Nutrients, not caloric restriction, extend lifespan in Queensland fruit flies (Bactrocera tryoni)

Caloric restriction (CR) has been widely accepted as a mechanism explaining increased lifespan (LS) in organisms subjected to dietary restriction (DR), but recent studies investigating the role of nutrients have challenged the role of CR in extending longevity. Fuelling this debate is the difficulty in experimentally disentangling CR and nutrient effects due to compensatory feeding (CF) behaviour. We quantified CF by measuring the volume of solution imbibed and determined how calories and nutrients influenced LS and fecundity in unmated females of the Queensland fruit fly, Bactocera tryoni (Diptera: Tephritidae). We restricted flies to one of 28 diets varying in carbohydrate:protein (C:P) ratios and concentrations. On imbalanced diets, flies overcame dietary dilutions, consuming similar caloric intakes for most dilutions. The response surface for LS revealed that increasing C:P ratio while keeping calories constant extended LS, with the maximum LS along C:P ratio of 21:1. In general, LS was reduced as caloric intake decreased. Lifetime egg production was maximized at a C:P ratio of 3:1. When given a choice of separate sucrose and yeast solutions, each at one of five concentrations (yielding 25 choice treatments), flies regulated their nutrient intake to match C:P ratio of 3:1. Our results (i) demonstrate that CF can overcome dietary dilutions; (ii) reveal difficulties with methods presenting fixed amounts of liquid diet; (iii) illustrate the need to measure intake to account for CF in DR studies and (iv) highlight nutrients rather than CR as a dominant influence on LS.     

Identification of nucleic acid adducts from trans-4-acetylaminostilbene

It has been proposed that trans-4-acetylaminostilbene (AAS) is an initiator for tumor formation in rat liver and that the metabolically formed hydroxamic acid ester ultimately reacts with nucleic acids in vivo. We have now studied the generation of a major adduct in vitro. trans-4-N-Acetoxy-N-acetylaminostilbene (N-acetoxy-AAS) was reacted with guanosine at pH 7.5 and reaction products were separated by chromatography on Sephadex LH-20 and RP18 HPLC. The major adduct isolated consists of four isomers which have been tentatively identified by mass- and 1H-NMR spectroscopy as (S,S)- and (R,R)-guanosine-N2,beta-N3,alpha-N-acetylaminobibenzyl and the respective regio isomers guanosine-N2,alpha-N3,beta-N-acetylaminobibenzyl. These adducts are formed in a ratio of 9:9:1:1. Under acidic conditions (pH 2) the ribose moiety is removed and two regio isomeric base adducts are formed in the ratio 9:1. Results to be published indicate that the adducts are also formed in vivo in rat liver RNA and DNA.     

Target-cell-induced anergy in natural killer cells: suppression of cytotoxic function

 Our earlier studies have demonstrated that natural killer (NK) cells are the effectors that participate during the spontaneous regression of AK-5 tumour in syngeneic hosts. We have shown that the tumour cells are killed by necrosis and apoptosis. In this study, we have examined the induction of functional anergy in NK cells following coculture with fixed AK-5 tumour cells at high ratio. NK cells, upon coculture with fixed AK-5 cells (1:1 ratio), showed loss of cytotoxic function against both AK-5 (antibody-dependent cell cytotoxicity) as well as YAC-1 targets. The response of these cells to the activation by recombinant interleukin-2 and recombinant interferon γ was poor. Induction of tumour necrosis factor α (TNFα) secretion was observed after coculture of NK cells with fixed AK-5 cells. The cocultured cell supernatant inhibited the cytotoxic activity of NK cells, which was partially restored with anti-TNFα antibody. In addition, NK cells, after treatment with fixed tumour cells showed overexpression of the Fas receptor. We have also observed induction of apoptosis in cocultured NK cells. These studies suggest that the fixed tumour cells (antigen) at high ratio are able to suppress NK cell function as well as induce death in NK cells. Received: 16 September 1999 / Accepted: 13 January 2000     

A sperm nuclear basic protein from the sperm of the marine worm Chaetopterus variopedatus with sequence similarity to the arginine-rich C-termini of chordate protamine-likes

