A subnanogram assay for phospholipase activity based on a long-chain radioiodinatable phosphatidylcholine

Here, we introduce a radioiodinatable long-chain phosphatidylcholine (BHC12PC) which serves as the base for a very sensitive phospholipase assay. This compound has a 4-hydroxyphenyl group attached at the end of the fatty acyl chain located in position sn-2. This feature enables this phospholipid to be radioiodinated. BHC12PC was tested as a substrate of Naja naja naja PLA(2) and Bacillus cereus PLC in a mixed micellar system with Triton X-100. The detection limit for the assays was 0.25ng of PLA(2) and 0.05ng of PLC, thus becoming one of the most sensitive methods described so far. A low specific radioactivity (500microCi/mmol) suffices to achieve this level of sensitivity. In both cases, the behavior of BHC12PC was indistinguishable from that shown by phospholipids with n-acyl chains of similar length. The choice of spacer prevents any unfavorable interaction of the bulky 4-hydroxyphenyl group at the active site of the enzymes. The progress of the reaction as monitored by thin-layer chromatography is compared side by side with an alternative method based on the selective adsorption of BHC12PC to silica gel, which renders identical results in a simpler fashion. An additional advantage of BHC12PC is that the cost per Ci of the radioiodinated derivative is significantly lower than that of other labeled phospholipids ((3)H, (14)C, or (32)P).     

Coralline algae indicate Pleistocene evolution from deep,open platform to outer barrier reef environments in the northern Great Barrier Reef margin

Coralline algae from a drill core in Ribbon Reef 5 have been used to interpret changes in the depositional palaeoenvironments in the northern Australian Great Barrier Reef over the last 790 ka. Three main coralline algal assemblages, each dominated by members of a particular subfamily, have been distinguished by quantitative analysis: (1) mastophoroid assemblages, usually occurring as crusts on corals, are typical of the shallowest reef settings; (2) lithophylloid assemblages within algal nodules may represent shallow-water, cooler environments or deeper reef subenvironments; and (3) melobesioid assemblages are characteristic of deeper-water platform areas. The algal assemblages between 96 and 210 m b.s.f. (metre below sea floor) record a fluctuating but progressive shallowing-upwards from deep, outer-platform to shallower, non-reefal depositional environments. Two intervening episodes dominated by mastophoroids represent two phases of reef growth. The section above 96 m b.s.f. comprises several stacked reefs in which mastophoroid crusts similar to the present-day shallow-water assemblages predominate.Electronic Supplementary Material Supplementary material is available for this article at .Communicated by: Geological Editor P.K. Swart     

Inhibition of Akt potentiates 2-DG-induced apoptosis via downregulation of UPR in acute lymphoblastic leukemia

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-D-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with D-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG-treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG-treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival.     

Three new species and a new record of Diplococcium from plant debris in Spain

Diplococcium dimorphosporum sp. nov., D. racemosum sp. nov., D. singulare sp. nov. and D. pulneyense Subram. & Sekar collected from plant debris in natural areas of Spain are described and illustrated. The first species is characterized principally by the production of branched conidiophores and short chains of conidia. Diplococcium singulare has unbranched conidiophores, and conidia produced usually at the tip of conidiophores and from lateral spherical conidiogenous cells. In addition, both species develop a Selenosporella synanamorph with narrow falcate conidia. Diplococcium racemosum produces branched, verrucose conidiophores, and verrucose conidia in long branched chains. Diplococcium pulneyense is the second record, being described for first time on the natural substratum and re-described in pure culture. A key to currently accepted species of Diplococcium is provided.     

Oral lactoferrin treatment resolves amoebic intracecal infection in C3H/HeJ mice

Entamoeba histolytica is a protozoan parasite that causes amoebiasis, an illness that affects many people around the world. We have previously reported that lactoferrin is able to kill E. histolytica in in vitro cultures. The aim of the present study was to evaluate the therapeutic effect of orally administered bovine lactoferrin in the control of intestinal amoebiasis of susceptible C3H/HeJ mice. The results showed that 20 mg lactoferrin/kg orally administered each day for 1 week was able to eliminate the infection in 63% of the mice, since neither trophozoites nor evidence of epithelial damage and (or) swelling were found in tissue sections of the cecum. The rest of the treated animals (37%) showed a decrease in trophozoite numbers and mucus secreted to the lumen, as compared with untreated and infected mice (p < 0.05). By immunohistochemistry, the profile of secreted cytokines in the cecum revealed that infected but untreated animals showed a mixed Th1/regulatory cytokines profile, whereas the cecum of mice treated (cured) showed a Th2 cytokine profile (IL-4) and expression of the multifunctional IL-6. In addition, cytokines and increasing cecal production of total IgA antibodies were found associated with little inflammation and disease control observed in the cecum of lactoferrin-treated animals. These results suggest that oral administration of lactoferrin can control intestinal amoebic infection probably by killing amoebas or favoring their removal and reestablish the antiinflammatory intestinal environment.     

