Immobilization of proteins on oxidized crosslinked Sepharose preparations by reductive amination

Mild periodate oxidation of certain commercially available crosslinked agarose beads (Sepharose CL-4B and CL-6B) results in the generation of aldehydo groups which were useful for immobilization of amino compounds by reductive amination using pyridine borane. Consumption of periodate ion and production of formaldehyde were only observed with crosslinked Sepharose preparations and were correlated with a binding capacity much greater than that of uncross-linked gels when subjected to the reductive amination reaction. Up to 50 mg (approximately 0.73 mumol) of bovine serum albumin and 30 mumol of glycylglycine were coupled per gram of moist oxidized Sepharose CL-6B. The immobilization reaction was shown to proceed at neutral pH requiring about 12 h for completion and to be relatively insensitive to temperature and pyridine borane concentration. The oxidized gel was shown to be stable for at least 2 months upon storage in 0.1 M acetic acid. This method has proven to be useful for the preparation of a variety of affinity matrices and immobilized enzymes.     

Feeding ecology of mackerel and dietary overlap with herring in Icelandic waters

The Northeast Atlantic mackerel (Scomber scombrus) has been extending its summer feeding distribution north and west, including around Iceland, since around 2006. The objective of this study is to quantify the weight gain and total food consumption of mackerel and to evaluate the food competition between mackerel and herring (Clupea harengus) feeding in the marine ecosystem around Iceland during the summers 2009–2011. Mackerel feeding in Icelandic waters gained around 43% on average in weight during these summers. Based on swept-area abundance estimates of mackerel from an international survey in 2011 and available estimates of food conversion efficiency in mackerel, the weight gain in Icelandic waters in 2011 corresponded to a total consumption of around 3.4 (2.4?4.5) million tonnes. Overall, 98% of 2314 mackerel, 91% of 398 Icelandic summer-spawning herring and 96% of 424 Norwegian spring-spawning herring stomachs, collected over the summers 2009?2011, contained food. The mean weight of the stomach content of mackerel was higher than for herring in all the years. While the stomach content weight of mackerel was generally highest in southeastern and southwestern areas, it was highest for herring in western, eastern and northern areas. Analysis of stomach contents showed that Copepoda were the most important food of mackerel in most areas, while Copepoda and Euphausiacea were the most important food items for herring. Fish prey contributed a higher proportion in stomachs of mackerel than herring, although relatively low for both species.     

Physical,Behavioural and Genetic Predictors of Adult Hypertension: The Findings of the Kaunas Cardiovascular Risk Cohort Study

Background

The roots of adult hypertension go back to childhood. This study aimed to examine the independent effects of physical, behavioural and genetic factors identified in childhood and mid-adulthood for prediction of adult hypertension.

Methods

The study subjects were participants of the Kaunas Cardiovascular Risk Cohort study started in 1977 (n = 1082, age 12–13 years). In 2012, a total of 507 individuals (63.9% of eligible sample) participated in the 35-year follow-up survey. Health examination involved measurements of blood pressure (BP), anthropometric parameters, and interview about health behaviours. Subjects were genotyped for AGT (M235T), ACE (I/D, rs4340), ADM (rs7129220), and CACNB2 (rs12258967) genes polymorphisms. A genetic risk score was calculated as the sum of the number of risk alleles at each of four single nucleotide polymorphisms.

Results

AGT TT genotype male carriers had the highest mean values of systolic BP in childhood. In females, ADM genotype AA was associated with the highest values of systolic and diastolic BP, while CACNB2 genotype CC carriers had the highest values of diastolic BP in childhood. Systolic and diastolic BP in childhood, gain in BMI from childhood to adulthood, and risky alcohol consumption predicted hypertension in middle-aged men. In women, genetic risk score together with diastolic BP in childhood and gain in BMI were significant predictors of adult hypertension. The comparison of four nested logistic regression models showed that the prediction of hypertension improved significantly after the addition of BMI gain. Genetic risk score had a relatively weak effect on the improvement of the model performance in women.

