In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion

Detailed structure-function information about human lipoprotein lipase (LPL) is unavailable because it is difficult to purify large amounts of the enzyme for study. To circumvent this problem, we constructed an in vitro LPL expression vector. Human LPL cDNA was cloned and inserted into the expression vector p91023(B). After transfection of COS M-6 cells with the human LPL cDNA construct, LPL enzyme activity was detected in cell extracts and culture medium. Purified human apolipoprotein C-II caused a 5-fold stimulation of the recombinant human LPL expressed in vitro. Using site-specific mutagenesis, Ala residues were substituted for Asn residues at two potential N-linked glycosylation sites (positions 43 and 359) and at a third unrelated Asn (position 257) in the LPL cDNA. RNA blot analysis demonstrated the presence of a single mRNA species in COS cells transfected with wild-type and mutant LPL expression vectors. Intracellular and secreted LPL activity was absent in the construct containing an Ala for Asn mutation at position 43, whereas the same substitutions at positions 257 and 359 did not appreciably affect activity. LPL activity was also absent in another construct containing a Gln for Asn mutation at position 43. Quantitation of LPL protein mass concomitant with measurement of enzyme activity showed that substitution of Ala or Gln for Asn at position 43 resulted in the production of an enzymatically inactive protein which accumulated intracellularly but was not secreted into the culture medium. Our report represents an initial documentation of the expression of cloned human LPL in vitro and of the importance of Asn-43 for both enzyme activity and secretion.     

Abamectin resistance in Drosophila is related to increased expression of P-glycoprotein via the dEGFR and dAkt pathways

Many insects have evolved resistance to abamectin but the mechanisms involved in this resistance have not been well characterized. P-glycoprotein (P-gp), an ATP-dependent drug-efflux pump transmembrane protein, may be involved in abamectin resistance. We investigated the role of P-gp in abamectin (ABM) resistance in Drosophila using an ABM-resistant strain developed in the laboratory. A toxicity assay, Western blotting analysis and a vanadate-sensitive ATPase activity assay all demonstrated the existence of a direct relationship between P-gp expression and ABM resistance in these flies. Our observations indicate that P-gp levels in flies' heads were higher than in their thorax and abdomen, and that both P-gp levels and LC₅₀ values were higher in resistant than in susceptible and P-gp-deficient strains. In addition, P-gp levels in the blood–brain barrier (BBB) of resistant flies were higher than in susceptible and P-gp-deficient flies, which is further evidence that a high level of P-gp in the BBB is related to ABM resistance. Furthermore, we found greater expression of Drosophila EGFR (dEGFR) in the resistant strain than in the susceptible strain, and that the level of Drosophila Akt (dAkt) was much higher in resistant than in susceptible flies, whereas that in P-gp-deficient flies was very low. Compared to susceptible flies, P-gp levels in the resistant strain were markedly suppressed by the dEGFR and dAkt inhibitors lapatinib and wortmannin. These results suggest that the increased P-gp in resistant flies was regulated by the dEGFR and dAkt pathways and that increased expression of P-gp is an important component of ABM resistance in insects.     

Psychometric properties of Youth Self-Rating Insomnia Scale (YSIS) in Chinese adolescents

Sleep and Biological Rhythms - Insomnia is prevalent in adolescents. Although several insomnia scales/questionnaires are available to assess insomnia symptoms and severity for adults, no insomnia...     

Tongxinluo Protects against Hypertensive Kidney Injury in Spontaneously-Hypertensive Rats by Inhibiting Oxidative Stress and Activating Forkhead Box O1 Signaling

Hypertension is an independent risk factor for the progression of chronic renal failure, and oxidative stress plays a critical role in hypertensive renal damage. Forkbox O1(FoxO1) signaling protects cells against oxidative stress and may be a useful target for treating oxidative stress-induced hypertension. Tongxinluo is a traditional Chinese medicine with cardioprotective and renoprotective functions. Therefore, this study aimed to determine the effects of Tongxinluo in hypertensive renal damage in spontaneously hypertensive rats(SHRs)and elucidate the possible involvement of oxidative stress and FoxO1 signaling in its molecular mechanisms. SHRs treated with Tongxinluo for 12 weeks showed a reduction in systolic blood pressure. In addition to increasing creatinine clearance, Tongxinluo decreased urinary albumin excretion, oxidative stress injury markers including malondialdehyde and protein carbonyls, and expression of nicotinamide adenine dinucleotide phosphate oxidase subunits and its activity in SHR kidneys. While decreasing phosphorylation of FoxO1, Tongxinluo also inhibited the phosphorylation of extracellular signal-regulated kinase1/2 and p38 and enhanced manganese superoxide dismutase and catalase activities in SHR kidneys. Furthermore, histology revealed attenuation of glomerulosclerosis and renal podocyte injury, while Tongxinluo decreased the expression of α-smooth muscle actin, extracellular matrixprotein, transforming growth factor β1 and small mothers against decapentaplegic homolog 3,and improved tubulointerstitial fibrosis in SHR kidneys. Finally, Tongxinluo inhibited inflammatory cell infiltration as well as expression of tumor necrosis factor-α and interleukin-6. In conclusion, Tongxinluo protected SHRs against hypertension-induced renal injury by exerting antioxidant, antifibrotic, and anti-inflammatory activities. Moreover, the underlying mechanisms of these effects may involve inhibition of oxidative stress and functional activation of FoxO1 signaling.     