The sperm nuclear basic proteins (SNBPs) of the marine annelid worm Chaetopterus variopedatus have been shown previously to consist of a mixture of two SNBPs: histone H1-like (CvH1) and C.variopedatus protamine-like (CvPL). Here, we report the structural characterization of CvPL. The protein has a molecular weight of 8370.5 Da, a K/R ratio of 0.34, and a secondary structure, which are intermediate between those of protamine (P) and protamine-like (PL) SNBPs. The N-terminal sequence of CvPL shows a high extent of similarity with the arginine-rich C-terminal domain of chordate PL-type SNBPs. Furthermore, the protein binds to DNA in a similar fashion as vertebrate PLs and their own CvH1, but in a way that is different from that of the lysine-rich somatic H1 histones. We have experimentally determined the molar ratio CvH1:CvPL to be ～1:6 in C. variopedatus sperm. Based on all of these, a model is proposed for the organization of the sperm chromatin by CvH1 and CvPL.     

Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition.

Clostridium butyricum has been grown in media devoid of biotin, to which long-chain fatty acids have been added to promote growth. We have shown previously that, under these conditions, exogenous fatty acids are extensively incorporated into the cellular phospholipids. Cells grown with elaidic acid, trans-9-18:1, have normal ratios of the glycerol acetal of plasmenylethanolamine (GAPlaE) to phosphatidylethanolamine (PE) plus plasmenylethanolamine (PlaE) compared with cells grown with biotin. When ethanol, cyclohexane, or n-octanol was added to elaidate-containing media, the ratio of GAPlaE to PE plus PlaE was significantly increased. Addition of dodecane and n-butanol did not affect this ratio. When cells were grown with oleic acid in the absence of biotin, the GAPlaE to PE plus PlaE ratio was increased 5.4-fold compared with elaidate-grown cells. In oleate-supplemented media, the addition of solvents or n-alcohols produced no further increase in this ratio. We conclude that these changes in lipid composition represent cellular responses to perturbation of the equilibria between the lamellar and nonlamellar liquid crystalline phases in the cell membrane.     

Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition.

Clostridium butyricum has been grown in media devoid of biotin, to which long-chain fatty acids have been added to promote growth. We have shown previously that, under these conditions, exogenous fatty acids are extensively incorporated into the cellular phospholipids. Cells grown with elaidic acid, trans-9-18:1, have normal ratios of the glycerol acetal of plasmenylethanolamine (GAPlaE) to phosphatidylethanolamine (PE) plus plasmenylethanolamine (PlaE) compared with cells grown with biotin. When ethanol, cyclohexane, or n-octanol was added to elaidate-containing media, the ratio of GAPlaE to PE plus PlaE was significantly increased. Addition of dodecane and n-butanol did not affect this ratio. When cells were grown with oleic acid in the absence of biotin, the GAPlaE to PE plus PlaE ratio was increased 5.4-fold compared with elaidate-grown cells. In oleate-supplemented media, the addition of solvents or n-alcohols produced no further increase in this ratio. We conclude that these changes in lipid composition represent cellular responses to perturbation of the equilibria between the lamellar and nonlamellar liquid crystalline phases in the cell membrane.     

Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production

In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.     

GvpCs with reduced numbers of repeating sequence elements bind to and strengthen cyanobacterial gas vesicles

We have previously shown that the gas-vesicle protein GvpC is present on the outer surface of the gas vesicle, can be reversibly removed and rebound to the surface, and increases the critical collapse pressure of the gas vesicle. The GvpC molecule, which contains five partially conserved repeats of 33 amino acids (33-RR) sandwiched between 18 N-terminal and 10 C-terminal amino acids, is present in a ratio of 1:25 with the GvpA molecule, which forms the ribs of the gas vesicle. By using recombinant techniques we have now made modified versions of GvpC that contain only the first two, three or four of the 33-amino-acid repeats. All of these proteins bind to and strengthen gas vesicles that have been stripped of their native GvpC. Recombinant proteins containing three or four repeats bind in amounts that give the same ratio of 33-RR:GvpA (i.e. 1:5) as the native protein, and they restore much of the strength of the gas vesicle; the protein containing only two repeats binds at a lower ratio (1:7.7), however, and restores less of the strength. Ancestral proteins with only two, three or four of the 33-amino-acid repeats would have been functional in strengthening the gas vesicle but the progressive increase in number of repeats would have provided strength with increased efficiency.     

