Hormonal regulation of uterine natural killer cells in mouse preimplantation uterus

Uterine Natural Killer (uNK) cells are the most abundant lymphocyte population recruited in the uteri during murine and human pregnancy. Previous investigation on uNK cells during mouse pregnancy focused more on its accumulation in postimplantation periods, which were believed to play important roles in regulating trophoblast invasion and angiogenesis towards successful placentation. However, by using recently developed methods of Dolichos biflorus agglutinin (DBA) lectin, a closer examination during mouse preimplantation revealed that there were also dynamic regulations of uNK cell, suggesting a major regulation by steroid hormones. Here we provide a detailed examination of uNK cells distribution during mouse early pregnancy by DBA lectin reactivity, with emphasis on preimplantation period and its hormonal regulation profiles. Our results showed that uNK precursor cells or its cell membrane specific components could be recruited in the uterus by estrogen or/and progesterone, and the effects could be completely abolished by specific antagonists of their nuclear receptors (estrogen and progesterone receptor). These results suggested that the preimplantation uterus, through concerted hormone regulation, could recruit uNK precursor cell or its specific cellular component, which might be conducive for uterine receptivity and further uNK construction/function during postimplantation.     

Metabolic flexibility and cell hierarchy in metastatic cancer

Cancer is characterized by disturbed homeostasis of self-renewing cell populations, and their ability to seed and grow in multiple microenvironments. This overarching cellular property of metastatic cancer emerges from the contentious cancer stem cell hypothesis that underpins the more generic hallmarks of cancer (Hanahan and Weinberg, 2000) and its subsequent add-ons. An additional characteristic, metabolic flexibility, is related to concepts developed by Warburg and to subsequent work by mid 20th century biochemists who elucidated the bioenergetic workings of mitochondria. Metabolic flexibility may circumvent limitations inherent in the increasingly popular but erroneous view that aerobic glycolysis is a universal property of cancer cells. Cancer research in the second half of the 20th century was largely the domain of geneticists and molecular biologists using reductionist approaches. Integrated approaches that address cancer cell hierarchy and complexity, and how cancer cells adapt their metabolism according to their changing environment are now beginning to emerge, and these approaches promise to address the poor mortality statistics of metastatic cancer.     

Fever-triggered ventricular arrhythmias in Brugada syndrome and type 2 long-QT syndrome

The risk for lethal ventricular arrhythmias is increased in individuals who carry mutations in genes that encode cardiac ion channels. Loss-of-function mutations in SCN5A, the gene encoding the cardiac sodium channel, are linked to Brugada syndrome (BrS). Arrhythmias in BrS are often preceded by coved-type ST-segment elevation in the right-precordial leads V1 and V2. Loss-of-function mutations in KCNH2, the gene encoding the cardiac ion channel that is responsible for the rapidly activating delayed rectifying potassium current, are linked to long-QT syndrome type 2 (LQT-2). LQT-2 is characterised by delayed cardiac repolarisation and rate-corrected QT interval (QTc) prolongation. Here, we report that the risk for ventricular arrhythmias in BrS and LQT-2 is further increased during fever. Moreover, we demonstrate that fever may aggravate coved-type ST-segment elevation in BrS, and cause QTc lengthening in LQT-2. Finally, we describe molecular mechanisms that may underlie the proarrhythmic effects of fever in BrS and LQT-2. (Neth Heart J 2010;18:165-9.)     

Separation and purification of echinacoside from Penstemon barbatus (Can.) Roth by recycling high-speed counter-current chromatography

Echinacoside is an important bioactive compound extracted from Cistanche tubulosa which was endangered by overexploitation. It is imperative to find an alternative source. Echinacoside was isolated from Penstemon barbatus (Can.) Roth for the first time. The peak contents of echinacoside are 9.09 ± 0.32 mg/g and 7.25 ± 0.36 mg/g respectively in the leaves and roots annually. The methanolic extracts from 20 g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography (HSCCC) and recycling HSCCC with the solvent system n-butanol–water (1:1, v/v). Totally 42.0 mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR, ¹H NMR and ¹³C NMR is identical to the standard sample. This indicates that P. barbatus might be ideal source for preparation of large scale of echinacoside.     

