Corticotropin releasing hormone and related peptides can act as bioregulatory factors in human keratinocytes

Summary Following previous findings in human skin of the functional expression of genes for the corticotropin releasing hormone (CHR) receptor type 1 (CRH-R1) and CRH itself, we searched for local phenotypic effects for peptides related to CRH. We now report that CRH, sauvagine, and urocortin inhibit proliferation of human HaCaT keratinocytes in a dose-dependent manner. The peptides produced variable cyclic adenosine 3′∶5′-monophosphate stimulation with CRH having the highest potency. Binding of iodine 125 CRH to intact keratinocytes was inhibited by increasing doses of CRH, sauvagine, or urocortin, all showing equal inhibitory potency. Immunocytochemistry identified CRH-R1 immunoreactivity in HaCaT keratinocytes. In conclusion, CRH (exogenous or produced locally) and the related urocortin and sauvagine peptides can modify human keratinocyte phenotype through a receptor-mediated pathway.     

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods—direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc—in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols.     

Cellular Distribution and Subcellular Localization of mCLCA1/2 in Murine Gastrointestinal Epithelia

mCLCA1/2 are members of the CLCA protein family that are widely expressed in secretory epithelia, but their putative physiological role still awaits elucidation. mCLCA1/2 have 95% amino acid identity, but currently no specific antibody is available. We have generated a rabbit polyclonal antibody (pAb849) against aa 424–443 of mCLCA1/2. In HEK293 cells transfected with mCLCA1; pAb849 detected two specific protein bands at ∼125 kDa and 90 kDa, representing full-length precursor and N-terminal cleavage product, respectively. pAb849 also immunoprecipitated mCLCA1 and labeled the protein by immunostaining. But pAb849 crossreacted with mCLCA3/4/6 despite ≤80% amino acid identity of the antigenic epitope. We therefore investigated the cellular localization of mCLCA1/2 in epithelial tissues, which do not express mCLCA3/4/6 (salivary glands, pancreas, kidney) or express mCLCA3/6 with known localization (mucus cells of stomach and small intestine; villi of small intestine). mCLCA1/2 mRNA and protein expression were found in both parotid and submandibular gland, and immunohistochemistry revealed labeling in parotid acinar cells, in the luminal membrane of parotid duct cells, and in the duct cells of submandibular gland. In exocrine pancreas, mCLCA1/2 expression was restricted to acinar zymogen granule membranes, as assessed by immunoblotting, immunohistochemistry, and preembedding immunoperoxidase and immunogold electron microscopy. Moreover, mCLCA1/2 immunolabeling was present in luminal membranes of gastric parietal cells and small intestinal crypt enterocytes, whereas in the kidney, mCLCA1/2 protein was localized to proximal and distal tubules. The apical membrane localization and overall distribution pattern of mCLCA1/2 favor a transmembrane protein implicated in transepithelial ion transport and protein secretion. (J Histochem Cytochem 58:653–668, 2010)     

Disparate patterns of age-related changes in lipid peroxidation in long-lived naked mole-rats and shorter-lived mice

A key tenet of the oxidative stress theory of aging is that levels of accrued oxidative damage increase with age. Differences in damage generation and accumulation therefore may underlie the natural variation in species longevity. We compared age-related profiles of whole-organism lipid peroxidation (urinary isoprostanes) and liver lipid damage (malondialdehyde) in long living naked mole-rats [maximum lifespan (MLS) > 28.3 years] and shorter-living CB6F1 hybrid mice (MLS approximately 3.5 years). In addition, we compared age-associated changes in liver non-heme iron to assess how intracellular conditions, which may modulate oxidative processes, are affected by aging. Surprisingly, even at a young age, concentrations of both markers of lipid peroxidation, as well as of iron, were at least twofold (P < 0.005) greater in naked mole tats than in mice. This refutes the hypothesis that prolonged naked mole-rat longevity is due to superior protection against oxidative stress. The age-related profiles of all three parameters were distinctly species specific. Rates of lipid damage generation in mice were maintained throughout adulthood, while accrued damage in old animals was twice that of young mice. In naked mole-rats, urinary isoprostane excretion declined by half with age (P < 0.001), despite increases in tissue iron (P < 0.05). Contrary to the predictions of the oxidative stress theory, lipid damage levels did not change with age in mole-rats. These data suggest that the patterns of age-related changes in levels of markers of oxidative stress are species specific, and that the pronounced longevity of naked mole-rats is independent of oxidative stress parameters.     

