Binding of N-terminal fragments of anthrax edema factor (EF(N)) and lethal factor (LF(N)) to the protective antigen pore

Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA(63), forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA(63)-channels. Here, in experiments with artificial lipid bilayer membranes, EF(N) and LF(N) show block of PA(63)-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF(N), increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA(63)-channel.     

Expression of serum amyloid A in chondrocytes and myoblasts differentiation and inflammation: possible role in cholesterol homeostasis.

Serum amyloid A (SAA) is synthesized by the liver during the acute phase. Local expression of SAA mRNA has been reported also in non-liver cells, a potential local source of SAA protein not related to the systemic acute phase response. SAA function has not been established yet. In the present study, we identified SAA as a protein expressed by chondrocytes and myoblasts in response to inflammatory stimula. In both cell systems, SAA mRNA and protein expression is strongly stimulated by bacterial lipopolysaccharide treatment. SAA mRNA expression is also enhanced during terminal differentiation of cells of the chondrogenic and myogenic lineage; mRNA is barely detectable in prechondrogenic cells and is highly expressed in differentiated hyperthrophic chondrocytes. An increased level of SAA mRNA was also observed in vivo when we compared mRNA extracted from tibiae of 10 day embryos, still fully cartilaginous, with tibiae from 18 day embryos, a stage when the endochondral ossification process has already started. p38 activation, a well-known event of the chondrogenesis signaling cascade, controls expression of SAA in cartilage following inflammatory stimuli. SAA secreted by stimulated chondrocytes is associated with cholesterol. Cholesterol is synthesized by the same chondrocytes and is also increased in inflammatory conditions. A role of SAA in cholesterol homeostasis in chondrocytes is proposed.     

miR-21 coordinates tumor growth and modulates KRIT1 levels

miR-21 is overexpressed in tumors and it displays oncogenic activity. Here, we show that expression of miR-21 in primary tumors anticorrelates with KRIT1/CCM1, an interacting partner of the Ras-like GTPase Rap1, involved in Cerebral Cavernous Malformations (CCM). We present evidences that miR-21 silences KRIT1 by targeting its mRNA 3′UTR and that this interaction is involved in tumor growth control. In fact, miR-21 over-expression or KRIT1 knock-down promote anchorage independent tumor cell growth compared to controls, whereas the opposite is observed when anti-miR-21 or KRIT1 overexpression are employed. Our findings suggest that miR-21 promotes tumor cell growth, at least in part, by down-modulating the potential tumor suppressor KRIT1.     

Refractive Errors Affect the Vividness of Visual Mental Images

The hypothesis that visual perception and mental imagery are equivalent has never been explored in individuals with vision defects not preventing the visual perception of the world, such as refractive errors. Refractive error (i.e., myopia, hyperopia or astigmatism) is a condition where the refracting system of the eye fails to focus objects sharply on the retina. As a consequence refractive errors cause blurred vision.We subdivided 84 individuals according to their spherical equivalent refraction into Emmetropes (control individuals without refractive errors) and Ametropes (individuals with refractive errors). Participants performed a vividness task and completed a questionnaire that explored their cognitive style of thinking before their vision was checked by an ophthalmologist. Although results showed that Ametropes had less vivid mental images than Emmetropes this did not affect the development of their cognitive style of thinking; in fact, Ametropes were able to use both verbal and visual strategies to acquire and retrieve information. Present data are consistent with the hypothesis of equivalence between imagery and perception.     

Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine.     