Studies on amikhellin. II.-Inhibition of dna-polymerase from murine sarcoma leukemia virus.

We have previously reported that amikhellin binds to double-stranded DNA by an intercalation process (1). We now report that this drug inhibits the DNA-polymerase from murine sarcoma leukemia virus. The extent of inhibition was found to vary with the nature of the primer-template used : maximum with poly(rA)n-oligo(dT)10 (nucleotide ratio 20:1), minimum with poly(rA)n-poly(dT)n and intermediate with native calf thymus DNA. Experiments performed with synthetic templates of the (rA)-(dT) type have led to the following conclusions as to the mechanism of inhibition: 1) Amikhellin acts at an early stage of the synthesis reaction because the drug is no longer inhibitory when a limited extension of the oligo(dT) primers has been allowed to occur. However, mere incubation of the enzyme with the template in the absence of dTTP is not sufficient to confer resistance to the drug. 2) Progression of enzyme molecules actively engaged in polymerization is stopped when they reach downstream duplex regions to which amikhellin is bound.     

青海省玉树、果洛州岩羊的种群结构及生命表初探

本文根据野外发现的4470只岩羊,就其栖息地、种群大小和年龄组成进行分析。在已分辨性别的2168只岩羊中,成年雌雄性比为1：0.5。此外,我们还利用54架自然死亡的雄性岩羊头骨,初步探讨年龄分布、存活曲线和生命表。     

Apolipoprotein E: phospholipid binding studies with synthetic peptides from the carboxyl terminus.

We have previously shown that the synthetic peptide apoE(129-169) forms lipid-peptide complexes with dimyristoylphosphatidylcholine (DMPC) with an L:P molar ratio of 125:1; the peptide in the isolated complex contains approximately 56% alpha-helicity. These results verify the presence of an amphipathic alpha-helix in this region of apoE as predicted by Chou-Fasman analysis and hydrophobicity calculations. To further define the lipid binding regions of apoE, we have synthesized four peptides, apoE(211-243), -(202-243), -(267-286), and -(263-286), from the carboxyl terminus of apoE and studied their lipid binding properties; apoE(202-243) contains two potential amphipathic helices. Although all four peptides formed alpha-helices in the helix-forming solvent 30% hexafluoropropanol, we found that only apoE(263-286) formed a stable complex with DMPC. The peptide contained approximately 80% alpha-helicity, and its Trp fluorescence spectrum was blue-shifted by 20 nm in the complex which had an L:P ratio of 163:1. We conclude that this sequence is a newly identified lipid binding region of apoE and that the amphipathic helices 203-221 and 226-243 are too hydrophilic to bind phospholipid.     

Effects of macronutrient intake on the lifespan and fecundity of the marula fruit fly,Ceratitis cosyra (Tephritidae): Extreme lifespan in a host specialist

In insects, lifespan and reproduction are strongly associated with nutrition. The ratio and amount of nutrients individuals consume affect their life expectancy and reproductive investment. The geometric framework (GF) enables us to explore how animals regulate their intake of multiple nutrients simultaneously and determine how these nutrients interact to affect life‐history traits of interest. Studies using the GF on host‐generalist tephritid flies have highlighted trade‐offs between longevity and reproductive effort in females, mediated by the protein‐to‐carbohydrate (P:C) ratio that individuals consume. Here, we tested how P and C intake affect lifespan (LS) in both sexes, and female lifetime (LEP), and daily (DEP) egg production, in Ceratitis cosyra, a host‐specialist tephritid fly. We then determined the P:C ratio that C. cosyra defends when offered a choice of foods. Female LS was optimized at a 0:1 P:C ratio, whereas to maximize their fecundity, females needed to consume a higher P:C ratio (LEP = 1:6 P:C; DEP = 1:2.5 P:C). In males, LS was also optimized at a low P:C ratio of 1:10. However, when given the opportunity to regulate their intake, both sexes actively defended a 1:3 P:C ratio, which is closer to the target for DEP than either LS or LEP. Our results show that female C. cosyra experienced a moderate trade‐off between LS and fecundity. Moreover, the diets that maximized expression of LEP and DEP were of lower P:C ratio than those required for optimal expression of these traits in host‐generalist tephritids or other generalist insects.     

