Cloning and expression of a cDNA encoding a catalytically active fragment of calf thymus DNA polymerase alpha

A calf thymus cDNA expression library was constructed in the EcoRI site of lambda gt11 and probed with an antibody raised against calf thymus DNA polymerase alpha. Three classes of antibody-reactive clones were isolated. The largest class carried a 1.9 kilobase calf cDNA insert and expressed a 165-175 kilodalton beta-galactosidase:calf fusion protein which displayed DNA polymerase activity. The characteristic responses of the polymerase activity to alpha-specific inhibitors and antibodies identified the 1.9 kilobase cDNA as a sequence specifically derived from the structural gene encoding the pol alpha catalytic core.     

Effects of dietary polyamines and clofibrate on metabolism of polyamines in the rat

The activities of catalase, polyamine oxidase, diamine oxidase, ornithine decarboxylase, and peroxisomal β-oxidation were assayed in homogenates from liver and small intestinal mucosa of rats which had been fed either a diet very low in polyamines or a diet containing five times the levels of dietary polyamines (putrescine, spermine, and spermidine) found in a standard rat diet. In rats fed the high polyamine diet, hepatic activities of catalase and polyamine oxidase were significantly decreased. Levels of the other activities were unchanged, except that intestinal ornithine decarboxylase was decreased. In rats treated simultaneously with clofibrate, the high polyamine diet restored activities of catalase, ornithine decarboxylase, and polyamine oxidase back to levels found in rats fed the low polyamine diet. The expected increase in activity of peroxisomal β-oxidation was observed, although this was somewhat diminished in rats fed the high polyamine diet. Intestinal diamine oxidase activity was stimulated by clofibrate, particularly in rats fed the high polyamine diet. For the duration of the experiment (20 days), levels of putrescine, spermine, and spermidine in blood remained remarkably constant irrespective of treatment, suggesting that polyamine homeostasis is essentially independent of dietary supply of polyamines. It is suggested that intestinal absorption/metabolism of polyamines is of significance in this respect. Treatment with clofibrate appeared to alter polyamine homeostasis.     

Initial events in infectious salmon anemia virus infection: evidence for the requirement of a low-pH step

We have investigated the initial steps in the interaction between infectious salmon anemia virus (ISAV) and cultured cells from Atlantic salmon (SHK-1 cell line). Using radioactively or fluorescently labelled viral particles we have studied the binding and fusion kinetics and the effect of pH on binding, uptake, and fusion of ISAV to SHK-1 cells and liposomes. As pH in the medium was reduced from 7.5 to 4.5, the association of virus to the cells was nearly doubled. The same effect of pH was observed when fusion between ISAV and liposomes was analyzed. In addition, the binding of ISAV to intact SHK-1 cells and to cell membrane proteins blotted onto filters was neuraminidase sensitive. However, the increased binding induced by low pH was not neuraminidase sensitive, probably reflecting activation of a fusion peptide at low pH. By using confocal fluorescence microscopy, the increased fusion of fluorescently labelled ISAV with the plasma membrane due to low pH could be demonstrated. When vacuolar pH in the cells was raised during inoculation with chloroquine or ammonium chloride, both electron and confocal microscopy showed accumulation of ISAV in endosomes and lysosomes. Production of infectious virus could be increased by lowering the extracellular pH during infection. Furthermore, chloroquine present during virus inoculation also caused a reduction in the synthesis of viral proteins in ISAV-infected cells as well as in the production of infective virus. These results indicate that ISAV binds to sialic acid residues on the cell surface and that the fusion between virus and cell membrane takes place in the acid environment of endosomes. This provides further evidence for a high degree of similarity between ISAV and influenza virus and extends the basis for the classification of this virus as a member of the Orthomyxoviridae family.     