Ferulic Acid Improves Cognitive Skills Through the Activation of the Heme Oxygenase System in the Rat

Over the last years, many studies reported on the antioxidant effects of ferulic acid (FA) in preclinical models of dementia through the activation of the heme oxygenase/biliverdin reductase (HO/BVR) system. However, only a few studies evaluated whether FA could improve neurological function under milder conditions, such as psychological stress. The aim of this study was to investigate the effects of FA (150 mg/kg intraperitoneal route) on cognitive function in male Wistar rats exposed to emotional arousal. Animals were randomly assigned to two experimental groups, namely not habituated or habituated to the experimental context, and the novel object recognition test was used to evaluate their cognitive performance. The administration of FA significantly increased long-term retention memory in not habituated rats. Ferulic acid increased the expression of HO-1 in the hippocampus and frontal cortex of not habituated rats only, whereas HO-2 resulted differently modulated in these cognitive brain areas. No significant effects on either HO-1 or HO-2 or BVR were observed in the cerebellum of both habituated and not habituated rats. Ferulic acid activated the stress axis in not habituated rats, as shown by the increase in hypothalamic corticotrophin-releasing hormone levels. Pre-treatment with Sn-protoporphyrin-IX [0.25 μmol/kg, intracerebroventricular route (i.c.v.)], a well-known inhibitor of HO activity through which carbon monoxide (CO) and biliverdin (BV) are generated, abolished the FA-induced improvement of cognitive performance only in not habituated rats, suggesting a role for HO-derived by-products. The CO-donor tricarbonyldichlororuthenium (II) (30 nmol/kg i.c.v.) mimicked the FA-related improvement of cognitive skills only in not habituated rats, whereas BV did not have any effect in any group. In conclusion, these results set the stage for subsequent studies on the neuropharmacological action of FA under conditions of psychological stress.     

Chromosomal Instability and Telomere Length Variations during the Life Span of Human Fibroblast Clones

Growth characteristics, karyotype changes, and telomere length variations were analyzed during the life span of 12 anchorage-independent clones isolated from a xeroderma pigmentosum fibroblast strain. After an initial period of comparable active growth, all the clones showed a decline in the growth rate and finally entered a phase of replicative senescence; however, the number of population doublings and the time required to enter senescence varied among the clones. Repeated cytogenetic analyses during culture propagation showed the appearance of chromosome anomalies, mainly telomeric association (tas) and unbalanced translocations. In all the clones the percentage of abnormal mitoses increased with culture passage, but reached different levels (from less than 10% to about 100%). This finding indicates that the replicative block may be associated with differently altered cytogenetic patterns. Specific chromosome arms (5p, 16q, 19q, and 20q) were preferentially involved intas,suggesting that alterations in chromosome ends may occur which predispose to fusion. In some clones it was possible to demonstrate the origin of marker chromosomes from the evolution oftas.Telomere length analysis by Southern blotting on DNA samples prepared from 7 clones and from the parental cell lines showed that the terminal restriction fragment (TRF) profiles were homogeneous in senescent parental cells and in the clones during the last part of their life in culture, regardless of the degree of karyotype abnormalities. The homogeneity of the TRF profiles supports the hypothesis of a critical telomere length at senescence.     

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.     

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Discrimination of Food-Borne Microorganisms

A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.     

Viral Proteins Formed in a Cell-Free RABBIT Reticulocyte System Programmed with RNA from a Temperature-Sensitive Mutant of Sindbis Virus

Viral messenger RNA was isolated from BHK cells infected with a temperature-sensitive mutant of Sindbis virus and was further purified using an oligo(dT) column. Addition of this mRNA cell-free extracts from rabbit reticulocytes led to formation of discrete authentic viral capsid protein when the reaction was performed at 29 C. However, this same protein-synthesizing system failed to make discrete viral capsid when incubated with the viral RNA at 39 C. Instead, larger-molecular-weight polypeptides that contained the viral capsid peptide sequences were produced. The inability to make a separate viral capside protein in vitro at elevated temperatures by the mRNA from this mutant exactly mimics the phenotype of this ts mutant in viral-infected cells. Three mechanisms are discussed that might account for a temperature-sensitive release of capsid. One of these is based on a model in which there are multiple sites for initiation of translation of polypeptides on a polycistronic viral mRNA.