Mechanism of CDK5 activation revealed by steered molecular dynamics simulations and energy calculations

In the current work, CDK5/p25 complexes were pulled apart by applying external forces with steered molecular dynamics (SMD) simulations. The crucial interactions between the kinase and the activation protein were investigated and the SMD simulations showed that several activation-relevant motifs of CDK5 leave p25 in sequence during the pulling and lead to an apo-CDK2 like CDK5 structure after separation. Based on systematic examination of hydrogen bond breaking and classical MD/molecular mechanics-generalized Born/surface area) (MM-GBSA) calculations, a CDK5 activation mechanism by p25 is suggested. This is the first step towards the systemic development of CDK inhibitors and the mechanism proposed could lead to a better understanding of the protein–protein recognition characteristics between the kinase and its activator.     

Quantitative proteomic analysis of G‐protein signalling in Stagonospora nodorum using isobaric tags for relative and absolute quantification

The G protein α‐subunit (Gna1) in the wheat pathogen Stagonospora nodorum has previously been shown to be a critical controlling element in disease ontogeny. In this study, iTRAQ and 2‐D LC MALDI‐MS/MS have been used to characterise protein expression changes in the S. nodorum gna1 strain versus the SN15 wild‐type. A total of 1336 proteins were identified. The abundance of 49 proteins was significantly altered in the gna1 strain compared with the wild‐type. Gna1 was identified as having a significant regulatory role on primary metabolic pathways, particularly those concerned with NADPH synthesis or consumption. Mannitol dehydrogenase was up‐regulated in the gna1 strain while mannitol 1‐phosphate dehydrogenase was down‐regulated providing direct evidence of Gna1 regulation over this enigmatic pathway. Enzymatic analysis and growth assays confirmed this regulatory role. Several novel hypothetical proteins previously associated with stress and pathogen responses were identified as positively regulated by Gna1. A short‐chain dehydrogenase (Sch3) was also significantly less abundant in the gna1 strains. Sch3 was further characterised by gene disruption in S. nodorum by homologous recombination. Functional characterisation of the sch3 strains revealed their inability to sporulate in planta providing a further link to Gna1 signalling and asexual reproduction. These data add significantly to the identification of the regulatory targets of Gna1 signalling in S. nodorum and have demonstrated the utility of iTRAQ in dissecting signal transduction pathways.     

Proteomic analysis of human gastric juice: a shotgun approach

Gastric juice is the most proximal fluid surrounding the stomach tissue. The analysis of gastric juice protein contents will thus be able to accurately reflect the pathophysiology of the stomach. This biological fluid is also a potential reservoir of secreted biomarkers in higher concentration as compared to the serum. Unlike the rest of the gastrointestinal fluids, there were very few studies reported on gastric juice proteome. To date, the proteins that routinely populate this biofluid are largely unknown. This is partly due to the technical difficulties in processing a sample that contains a collection of other gastrointestinal fluids, especially saliva. In this study, we attempt to profile the protein components of the gastric fluids from chronic gastritis patients using a direct shotgun proteomics approach. These data represent the first report of the proteome of human gastric juice with gastritis background.     