Sampling issues in quantitative analysis of dendritic spines morphology

ABSTRACT: BACKGROUND: Quantitative analysis of changes in dendritic spine morphology has become an interesting issue in contemporary neuroscience. However, the diversity in dendritic spines population might seriously influence the results of measurements in which their morphology is studied, the detection of differences in spine morphology between control and test group is often compromised by the number of dendritic spines taken for analysis. In order to estimate how severe is such an impact we have performed Monte Carlo simulations examining various experimental setups and statistical approaches. The confocal images of dendritic spines from hippocampal dissociated cultures have been used to create a set of variables exploited as the simulation resources. RESULTS: The tabulated results of simulations are given, providing the number of dendritic spines required for the detection of hidden morphological differences between control and test group, in spine head-width, length and area. It turns out that this is the head-width among these three variables, where the changes are most easily detected. Simulation of changes occurring in a subpopulation of spines reveal the strong dependence of detectability on the statistical approach applied. The analysis based on comparison of percentage of spines in subclasses is less sensitive than the direct comparison of relevant variables describing spines morphology. CONCLUSIONS: We evaluated the sampling aspect and effect of systematic morphological variation on detecting the differences in spine morphology. Provided results may serve as a guideline in selecting the number of samples to be studied in a planned experiment. Our simulations might be a step towards the development of a standardized method of quantitative comparison of dendritic spines morphology, in which different sources of errors are considered.     

Regulation of arsenic trioxide-induced cellular responses by Mnk1 and Mnk2

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As(2)O(3) induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38alpha MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1(-/-) or Mnk2(-/-), or double Mnk1(-/-)Mnk2(-/-) knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.     

Endothelin receptor blockade does not improve hypoxemia following acute pulmonary thromboembolism.

We studied the roles of endothelins in determining ventilation (Va) and perfusion (Q) mismatch in a porcine model of acute pulmonary thromboembolism (APTE), using a nonspecific endothelin antagonist, tezosentan. Nine anesthetized piglets (approximately 23 kg) received autologous clots (approximately 20 g) via a central venous catheter at time = 0 min. The distribution of Va and Q at five different time points (-30, -5, 30, 60, 120 min) was mapped by fluorescent microspheres of 10 different colors. Five piglets (group 1) received tezosentan (courtesy of Actelion) starting at time = 40 min for 2 h, and four piglets (group 2) received only saline and served as control. Our results showed that, in all of the animals at 30 min following APTE but before tezosentan, the mean Va/Q was increased, as was Va/Q heterogeneity (log SD Va/Q), which represented a widening of its main peak. Afterwards, tezosentan attenuated the pulmonary hypertension in group 1 but also produced moderate systemic hypotension. However, it did not improve arterial PO2 or Va/Q mismatch. We concluded that endothelin antagonism had minimal impact on gas exchange following APTE and confirmed our earlier observation that the main mechanism for hypoxemia in APTE was due to the mechanical redistribution of pulmonary regional blood flow away from the embolized vessels, resulting in the creation of many divergent low and high Va/Q regions.     