Extra-Pair Mating and Evolution of Cooperative Neighbourhoods

A striking but unexplained pattern in biology is the promiscuous mating behaviour in socially monogamous species. Although females commonly solicit extra-pair copulations, the adaptive reason has remained elusive. We use evolutionary modelling of breeding ecology to show that females benefit because extra-pair paternity incentivizes males to shift focus from a single brood towards the entire neighbourhood, as they are likely to have offspring there. Male-male cooperation towards public goods and dear enemy effects of reduced territorial aggression evolve from selfish interests, and lead to safer and more productive neighbourhoods. The mechanism provides adaptive explanations for the common empirical observations that females engage in extra-pair copulations, that neighbours dominate as extra-pair sires, and that extra-pair mating correlates with predation mortality and breeding density. The models predict cooperative behaviours at breeding sites where males cooperate more towards public goods than females. Where maternity certainty makes females care for offspring at home, paternity uncertainty and a potential for offspring in several broods make males invest in communal benefits and public goods. The models further predict that benefits of extra-pair mating affect whole nests or neighbourhoods, and that cuckolding males are often cuckolded themselves. Derived from ecological mechanisms, these new perspectives point towards the evolution of sociality in birds, with relevance also for mammals and primates including humans.     

Correlates of complete brood failure in blue tits: could extra‐pair mating provide unexplored benefits to females?

Behavioural ecologists have for decades investigated the adaptive value of extra‐pair copulation (EPC) for females of socially monogamous species. Despite extensive effort testing for genetic benefits, there now seems to be a consensus that the so‐called ‘good genes’ effects are at most weak. In parallel the search for direct benefits has mostly focused on the period surrounding egg laying, thus neglecting potential correlates of EPC that might be expressed at later stages in the breeding cycle. Here we used Bayesian methods to analyse data collected over four years in a population of blue tits Cyanistes caeruleus, where no support was previously found for ‘good genes’ effects. We found that broods with mixed paternity experienced less brood failure at the nestling stage than broods with single paternity, and that females having experienced complete brood failure in their previous breeding attempt had higher rates of mixed paternity than either yearling or previously successful females. To better understand these observations we also explored relationships between extra‐pair mating, male and female phenotype, and local breeding density. We found that in almost all cases the sires of extra‐pair offspring were close neighbours, and that within those close neighbourhoods extra‐pair sires were older than other males not siring extra‐pair offspring. Also, females did not display consistent EPC status across years. Taken together our results suggest that multiple mating might be a flexible female behaviour influenced by previous breeding experience, and motivate further experimental tests of causal links between extra‐pair copulation and predation.     

Effect of genetic polymorphisms involved in folate metabolism on the concentration of serum folate and plasma total homocysteine (p-tHcy) in healthy subjects after short-term folic acid supplementation: a randomized,double blind,crossover study

Data on the effect of combined genetic polymorphisms, involved in folate metabolism, on the concentration of serum folate after folic acid supplementation are scarce. Therefore, we investigated the impact of seven gene polymorphisms on the concentration of serum folate and p-tHcy in healthy subjects after short-term folic acid supplementation. In a randomized, double blind, crossover study, apparently healthy subjects were given either 0.8 mg folic acid per day (n = 46) or placebo (n = 45) for 14 days. The washout period was 14 days. Fasting blood samples were collected on day 1, 15, 30 and 45. Data on subjects on folic acid supplementation (n = 91) and on placebo (n = 45) were used for the statistical analysis. The concentration of serum folate increased higher in subjects with higher age (53.5 ± 7.0 years) than in subjects with lower age (24.3 ± 3.2 years) after folic acid supplementation (p = 0.006). The baseline concentration of serum folate in subjects with polymorphism combination, reduced folate carrier protein, RFC1-80 GA and methylenetetrahydrofolate reductase, MTHFR677 CT+TT, was lower than RFC1-80 AA and MTHFR677 CT+TT (p = 0.002). After folic acid supplementation, a higher increase in the concentration of serum folate was detected in subjects with polymorphism combination RFC1-80 GA and MTHFR677 CC than RFC1-80 GG and MTHFR CT+TT combination (p < 0.0001). The baseline concentration of plasma total homocysteine (p-tHcy) was altered by combined polymorphisms in genes associated with folate metabolism. After folic acid supplementation, in subjects with combined polymorphisms in methylenetetrahydrofolate dehydrogenase, MTHFD1-1958 and MTHFR-677 genes, the concentration of p-tHcy was changed (p = 0.002). The combination of RFC1-80 and MTHFR-677 polymorphisms had a profound affect on the concentration of serum folate in healthy subjects before and after folic acid supplementation.     

