentration of riboflavin-binding hexamerin rises to 15-30 mg/ml in pupae; relative to other hexamerins, very little is stored in the fat body. All of the apoprotein and 75% of riboflavin disappear from the hemolymph during adult development. An amount of flavin at least equal to that stored in pupal hemolymph is transferred to the eggs formed during this period.

Animals↗
PubMed1992

Selectivity in storage hexamerin clearing demonstrated with hemolymph transfusions between Hyalophora cecropia and Actias luna.

When Hyalophora cecropia hemolymph was injected into wandering Actias luna larvae, a methionine-rich hexamerin was selectively transferred to the host's fat body, and completely cleared from the hemolymph by the time of pupal eclosion. Donor arylphorin was 30-40% removed from the hemolymph, and riboflavin-binding hexamerin was even less completely cleared. During the pupal-adult molt, these rates were reversed: methionine-rich hexamerin disappeared no faster than bovine serum albumin, while riboflavin-binding hexamerin was rapidly and completely cleared from the hemolymph, even though A. luna hemolymph lacks a homologue of this protein; arylphorin, again, was cleared at an intermediate rate. Selective clearing of the three hexamerins occurred at similar stages in H. cecropia, their species of origin. Developmentally programmed clearing, with selectivity at least partially conserved between genera, was also demonstrated with transfused vitellogenin: in A. luna females that were forming yolk, H. cecropia vitellogenin was cleared more rapidly than bovine serum albumin; but in younger females, and in males at all stages of metamorphosis, this Mr 510,000 molecule was instead an indicator of nonselective, large protein clearing. Nonselective clearing was more complete during adult development than during pupation. It also showed signs of being more effective for small than for large proteins, insensitive to carbohydrate conjugates, and unsaturated at the protein levels used.

Animals↗
PubMed1990

Lipophorin as a yolk precursor in Hyalophora cecropia: uptake kinetics and competition with vitellogenin.

Vitellogenic follicles of Hyalophora cecropia were incubated in metabolically radiolabeled, high-density lipophorin isolated from pharate adult hemolymph by KBr density gradient centrifugation. The follicles transferred this probe from the incubation medium to the cortical yolk spheres in the oocyte by an energy-dependent and saturable mechanism. Vitellogenin and high-density lipophorin competed with each other for uptake, and are therefore concentrated by the follicle with a common mechanism. Microvitellin and lipophorin, in contrast, did not compete for uptake. The K(uptake) for the accumulation of high-density lipophorin was substantially higher than the value estimated earlier for vitellogenin (133 microM vs. 18 microM). This relationship helps explain why the shared concentrating mechanism does not deplete the lipid transport capacity of the hemolymph, and how a low vitellogenin: lipophorin molar ratio in the hemolymph yields a high ratio in the mature egg.

Animals↗
PubMed1990

Activation of a new physiological state at the onset of vitellogenesis in Hyalophora follicles.

Ovarian follicles of Hyalophora cecropia are shown here to undergo a comprehensive transformation about 2 days after they are first formed and several hours before the first yolk spheres are visible. (1) Electrical coupling initiates between the follicle cells and the oocyte-nurse cell complex, as well as between adjacent follicles. (2) Oocyte and nurse cell membranes begin to hyperpolarize, adding an azide- and vanadate-sensitive component to a basal potential that is unaffected by these inhibitors. (3) The cytoplasmic pH of the oocyte rises from 6.7 to 7.4. (4) The nurse cells hyperpolarize more strongly than the oocyte, so that the charge-dependent restrictions on protein movement across the cytoplasmic bridges that connect these cells arise at this time. (5) The follicle swells and becomes more turgid. (6) Uridine incorporation shuts down in the germinal vesicle and accelerates in the other nuclei of the follicle. The changes are sufficiently synchronous to suggest that they may be responses to a single, branching cascade of activation. The altered cell potentials, cytoplasmic pH, and turgidity implicate the cell membrane in an early stage of activation.

Animals↗
PubMed1980

Electrophoresis of proteins in intercellular bridges.

Cytoplasmic polarity giving rise at mitosis to daughter cells with distinct but complementary morphogenetic functions has been proposed by Jaffe and co-workers to have transcellular ion currents as one of its essential physiological steps. The clearest evidence is from fucoid eggs in which such currents have been shown to parallel the prospective axis of germination. The currents are caused by stabilized accumulations of cation pumps on one side of the cell, and of permeability channels on the opposite side; they are strong enough to suggest that the accompanying gradient in electrical potential could have an electrophoretic effect on the distribution of cytoplasmic constituents, including those that serve as morphogenetic determinants. Although ion currents correlated with axes of morphogenesis have thus been clearly established, their effects on intracellular localizations and morphogenetic activity have remained speculative. We report here evidence that an endogenously generated gradient in electrical potential in the oocyte-nurse cell syncytium of Hyalophora cercropia can, in fact, influence the distribution of soluble proteins in a cytoplasmic continuum.

Animals↗
PubMed1979

The function of the follicular epithelium in vitellogenic Oncopeltus follicles.

Autoradiographic and electrophoretic methods that have detected an endogenously synthesized protein yolk component in Hyalophora cecropia follicles failed to reveal such a protein in Oncopeltus fasciatus. Similarly, neither sulfate nor glucosamine was incorporated into the intercellular matrix of vitellogenic follicles, though both label these regions intensely in Hyalophora. While both insects produce yolk from hemolymph vitellogenin, their follicles thus appear to support the process by very different synthetic means.

Animals↗