Helicobacter pylori Infection and Family History of Gastric Cancer Decrease Expression of FHIT Tumor Suppressor Gene in Gastric Mucosa of Dyspeptic Patients

Background:  The expression of a fragile histidine triad (FHIT) protein is lost in stomach tumors. The study aimed at determining whether FHIT expression is affected by Helicobacter pylori infection, strain virulence ( vacA and cagA genes) and histopathological changes in the gastric mucosa of patients with functional dyspepsia having first-degree relatives with gastric cancer.
Materials and Methods:  Eighty-eight never-smoking patients with functional dyspepsia were selected for the study, and 48 of them had first-degree relatives with gastric cancer. Bacterial DNA amplification was used to identify H. pylori colonization. The level of FHIT gene expression was determined by qRT-PCR (mRNA) and Western blot (FHIT protein) analyses.
Results:  For patients having first-degree relatives with gastric cancer FHIT expression was lower (mRNA by ca. 40–45% and protein by 30%) compared with the control patients ( p  < .05). H. pylori infection decreased the FHIT mRNA level by 10–35% and the protein level by 10–20%. Bacterial strain vacA (+) cagA (+) lowered FHIT mRNA by ca. 30–35% in the antrum samples of both groups and in corpus samples of patients with first-degree relatives with gastric cancer ( p  < .05). The FHIT mRNA level was twice as high in control H. pylori- negative patients with intestinal metaplasia, compared with those with non-atrophic gastritis.
Conclusions:  The decreased FHIT gene expression associated with hereditary factors and with H. pylori infection, especially with vacA (+) cagA (+)-positive strains, may be related to gastric carcinoma development.     

Synaptically Released Matrix Metalloproteinase Activity in Control of Structural Plasticity and the Cell Surface Distribution of GluA1-AMPA Receptors

Synapses are particularly prone to dynamic alterations and thus play a major role in neuronal plasticity. Dynamic excitatory synapses are located at the membranous neuronal protrusions called dendritic spines. The ability to change synaptic connections involves both alterations at the morphological level and changes in postsynaptic receptor composition. We report that endogenous matrix metalloproteinase (MMP) activity promotes the structural and functional plasticity of local synapses by its effect on glutamate receptor mobility and content. We used live imaging of cultured hippocampal neurons and quantitative morphological analysis to show that chemical long-term potentiation (cLTP) induces the permanent enlargement of a subset of small dendritic spines in an MMP-dependent manner. We also used a superresolution microscopy approach and found that spine expansion induced by cLTP was accompanied by MMP-dependent immobilization and synaptic accumulation as well as the clustering of GluA1-containing AMPA receptors. Altogether, our results reveal novel molecular and cellular mechanisms of synaptic plasticity.     

Influenza Pneumonia Surveillance among Hospitalized Adults May Underestimate the Burden of Severe Influenza Disease

Background

Studies seeking to estimate the burden of influenza among hospitalized adults often use case definitions that require presence of pneumonia. The goal of this study was to assess the extent to which restricting influenza testing to adults hospitalized with pneumonia could underestimate the total burden of hospitalized influenza disease.

Methods

We conducted a modelling study using the complete State Inpatient Databases from Arizona, California, and Washington and regional influenza surveillance data acquired from CDC from January 2003 through March 2009. The exposures of interest were positive laboratory tests for influenza A (H1N1), influenza A (H3N2), and influenza B from two contiguous US Federal Regions encompassing the study area. We identified the two outcomes of interest by ICD-9-CM code: respiratory and circulatory hospitalizations, as well as critical illness hospitalizations (acute respiratory failure, severe sepsis, and in-hospital death). We linked the hospitalization datasets with the virus surveillance datasets by geographic region and month of hospitalization. We used negative binomial regression models to estimate the number of influenza-associated events for the outcomes of interest. We sub-categorized these events to include all outcomes with or without pneumonia diagnosis codes.

Results

We estimated that there were 80,834 (95% CI 29,214–174,033) influenza-associated respiratory and circulatory hospitalizations and 26,760 (95% CI 14,541–47,464) influenza-associated critical illness hospitalizations. When a pneumonia diagnosis was excluded, the estimated number of influenza-associated respiratory and circulatory hospitalizations was 24,816 (95% CI 6,342–92,624). The estimated number of influenza-associated critical illness hospitalizations was 8,213 (95% CI 3,764–20,799). Around 30% of both influenza-associated respiratory and circulatory hospitalizations, as well as influenza-associated critical illness hospitalizations did not have pneumonia diagnosis codes.

Conclusions

Surveillance studies which only consider hospitalizations that include a diagnosis of pneumonia may underestimate the total burden of influenza hospitalizations.