Therapeutic vaccination against a murine lymphoma by intratumoral injection of a cationic anticancer peptide

Cationic antimicrobial peptides (CAPs) exhibit promising anticancer activities. In the present study, we have examined the in vivo antitumoral effects of a 9-mer peptide, LTX-302, which is derived from the CAP bovine lactoferricin (LfcinB). A20 B cell lymphomas of BALB/c origin were established by subcutaneous inoculation in syngeneic mice. Intratumoral LTX-302 injection resulted in tumor necrosis and infiltration of inflammatory cells followed by complete regression of the tumors in the majority of the animals. This effect was T cell dependent, since the intervention was inefficient in nude mice. Successfully treated mice were protected against rechallenge with A20 cells, but not against Meth A sarcoma cells. Tumor resistance could be adoptively transferred with spleen cells from LTX-302-treated mice. Resistance was abrogated by depletion of T lymphocytes, or either the CD4⁺ or CD8⁺ T cell subsets. Taken together, these data suggest that LTX-302 treatment induced long-term, specific cellular immunity against the A20 lymphoma and that both CD4⁺ and CD8⁺ T cells were required. Thus, intratumoral administration of lytic peptide might, in addition to providing local tumor control, confer a novel strategy for therapeutic vaccination against cancer.     

Identification and molecular cloning of a neuropeptide Y homolog that produces prolonged inhibition in Aplysia neurons.

The neuroendocrine bag cell neurons of the marine mollusk Aplysia produce prolonged inhibition that lasts for more than 2 hr. We purified a peptide from the abdominal ganglion that mimics this inhibition. Mass spectrometry and microsequence analysis indicate that the peptide is 40 aa long and is amidated at its carboxyl terminus. It is highly homologous to vertebrate neuropeptide Y (NPY) and other members of the pancreatic polypeptide family. As determined from cloned cDNA, the gene coding for the precursor protein shares a common structural organization with genes encoding precursors of the vertebrate family. The peptides may therefore have arisen from a common ancestral gene. Bag cell neurons are immunoreactive for Aplysia NPY, and Northern blot analysis indicates that as with its vertebrate counterparts, the peptide is abundantly expressed in the CNS. This suggests that peptides related to NPY may have important functions in the nervous system of Aplysia as well as in other invertebrates.     

Endogenous estrogen, testosterone and progesterone levels in relation to breast cancer risk

Multiple lines of evidence support a central role of hormones in the etiology of breast cancer. In epidemiologic studies, considerable effort has focused on delineating the role of endogenous hormones in risk of breast cancer among postmenopausal women. Recently, substantial additional data has accrued from prospective studies where endogenous hormones are measured in study subjects prior to disease diagnosis. In this review, the epidemiologic evidence linking sex steroids—estrogens, testosterone, and progesterone, specifically—with subsequent risk of breast cancer in both premenopausal and postmenopausal women is summarized. Overall, a strong positive association between breast cancer risk and circulating levels of both estrogens and testosterone has now been well confirmed among postmenopausal women; women with hormone levels in the top 20% of the distribution (versus bottom 20%) have a two- to three-fold higher risk of breast cancer. Evidence among premenopausal women is more limited, though increased risk associated with higher levels of testosterone is consistent. However, both positive and null associations have been observed with estrogens and progesterone and clearly more evaluation is needed.