Membrane associated events during proliferative inhibition of granulocyte precursors

A new way of assessing the significance of intracellular signals that may regulate cellular proliferation, would be to analyze possible 'second messengers' when proliferation is slowed down, rather than stimulated. Therefore, we examined proliferating mononuclear blood cells from leukaemic patients which had been exposed to an inhibitory ox leucocyte extract. The extract decreased 3H-thymidine incorporation in leukaemic cells in short-term cultures. The inhibition was not cell-line specific, but was nevertheless non-toxic and not due to endotoxin. The K+ flux into the leukaemic cells was assessed with 86Rb+, a K+ analogue. An inverse relationship was found between 86Rb+ uptake and 3H-thymidine incorporation. The increased 86Rb+ influx was probably due to leakage or exchange mechanisms other than the Na+/K+ membrane pump, as suggested by ouabain inhibition experiments. However, the long lag time (greater than 45 min) between addition of inhibitor and a marked increase in 86Rb+ uptake does not support a role for the K+ flux as an early mediator of the inhibitory signal.     

Production of proliferation inhibitors by mature granulocytes

The aim of the experiments was to find ways of increasing the yield of small molecular weight inhibitors of cell proliferation released by granulocytes. Almost pure populations of granulocytes from pig or human blood, or from sterile inflammatory exudates in rats were treated in various ways and then spun down. Molecules below approximately 10 000 dalton (Diaflo ultrafiltration or Sephadex G 25 filtration) in the supernatants were tested for inhibitory activity by measuring 3H-thymidine incorporation in 5 to 6-h coverslip cultures of rat bone marrow cells. The different granulocyte treatments were: Freeze-thawing, sonication, incubation (at +4 degrees -37 degrees C) in hypotonic media (0-200 mosm/kg), storage in vitro overnight (at +4 degrees C) before incubation, incubation at 37 degrees C in complete and buffered tissue culture medium (Fischer's with 10 mmol/1 HEPES), incubation in saline only (2-h periods, approximately 70 X 10(6) cells/ml), or with lidocaine added, with Ca++ and the Ca++ ionophore A-23187, with K+ and the K+ ionophore Valinomycin, with a high K+ concentration (50 mmol/1), with arachidonic acid, with a cAMP analogue, or with a protease inhibitor added during or at the end of the incubation. On a per cell basis rat peritonitis granulocytes released more inhibitor than pig blood granulocytes, whereas human blood granulocytes were not detectably inhibitory at all. Arachidonic acid was the most promising agent tested to increase inhibitor release above that occurring spontaneously from granulocytes incubated in saline.     

Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius

Summary Microsomal and soluble fractions of Pleurotus pulmonarius exhibited a reduced carbon monoxide difference spectrum with P450 maxima at 448nm and 450–452nm respectively. Substrate induced Type I spectra were observed on addition of benzo(a)pyrene to both fractions. Benzo(a)pyrene hydroxylation was measured using the aryl hydrocarbon hydroxylase assay and was observed to be P450 dependent as indicated by carbon monoxide inhibition together with the substrate binding characteristics. The activity of the fractions were observed to give K_m of 200mM and 660mM and V_max of 1.25 nmol/min/nmol P450 and 0.57 nmol/min/nmol P450 for the microsomal and cytosolic fractions respectively.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Effectiveness of larvae ofSyrphus ribesii andS. Corollae [Diptera: Syrphidae] as predators onMyzus persicae [Homoptera: Aphididae]

Capture efficiency, handling time and functonal response to prey density were studied in larvae ofSyrphus ribesii (L.) andS. corrollae (Fabr.) eatingMyzus persicae Sulz. at 20°C, 16 hrs light. First instar larvae ofS. ribesii had distinctly higher capture efficiency than 1st instar larvae ofS. corollae, both versus 1st instar and adult aphids. Second and 3rd instar larvae of both species seemed to prefer adult rather than 1st instar aphids, but no distinct difference in capture efficiency between the species was found. On comparable stages,S. ribesii always had shorter handling time thanS. corollae and it appeared in both species to be correlated with size of prey and predator. Handling time was thus shortest when 3rd instar larvae consumed 1st instar aphids. During one hour, 3rd instar larvae ofS. ribesii consumed aphids in quantities almost linearly dependent on aphid density (5, 10, 20 and 40 adult aphids/100 cm²), although the response also could roughly be described byHolling's “basic functional response curve”. On the contrary, 3rd instar larvae ofS. corollae soon reached a maximum consumption during one hour, being almost constant (5–7 aphids) at prey densities ≥10 aphids/100 cm².

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Résumé L'efficacité prédatrice (proportion de toutes les rencontres entre larves de syrphes et pucerons se terminant par la capture et la succion de pucerons), la durée d'activité et la relation entre l'absorption de nourriture et la densité des proies (5, 10, 20 et 40Myzus persicae Sulz. aptères au 5^e stade larvaire/100 cm²) ont été étudiées chez des larves deSyrphus ribesii (L.) etS. corollae (Fabr.) à 20°C et 16 h de photopériode. Les L₁ deS. ribesii présentent une efficacité prédatrice des pucerons des premier et dernier stades nettement plus élevée que les L₁ deS. corollae (S. ribesii: 71 et 38%;S. corollae: 42 et 0%). Chez les larves des 2^e et 3^e stades, on n'a observé aucune différence d'efficacité prédatrice entre les 2 espèces, mais une préférence pour les pucerons adultes. La durée d'activité deS. ribesii est dans tous les cas plus courte que celle deS. corollae pour un même stade de larves et de pucerons. Une corrélation positive semble exister entre la taille des proies et celle des prédateurs. La durée d'activité est la plus courte pour des prédateurs en L₃ s'alimentant de pucerons au premier stade (1,3 mn chezS. ribesii et 2,3 mn chezS. corollae). L'absorption de nourriture enregistrée en une heure chezS. ribesii varie presque linéairement avec la densité des pucerons; elle correspond d'autre part assez bien à la formule deHolling. L'absorption de nourriture parS. corollae atteint un maximum de 7 pucerons à l'heure et demeure pratiquement constante à partir d'une densité≥10 pucerons/100 cm².

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The author wishes to thank techn. ass.Bodil Horgen for assistance during the experiments     

Prions in Milk from Ewes Incubating Natural Scrapie

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrP^Sc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.