Reconstructing the History of Mesoamerican Populations through the Study of the Mitochondrial DNA Control Region

The study of genetic information can reveal a reconstruction of human population’s history. We sequenced the entire mtDNA control region (positions 16.024 to 576 following Cambridge Reference Sequence, CRS) of 605 individuals from seven Mesoamerican indigenous groups and one Aridoamerican from the Greater Southwest previously defined, all of them in present Mexico. Samples were collected directly from the indigenous populations, the application of an individual survey made it possible to remove related or with other origins samples. Diversity indices and demographic estimates were calculated. Also AMOVAs were calculated according to different criteria. An MDS plot, based on FST distances, was also built. We carried out the construction of individual networks for the four Amerindian haplogroups detected. Finally, barrier software was applied to detect genetic boundaries among populations. The results suggest: a common origin of the indigenous groups; a small degree of European admixture; and inter-ethnic gene flow. The process of Mesoamerica’s human settlement took place quickly influenced by the region’s orography, which development of genetic and cultural differences facilitated. We find the existence of genetic structure is related to the region’s geography, rather than to cultural parameters, such as language. The human population gradually became fragmented, though they remained relatively isolated, and differentiated due to small population sizes and different survival strategies. Genetic differences were detected between Aridoamerica and Mesoamerica, which can be subdivided into “East”, “Center”, “West” and “Southeast”. The fragmentation process occurred mainly during the Mesoamerican Pre-Classic period, with the Otomí being one of the oldest groups. With an increased number of populations studied adding previously published data, there is no change in the conclusions, although significant genetic heterogeneity can be detected in Pima and Huichol groups. This result may be explained because populations historically assigned as belonging to the same group were, in fact, different indigenous populations.     

Risk factors associated with negative in-vivo diagnostic results in bovine tuberculosis-infected cattle in Spain

Background

Despite great effort and investment incurred over decades to control bovine tuberculosis (bTB), it is still one of the most important zoonotic diseases in many areas of the world. Test-and-slaughter strategies, the basis of most bTB eradication programs carried out worldwide, have demonstrated its usefulness in the control of the disease. However, in certain countries, eradication has not been achieved due in part to limitations of currently available diagnostic tests. In this study, results of in-vivo and post-mortem diagnostic tests performed on 3,614 animals from 152 bTB-infected cattle herds (beef, dairy, and bullfighting) detected in 2007–2010 in the region of Castilla y León, Spain, were analyzed to identify factors associated with positive bacteriological results in cattle that were non-reactors to the single intradermal tuberculin test, to the interferon-gamma (IFN-γ) assay, or to both tests applied in parallel (Test negative/Culture + animals, T-/C+). The association of individual factors (age, productive type, and number of herd-tests performed since the disclosure of the outbreak) with the bacteriology outcome (positive/negative) was analyzed using a mixed multivariate logistic regression model.

Results

The proportion of non-reactors with a positive post-mortem result ranged from 24.3% in the case of the SIT test to 12.9% (IFN-γ with 0.05 threshold) and 11.9% (95% CI 9.9-11.4%) using both tests in parallel. Older (>4.5 years) and bullfighting cattle were associated with increased odds of confirmed bTB infection by bacteriology, whereas dairy cattle showed a significantly lower risk. Ancillary use of IFN-γ assay reduced the proportion of T-/C + animals in high risk groups.

Conclusions

These results demonstrate the likelihood of positive bacteriological results in non-reactor cattle is influenced by individual epidemiological factors of tested animals. Increased surveillance on non-reactors with an increased probability of being false negative could be helpful to avoid bTB persistence, particularly in chronically infected herds. These findings may aid in the development of effective strategies for eradication of bTB in Spain.     

Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h⁻¹) and low dilution rate (0.012 h⁻¹) at two cultivation temperatures (37 and 33°C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33°C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.