Induction of pure and sectored mutant clones in excision-proficient and deficient strains of yeast.

We have found that UV-induced mutation frequency in a forward non-selective assay system (scoring white adex ade2 double auxotroph mutants among the red pigmented ade2 clones) increases linearly with dose up to a maximum frequency of about 3 X 10(-3) mutants per survivor and then declines in both RAD wild-type and rad2 excision deficient strains of Saccharomyces cerevisiae. Mutation frequencies of the RAD and the rad2 strains plotted against survival are nearly identical over the entire survival range. On this basis we conclude that unexcised pyrimidine dimers are the predominant type of pre-mutational lesions in both strains. In the RAD wild-type strain pure mutant clones outnumber sectors in a 10:1 ratio at all doses used; in rad2 this ratio varies from 1:1 at low doses up to 10:1 at high doses. As others have concluded for wild-type strains we find also in the rad2 strain that pure clone formation cannot be accounted for quantitatively by lethal sectoring events alone. We conclude that heteroduplex repair is a crucial step in pure mutant clone formation and we examine the plausibility of certain macromolecular mechanisms according to which heteroduplex repair may be coupled with replication, repair and sister strand exchange in yeast mutagenesis.     

Effect on the epigallocatechin gallate/epigallocatechin ratio in a green tea (Camellia sinensis L.) extract of different extraction temperatures and its effect on IgA production in mice

We found that the epigallocatechin gallate (EGCG)/epigallocatechin (EGC) ratio in a green tea (Camellia sinensis L.) extract was affected by the extraction temperature. The EGCG/EGC ratio in the 4 °C extract was around 1:3-4, whereas in the 100 °C extract, it was around 1:0.7. Oral administration of the mixture with a high EGC ratio (1:2-3 = EGCG/EGC) resulted in greater IgA production by murine Peyer's patch cells.     

Triglyceride levels are ethnic-specifically associated with an index of stearoyl-CoA desaturase activity and n-3 PUFA levels in Asians

Accumulated evidence suggests that hypertriglyceridemia (HTG) is independently associated with an increased incidence of cardiovascular disease. The hypotriglyceridemic effects of n-3 PUFAs have been confirmed in Caucasians, but the effect in Asians is less clear. Recent evidence indicates that stearoyl-CoA desaturase (SCD) activity induced with high-carbohydrate diets increases plasma triglyceride levels. We investigated the relationship between triglyceride levels and the ratio of plasma oleic acid to stearic acid (the 18:1/18:0 ratio), a plasma marker of SCD activity, and n-3 PUFAs in 411 Japanese, 418 Korean, and 251 Mongolian adults. The Japanese and Koreans had higher values for triglyceride than their Mongolian counterparts, despite lower body mass index values for the Japanese and Koreans. The Japanese and Koreans ate fish more frequently and had remarkably higher values for n-3 PUFAs than did the Mongolians. Multiple regression analysis showed that triglyceride levels had a great magnitude of correlation with the increases in 18:1/18:0 ratio for the Japanese and Mongolians, and n-3 PUFAs remained significant for the Mongolians. HTG is ethnicity-specifically associated with an increase in the 18:1/18:0 ratio and a decrease in n-3 PUFA in plasma for Japanese, Koreans, and Mongolians.     