The loss of phenol sulfotransferase 1 in hepatocellular carcinogenesis

Biomarkers for the detection of early hepatocellular carcinoma (HCC) are urgently needed. To identify biomarkers of HCC, we performed a comparative proteomics analysis, based on 2‐DE of HCC tissues and surrounding non‐tumor tissues. Six xenobiotic enzymes were significantly down‐regulated in the HCC tissue. Among these, phenol sulfotransferase (SULT1A1) was confirmed by Western blot analysis in 105 HCC patients. SULT1A1 showed a significant decrease in 98.1% of the HCC tissues, with 88.6% sensitivity and 66.7% specificity for the detection of HCC. Immunohistochemistry for SULT1A1 was performed and compared with glypican‐3, which is a well‐known marker of HCC. The results showed down‐regulation of SULT1A1 and up‐regulation of glypican‐3 in 52.6 and 71.9% of the HCCs, and the use of both markers improved the sensitivity up to 78.9%. Moreover, SULT1A1 was useful in differentiating early HCC from benign dysplastic nodules. Clinically, the down‐regulation of SULT1A1 was closely associated with an advanced International Union Against Cancer stage and high levels of serum α‐fetoprotein. In conclusion, the results of this study demonstrate that the loss of SULT1A1 appears to be a characteristic molecular signature of HCC. SULT1A1 might be a useful biomarker for the detection of early HCC and help predict the clinical outcome of patients with HCC.     

Biochemical studies on malathion resistance,inheritance and association of carboxylesterase activity in brown planthopper,Nilaparvata lugens complex in Peninsular Malaysia

Abstract Two sympatric populations of brown planthopper (BPH), one from rice and the other from Leersia hexandra were collected from each of five locations in Malaysia. All the tested malathion-resistant individuals of the rice BPH population and F₁ generation (cross between malathion-resistant [usually caught on rice] and malathion-susceptible [usually caught on Leersia]) showed high esterase activity, while all malathion-susceptible individuals on L. hexandra showed low esterase activity. In the F₂ generation, all the individuals tested against malathion were approximately 75% resistant and 25% susceptible and the inheritance pattern of esterase activity (high and low esterase activity) segregated in the same manner to a 3: 1 ratio. This confirms that resistance to malathion is mono-factorial and inheritance pattern of esterase activity is also linked to malathion resistance. Carboxylesterase or total esterase activity in BPH is inherited in a simple Mendelian fashion that is encoded by a single dominant gene. For the total esterase assay, average esterase activity levels in the rice-infesting population ranged from 17.64 to 19.37 nmoles 1-napthol/mg protein while that in the Leersia-infesting population ranged from 5.29 to 6.11 nmoles 1-napthol/mg protein. In terms of esterase activity, the two sympatric Nilaparvata lugens populations separated into two distinct groups. Results based on the tube color intensity test showed 96% and 98% resistant and susceptible individuals were present in the rice- and Leersia-infesting populations, respectively. In a filter paper test, the rice-infesting population had 94% with high esterase activity while the Leersia-infesting population had 96% with low esterase activity.     

Markers of squamous cell carcinoma in sarco/endoplasmic reticulum Ca ATPase 2 heterozygote mice keratinocytes

A mutation of Atp2a2 gene encoding the sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2) causes Darier's disease in human and null mutation in one copy of Atp2a2 leads to a high incidence of squamous cell tumor in a mouse model. In SERCA2 heterozygote (SERCA2^+/−) mice keratinocytes, mechanisms involved in partial depletion of SERCA2 gene and its related tumor induction have not been studied. In this study, we investigated Ca²⁺ signaling and differential gene expression in primary cultured keratinocytes from SERCA2^+/− mice. SERCA2^+/− keratinocytes showed reduced initial increases in intracellular concentration of calcium in response to ATP, a G-protein coupled receptor agonist, and higher store-operated Ca²⁺ entry with the treatment of thapsigargin, an inhibitor of SERCA, compared to wild type kerationcytes. Protein expressions of plasma membrane Ca²⁺ ATPases, NFATc1, phosphorylated ERK, JNK, and phospholipase γ1 were increased in SERCA2^+/− keratinocytes. Using the gene fishing system, we first found in SERCA2^+/− keratinocytes that gene level of tumor-associated calcium signal transducer 1, crystalline αB, procollagen XVIII α1, and nuclear factor I-B were increased. Expression of involucrin, a marker of keratinocyte differentiation, was decreased in SERCA2^+/− keratinocytes. These results suggest that the alterations of Ca²⁺ signaling by SERCA2 haploinsufficiency alternate the gene expression of tumor induction and differentiation in keratinocytes.