Conclusions

BP in childhood and the gain in BMI from childhood to adulthood were significant predictors of adult hypertension in both genders. Genetic risk score in women and risky alcohol consumption in men were independently related with the risk of adult hypertension.     

Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung

We sought to establish whether the cystic fibrosis transmembrane conductance regulator (CFTR) regulates the activity of amiloride-sensitive sodium channels (ENaC) in alveolar epithelial cells of wild-type, heterozygous (Cftr(+/-)), knockout (Cftr(-/-)), and ΔF508-expressing mice in situ. RT-PCR studies confirmed the presence of CFTR message in freshly isolated alveolar type II (ATII) cells from wild-type mice. We patched alveolar type I (ATI) and ATII cells in freshly prepared lung slices from these mice and demonstrated the presence of 4-pS ENaC channels with the following basal open probabilities (P(o)): wild-type=0.21 ± 0.015: Cftr(+/-)=0.4 ± 0.03; ΔF508=0.55 ± 0.01; and Cftr(-/-)=and 0.81 ± 0.016 (means ± SE; n ≥ 9). Forskolin (5 μM) or trypsin (2 μM), applied in the pipette solution, increased the P(o) and number of channels in ATII cells of wild-type, Cftr(+/-), and ΔF508, but not in Cftr(-/-) mice, suggesting that the latter were maximally activated. Western blot analysis showed that lungs of all groups of mice had similar levels of α-ENaC; however, lungs of Cftr(+/-) and Cftr(-/-) mice had significantly higher levels of an α-ENaC proteolytic fragment (65 kDa) that is associated with active ENaC channels. Our results indicate that ENaC activity is inversely correlated to predicted CFTR levels and that CFTR heterozygous and homozygous mice have higher levels of proteolytically processed ENaC fragments in their lungs. This is the first demonstration of functional ENaC-CFTR interactions in alveolar epithelial cells in situ.     

Interacting forces of predation and fishing affect species’ maturation size

1. Fishing is a strong selective force and is supposed to select for earlier maturation at smaller body size. However, the extent to which fishing‐induced evolution is shaping ecosystems remains debated. This is in part because it is challenging to disentangle fishing from other selective forces (e.g., size‐structured predation and cannibalism) in complex ecosystems undergoing rapid change.
2. Changes in maturation size from fishing and predation have previously been explored with multi‐species physiologically structured models but assumed separation of ecological and evolutionary timescales. To assess the eco‐evolutionary impact of fishing and predation at the same timescale, we developed a stochastic physiologically size‐structured food‐web model, where new phenotypes are introduced randomly through time enabling dynamic simulation of species'' relative maturation sizes under different types of selection pressures.
3. Using the model, we carried out a fully factorial in silico experiment to assess how maturation size would change in the absence and presence of both fishing and predation (including cannibalism). We carried out ten replicate stochastic simulations exposed to all combinations of fishing and predation in a model community of nine interacting fish species ranging in their maximum sizes from 10 g to 100 kg. We visualized and statistically analyzed the results using linear models.
4. The effects of fishing on maturation size depended on whether or not predation was enabled and differed substantially across species. Fishing consistently reduced the maturation sizes of two largest species whether or not predation was enabled and this decrease was seen even at low fishing intensities (F = 0.2 per year). In contrast, the maturation sizes of the three smallest species evolved to become smaller through time but this happened regardless of the levels of predation or fishing. For the four medium‐size species, the effect of fishing was highly variable with more species showing significant and larger fishing effects in the presence of predation.
5. Ultimately our results suggest that the interactive effects of predation and fishing can have marked effects on species'' maturation sizes, but that, at least for the largest species, predation does not counterbalance the evolutionary effect of fishing. Our model also produced relative maturation sizes that are broadly consistent with empirical estimates for many fish species.

     

Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in?vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs?form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.     