Factors controlling the biosynthesis of chlorosome antenna bacteriochlorophylls in green filamentous anoxygenic phototrophic bacteria of the family Oscillochloridaceae

We determined the concentrations of bacteriochlorophylls (BChl) in the light-harvesting antennae of Oscillochloris trichoides (of the family Oscillochloridaceae belonging to green filamentous mesophilic bacteria) cultivated either with gabaculine, an inhibitor of the C-5 pathway of BChl biosynthesis in a number of bacteria, or at various illumination intensities. We determined the BChl c: BChl a molar ratios in intact cells, in chlorosome-membrane complexes, and in isolated chlorosomes. We revealed that BChl c synthesis in Osc. trichoides was more gabaculine-sensitive than BChl a synthesis. Accordingly, an increase in gabaculine concentrations in the medium resulted in a decrease in the BChl c: BChl a ratio in the tested samples. We suggest that BChl synthesis in Osc. trichoides proceeds via the C-5 pathway, similar to representatives of other families of green bacteria (Chlorobium limicola and Chloroflexus aurantiacus). We demonstrated that the BChl c: BChl a ratio in the chlorosomes varied from 55 : 1 to 110 : 1, depending on light intensity. This ratio is, therefore, closer to that of Chlorobiaceae, and it significantly exceeds the BChl c: BChl a ratio in Chloroflexaceae.     

The effect of host Chlorella NC64A carbon : phosphorus ratio on the production of Paramecium bursaria Chlorella Virus-1

1. We used the freshwater alga Chlorella NC64A (Division Chlorophyta) and its virus Paramecium bursaria Chlorella virus‐1 (PBCV‐1) as a model system to test for potential stoichiometric constraints on a virus–host interaction. 2. Media phosphorus concentrations were manipulated to create Chlorella NC64A host cells with low (91 ± 23) or high (453 ± 246) C : P ratio. In contrast, the C : P ratio of PBCV‐1, calculated from its biochemical composition, was 17 : 1. 3. Stoichiometric theory predicts that infection success and postinfection viral production should be depressed in high C : P cultures due to insufficient intracellular P for production of P‐rich viral particles. 4. Consistent with this hypothesis, viral production was strongly affected by host C : P ratio. While host C : P ratio did not affect viral attachment or the percentage of new viral particles that were infectious, in the low C : P Chlorella NC64A treatment, nine times more viruses were produced per infected cell than in the high C : P treatment (158 ± 138 versus 18 ± 18), indicating that the low C : P cells were higher quality for PBCV‐1 proliferation. 5. This result implies that the stoichiometric quality of algal cells can have a major effect on host–virus population dynamics.     

Effect of male age structure on reproduction in white-tailed deer

Selective harvest regimes that create female-biased sex ratios can potentially lead to delayed breeding, reduced breeding synchrony, reduced productivity, and a female-biased sex ratio of offspring. These resulting changes in breeding behavior and population dynamics have potential to adversely affect population growth. In 2002, Pennsylvania implemented harvest regulation changes that reduced deer density (increased harvest of antlerless deer) and increased the number and age of antlered deer (implemented antler point restriction regulations) that resulted in a less female-biased sex ratio. We monitored date of conception, productivity (embryos/female), and sex ratio of embryos during 1999–2006 to test if timing of breeding occurred earlier and with greater synchrony, if productivity of females increased, and if the sex ratio of offspring would shift towards more males. Deer density decreased 23% and the adult (≥1.5 yr old) sex ratio declined from 2.30 to 1.95 females/male. The ratio of ≥2.5-year-old to 1.5-year-old males shifted towards more older males (1:3.7 in 2002 to 1:1.59 in 2006) and the ≥2.5-year-old male population increased from 41,853 during 1999–2001 to 54,064 by 2006. We found no evidence of any change in the timing or variability of date of conception, productivity, or offspring sex ratio. We conclude that harvest regulation changes implemented in Pennsylvania, USA, were insufficient to affect timing of breeding or population dynamics and that efforts by managers to identify a desired sex ratio or manipulate sex ratios to achieve management goals on a statewide scale will be challenging. © 2019 The Wildlife Society.     

Novel DNA-binding ligands with sequence selectivity based on hydrophobic structure.

We have developed diamino-bistetrahydrofuran compounds (diamino-bisTHF) as new DNA binding molecules. Diamino-bisTHF (3:RR8) stabilized GC-rich duplex DNA with sequence specificity. DNA binding affinity increased as the alkyl chain was lengthened, indicating that the hydrophobic interaction is essential for DNA binding. It was also found that DNA binding affinity of the ligands depends on the stereochemistry of the amino group. In thermodynamic evaluation, diamino-bisTHF (3:RR8) showed a high affinity to the 12 bp duplex at a molar ratio of 1:1.