The porcine chloride channel calcium-activated family member pCLCA4a mirrors lung expression of the human hCLCA4

Pig models of cystic fibrosis (CF) have recently been established that are expected to mimic the human disease closer than mouse models do. The human CLCA (originally named chloride channels, calcium-activated) member hCLCA4 is considered a potential modifier of disease severity in CF, but its murine ortholog, mCLCA6, is not expressed in the mouse lung. Here, we have characterized the genomic structure, protein processing, and tissue expression patterns of the porcine ortholog to hCLCA4, pCLCA4a. The genomic structure and cellular protein processing of pCLCA4a were found to closely mirror those of hCLCA4 and mCLCA6. Similar to human lung, pCLCA4a mRNA was strongly expressed in porcine lungs, and the pCLCA4a protein was immunohistochemically detected on the apical membranes of tracheal and bronchial epithelial cells. This stands in sharp contrast to mouse mCLCA6, which has been detected exclusively in intestinal epithelia but not the murine lung. The results may add to the understanding of species-specific differences in the CF phenotype and support the notion that the CF pig model may be more suitable than murine models to study the role of hCLCA4.     

Binding of trastuzumab to ErbB2 is inhibited by a high pericellular density of hyaluronan

Although trastuzumab is an efficient drug, primary and acquired resistance is a challenging problem. The authors have previously shown in mouse xenograft experiments that masking ErbB2 by hyaluronan leads to diminished binding of the antibody and consequent resistance. In the current work, they correlated trastuzumab binding with the pericellular density of hyaluronan in ErbB2-overexpressing human breast cancer samples. A method for quantifying the relative binding of trastuzumab was developed involving constant and low-frequency background subtraction, segmenting the image to membrane and background pixels followed by evaluation of trastuzumab fluorescence, normalized with the expression level of ErbB2, only in the membrane. The normalized binding of trastuzumab showed a negative correlation with the pericellular density of hyaluronan (r = -0.52) with the effect being the most pronounced in the extreme cases (i.e., low and high hyaluronan densities predicted strong and weak binding of trastuzumab, respectively). Removal of hyaluronan by hyaluronidase digestion unmasked the trastuzumab binding epitope of ErbB2 demonstrated by a significantly increased normalized binding of the antibody. The results show that the accumulation of pericellular hyaluronan plays a crucial role in masking ErbB2.     

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.     

Effect of reactivity on cellular accumulation and cytotoxicity of oxaliplatin analogues

The purpose of this study was to systematically investigate the relationships between reactivity, cellular accumulation, and cytotoxicity of a panel of oxaliplatin analogues with different leaving groups in human carcinoma cells. The reactivity of the complexes towards the nucleotides 2'-deoxyguanosine 5'-monophosphate and 2'-deoxyadenosine 5'-monophosphate was studied using capillary electrophoresis. Cellular accumulation and cytotoxicity were measured in an oxaliplatin-sensitive and oxaliplatin-resistant ileocecal colorectal adenocarcinoma cell line pair (HCT-8/HCT-8ox). Platinum concentrations were determined by flameless atomic absorption spectrometry. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess cytotoxicity. Early cellular platinum accumulation was predominantly affected by lipophilicity. A relationship between reactivity and cellular accumulation was observed for three of four platinum complexes investigated, whereas the most lipophilic oxaliplatin analogue was an exception. Increased reactivity and reduced lipophilicity were associated with high cytotoxic activity. Resistance was influenced by lipophilicity but not by reactivity. The observed relationships may help in the design of analogues with high antitumoral activity in oxaliplatin-sensitive as well as oxaliplatin-resistant cells.     

Combination of chemometrically assisted voltammetry, calorimetry, and circular dichroism as a new method for the study of bioinorganic substances: application to selenocystine metal complexes

Selenium-containing compounds play an important role in antioxidant defense systems, binding to toxic metals, preventing their uptake into cells, and thus protecting cells from metal-induced formation of reactive oxygen species. Here, we present a proposal for a relatively new method as a complement to the more usual methods used in selenium studies. A systematic study of the metal-binding properties of selenocystine (SeCyst) in the presence of divalent metal cations (Cd, Co, Hg, Ni, and Zn) is reported. Isothermal titration calorimetry provides thermodynamic parameters of the systems. Titrations produced curves that could be fit reasonably well to the one set of sites model. The data clearly demonstrate that one M²⁺ binds one SeCyst molecule, and the stable M(SeCyst) complex is formed under these conditions. The order of the SeCyst binding constant for the metal ions is Hg²⁺ > Cd²⁺ ~ Zn²⁺ > Ni²⁺> Co²⁺. Cadmium ion was selected as a modulator for the behavior of SeCyst in the presence of a nonessential metal, and zinc was selected for the case of an essential element. These interactions of SeCyst with Cd²⁺ and Zn²⁺, either individually or combined, were studied in aqueous buffered solutions at physiological pH by differential pulse polarography and circular dichroism spectroscopy. Furthermore, recently developed chemometric tools were applied to differential pulse polarography data obtained in mixtures of SeCyst and glutathione in the presence of Cd²⁺ at physiological pH.     