Real-Time PCR Quantification of the Plant Growth Promoting Bacteria Herbaspirillum seropedicae Strain SmR1 in Maize Roots

The plant growth promoting bacteria Herbaspirillum seropedicae SmR1 is an endophytic diazotroph found in several economically important crops. Considering that methods to monitor the plant–bacteria interaction are required, our objective was to develop a real-time PCR method for quantification of PGPB H. seropedicae in the rhizosphere of maize seedlings. Primer pairs were designed, and their specificity was verified using DNA from 12 different bacterial species. Ten standard curves of qPCR assay using HERBAS1 primers and tenfold serial dilutions of H. seropedicae SmR1 DNA were performed, and PCR efficiency of 91 % and correlation coefficient of 0.99 were obtained. H. seropedicae SmR1 limit of detection was 10¹ copies (corresponding to 60.3 fg of bacterial DNA). qPCR assay using HERBAS1 was used to detect and quantify H. seropedicae strain SmR1 in inoculated maize roots, cultivated in vitro and in pots, harvested 1, 4, 7, and 10 days after inoculation. The estimated bacterial DNA copy number per gram of root was in the range 10⁷–10⁹ for plants grown in vitro and it was around 10⁶ for plants grown in pots. Primer pair HERBAS1 was able to quantify H. seropedicae SmR1, and this assay can be useful for monitoring plant–bacteria interaction.     

5-Methyltetrahydrofolate is photosensitive in the presence of riboflavin.

5-Methyltetrahydrofolate (5MTHF) is the main form of folate in human plasma, and an important vitamin for human health. Photodegradation of folates may have played a role in the development of different human skin colours. 5MTHF can be degraded directly by exposure to ultraviolet radiation or by exposure to visible light in the presence of endogenous sensitizers like riboflavin (RF). These photochemical reactions were studied by absorption spectroscopy. While 5MTHF is stable under UV and visible light exposure in pure aqueous media, it is quickly degraded in the presence of RF during UVA and blue light exposure. The degradation of 5MTHF is dependent on the concentration of RF, but not on the concentration of 5MTHF itself. UVA and blue light gave similar reactions. Further investigations are necessary to evaluate the consequences of large light exposures in vivo in humans. Our findings should be taken into the ongoing discussion about the development of human skin colours. Due to the presence of RF in human blood, folate can be significantly degraded during prolonged or intense blue light exposure. Thus, a dark skin colour may be favourable for prevention of folate degradation under high solar fluence rates, such as in equatorial areas.     

Photodynamic inhibition of enzymatic detachment of human cancer cells from a substratum

The metabolism of poly(ADP-ribose) is known to play important roles in the nuclear function of the mammalian cells. In this study, changes in the activities and gene expressions of poly(ADP-ribose) glycohydrolases (PARG) in HL-60 cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or a PARG inhibitor, tannic acid, were investigated. Nuclear PARG activities of HL-60 cells treated with TPA were reduced to 30-40% of the activity in untreated cells at 24 h, while PARG activities in the cytoplasm remained unchanged. The transient decrease in the nuclear PARG activity by TPA treatment was accompanied by differentiation as measured by the nitroblue tetrazolium (NBT) reducing activity and adhesion to the culture dishes. In the presence of H7, an inhibitor of protein kinase C (PKC), both the decrease in nuclear PARG activity and the induction of differentiation by TPA treatment were suppressed. On the other hand, treatment with tannic acid caused the nuclear PARG activity to decrease continuously while the NBT reducing activity increased, but no morphological differentiation to macrophage-like cells was apparent. In order to analyze PARG gene expression, we isolated the human PARG cDNA by the RT-PCR technique. RT-PCR analysis revealed that TPA treatment leads to a reduction in the PARG gene expression prior to the phenotypic expression of macrophage-like cell differentiation, which was diminished by the presence of H7. Also, PARG gene expression was reduced by tannic acid treatment. These results provide the first evidence that a transient decrease in nuclear PARG activity is important for the onset of differentiation of HL-60 cells to macrophage-like cells.