Organismal Aging and Oxidants beyond Macromolecules Damage

The relationship between oxidants and organismal aging was first articulated through the free radical theory of aging. One of the major predictions of the free radical theory of aging is that oxidative stress shortens organisms’ lifespan because of an increased level of oxidants, which are damaging to macromolecules. However, challenging the role of oxidants in age‐related diseases, there is now sufficient evidence that antioxidant supplements do not provide significant health benefits. Interestingly, in addition to an increase in oxidant‐mediated macromolecules damage, there is convincing experimental data to support the role of senescent cells in the process of aging. Here, the current knowledge regarding the role of oxidants and cellular senescence in organismal aging is reviewed and it is proposed that, in addition to the role of oxidants as inducers of macromolecular damage, oxidants may also function as regulators of signaling pathways involved in the establishment of cellular senescence. If this role for oxidants is established, it may be necessary to modify the free radical theory of aging from “Organisms age because cells accumulate reactive oxygen species‐dependent damage over time” to: “Organisms age because cells accumulate oxidants’‐dependent damage and oxidants’‐dependent senescent characteristics over time.”     

PDMS Microwell Stencil Based Multiplexed Single‐Cell Secretion Analysis

Multiplexed single‐cell protein secretion analysis provides an in‐depth understanding of cellular heterogeneity in intercellular communications mediated by secreted proteins in both fundamental and clinical research. However, it has been challenging to increase the proteomic parameters co‐profiled from every single cell in a facile way. Herein, a simple method to improve the multiplexed proteomic parameters of PDMS microwell based single‐cell secretion analysis platform by sandwiching PDMS stencil in between two antibody‐coated glass slides is introduced. Two different antibody panels can be immobilized easily by static coating, without using sophisticated fluid handling or bulky equipment. 5‐plexed, 3‐fluorescence color single‐cell secretion assay is demonstrated with this platform to investigate human monocytic U937 cells in response to lipopolysaccharide and phorbol myristate acetate stimulation, which identified the existence of functional subsets dictated by different cytokine profiles. The technology introduced here is simple, easy to operate, which holds great potential to become a powerful tool for profiling multiplexed single‐cell cytokine secretion at high throughput to dissect cellular heterogeneity in secretome signatures.     

Experiencing winter for spring flowering: A molecular epigenetic perspective on vernalization

Many over-wintering plants, through vernalization, overcome a block to flowering and thus acquire competence to flower in the following spring after experiencing prolonged cold exposure or winter cold. The vernalization pathways in different angiosperm lineages appear to have convergently evolved to adapt to temperate climates. Molecular and epigenetic mechanisms for vernalization regulation have been well studied in the crucifer model plant Arabidopsis thaliana.Here, we review recent progresses on the vernalization pathway in Arabidopsis. In addition, we summarize current molecular and genetic understandings of vernalization regulation in temperate grasses including wheat and Brachypodium, two monocots from Pooideae, followed by a brief discussion on divergence of the vernalization pathways between Brassicaceae and Pooideae.     

Cardiac telocytes exist in the adult Xenopus tropicalis heart

Recent research has revealed that cardiac telocytes (CTs) play an important role in cardiac physiopathology and the regeneration of injured myocardium. Recently, we reported that the adult Xenopus tropicalis heart can regenerate perfectly in a nearly scar‐free manner after injury via apical resection. However, whether telocytes exist in the X tropicalis heart and are affected in the regeneration of injured X tropicalis myocardium is still unknown. The present ultrastructural and immunofluorescent double staining results clearly showed that CTs exist in the X tropicalis myocardium. CTs in the X tropicalis myocardium were mainly twined around the surface of cardiomyocyte trabeculae and linked via nanocontacts between the ends of the telopodes, forming a three‐dimensional network. CTs might play a role in the regeneration of injured myocardium.