The alkenyl migration mechanism catalyzed by extradiol dioxygenases: a hybrid DFT study

6-Hydroxymethyl-6-methylcyclohexa-2,4-dienone is a mechanistic probe which when incubated with an extradiol dioxygenase yields a 2-tropolone product. This observation was originally interpreted as evidence supporting a direct heterolytic 1,2-alkenyl migration mechanism for a ring expansion reaction catalyzed by this class of Fe(II)-dependent nonheme enzymes (Xin and Bugg in J Am Chem Soc 130:10422-10430, 2008). In the work reported in this contribution we used quantum chemical methods to test whether such a mechanism is energetically possible and we found that it is not, neither for the mechanistic probe nor for the native catalytic cycle intermediate. Models of increasing complexity were used to calculate energy barriers to the heterolytic 1,2-alkenyl migration and alternative radical mechanisms. It was found that the former involves substantially higher barriers than the latter. A tentative radical mechanism that accounts for the transformation of the probe substrate to 2-tropolone was also proposed, and it involves acceptable barriers.     

Acute inhibition of iron bioavailability by zinc: studies in humans

Iron (Fe) and zinc (Zn) deficiencies constitute two of the most important nutritional and public health problems affecting developing countries. Combined supplementation or fortification with Zn and Fe are strategies that can be used to improve the Zn and Fe status of a population. However, there is concern about potential negative interactions between these two micronutrients due to a competitive binding to DMT1 and Zip14 transporter. Studies performed in humans have shown an inhibitory effect of Zn on Fe absorption when both minerals are given together as a solution in fasting conditions. We found that at low doses of iron (0.5 mg) the threshold for the inhibition of iron bioavailability was at a Zn:Fe wt/wt ratio ≥5.9:1, whereas at higher doses of Fe (10 mg) this inhibition occurred at 1:1 Zn:Fe wt/wt ratio. This differential response could be explained by the variation in the abundance of both cations as they compete for a limited number of shared transporters at the enterocyte. Conflicting results have been obtained when this interaction was studied in different food matrices. A negative interaction was not observed when Fe and Zn were provided in a composite hamburger meal, premature formula, human milk, or cow milk. A decrease on Fe absorption was observed in only 1 of 3 studies when Fe and Zn were supplied in wheat flour. The possibility of a negative interaction should be considered for supplementation or fortification programs with both microminerals.     

Iron toxicity in neurodegeneration

Iron is an essential element for life on earth, participating in a plethora of cellular processes where one-electron transfer reactions are required. Its essentiality, coupled to its scarcity in aqueous oxidative environments, has compelled living organisms to develop mechanisms that ensure an adequate iron supply, at times with disregard to long-term deleterious effects derived from iron accumulation. However, iron is an intrinsic producer of reactive oxygen species, and increased levels of iron promote neurotoxicity because of hydroxyl radical formation, which results in glutathione consumption, protein aggregation, lipid peroxidation and nucleic acid modification. Neurons from brain areas sensitive to degeneration accumulate iron with age and thus are subjected to an ever increasing oxidative stress with the accompanying cellular damage. The ability of these neurons to survive depends on the adaptive mechanisms developed to cope with the increasing oxidative load. Here, we describe the chemical and thermodynamic peculiarities of iron chemistry in living matter, review the components of iron homeostasis in neurons and elaborate on the mechanisms by which iron homeostasis is lost in Parkinson's disease, Alzheimer's disease and other diseases in which iron accumulation has been demonstrated.     

Iron deficiency on neuronal function

Because of the intrinsic ability of iron to catalyze the formation of reactive oxygen species, it has been associated with oxidative stress and neurodegenerative diseases. However, iron deficiency (ID) also negatively impacts various functions of the brain, suggesting that iron plays an important physiological role in neuronal processes such as myelination, synaptogenesis, behavior and synaptic plasticity (SP). ID not only produces changes in the hippocampus, striatum, amygdale or prefrontal cortex, it also affects the interaction among these systems. In both humans and rodents, the perturbations of these structures are associated to cognitive deficits. These cognitive alterations have been well correlated with changes in neural plasticity, the possible cellular substrate of memory and learning. Given that SP is strongly affected by early ID and the lasting-neurological consequences remain even after ID has been corrected, it is important to prevent ID as well as to seek effective therapeutic interventions that reduce or reverse the long-term effects of the ID in the nervous system. This review will give an overview of the literature on the effects of iron deficit in neuronal functions such as behavior, neurotransmission and SP. We also discuss our recent data about the possible oxidative effect of iron on the mechanisms involved in neural plasticity.     

基层医院重症医学科运行模式的研究

??????? 目的 建立健全适于基层医疗单位重症医学科发展的运行模式。方法 以重症胰腺炎为例,比较普通模式和协作小组模式下参与治疗的专业人数、住ICU天数、ICU治疗费用、住院总费用、呼吸机天数、血液滤过总时间、住院期间入液总量和药物占住院费用比例等方面的差异。结果 与普通模式相比,协作小组模式下,参与治疗的专业人数增加、住ICU天数、ICU治疗费用和住院总费用下降,血液滤过总时间延长、住院期间入液总量减少和药物占住院费用比例降低。结论 协作小组是基层医院重症医学科运行的好模式,值得进一步完善。