The role of reelin in the development and evolution of the cerebral cortex

Reelin is an extracellular matrix protein that is defective in reeler mutant mice and plays a key role in the organization of architectonic patterns, particularly in the cerebral cortex. In mammals, a "reelin signal" is activated when reelin, secreted by Cajal-Retzius neurons, binds to receptors of the lipoprotein receptor family on the surface of cortical plate cells, and triggers Dab1 phosphorylation. As reelin is a key component of cortical development in mammals, comparative embryological studies of reelin expression were carried out during cortical development in non-mammalian amniotes (turtles, squamates, birds and crocodiles) in order to assess the putative role of reelin during cortical evolution. The data show that reelin is present in the cortical marginal zone in all amniotes, and suggest that reelin has been implicated in the evolution of the radial organization of the cortical plate in the synapsid lineage leading from stem amniotes to mammals, as well as in the lineage leading to squamates, thus providing an example of homoplastic evolution (evolutionary convergence). The mechanisms by which reelin instructs radial cortical organization in these two lineages seem different: in the synapsid lineage, a drastic amplification of reelin production occurred in Cajal-Retzius cells, whereas in squamates, in addition to reelin-secreting cells in the marginal zone, a second layer of reelin-producing cells developed in the subcortex. Altogether, our results suggest that the reelin-signaling pathway has played a significant role in shaping the evolution of cortical development.

Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses

Molecular oxygen (O2) is the premier biological electron acceptor that serves vital roles in fundamental cellular functions. However, with the beneficial properties of O2 comes the inadvertent formation of reactive oxygen species (ROS) such as superoxide (O2·-), hydrogen peroxide, and hydroxyl radical (OH·). If unabated, ROS pose a serious threat to or cause the death of aerobic cells. To minimize the damaging effects of ROS, aerobic organisms evolved non-enzymatic and enzymatic antioxidant defenses. The latter include catalases, peroxidases, superoxide dismutases, and glutathione S-transferases (GST). Cellular ROS-sensing mechanisms are not well understood, but a number of transcription factors that regulate the expression of antioxidant genes are well characterized in prokaryotes and in yeast. In higher eukaryotes, oxidative stress responses are more complex and modulated by several regulators. In mammalian systems, two classes of transcription factors, nuclear factor kB and activator protein-1, are involved in the oxidative stress response. Antioxidant-specific gene induction, involved in xenobiotic metabolism, is mediated by the "antioxidant responsive element" (ARE) commonly found in the promoter region of such genes. ARE is present in mammalian GST, metallothioneine-I and MnSod genes, but has not been found in plant Gst genes. However, ARE is present in the promoter region of the three maize catalase (Cat) genes. In plants, ROS have been implicated in the damaging effects of various environmental stress conditions. Many plant defense genes are activated in response to these conditions, including the three maize Cat and some of the superoxide dismutase (Sod) genes.

Enucleated L929 mouse fibroblasts support invasion and multiplication of Shigella flexneri 5a

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

Synthesis and secretion of transferrin by a bovine trabecular meshwork cell line

The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). β-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Role of high mobility group box-1 and protection of growth hormone and somatostatin in severe acute pancreatitis

In this study, we investigated the potential role of high-mobility group box 1 (HMGB1) in severe acute pancreatitis (SAP) and the effects of growth hormone (G) and somatostatin (S) in SAP rats. The rats were randomly divided into 6 groups of 20 each: sham-operated, SAP, SAP+saline, SAP+G, SAP+S and SAP+G+S. Ileum and pancreas tissues of rats in each group were evaluated histologically. HMGB1 mRNA expression was measured by reverse transcription-PCR. Levels of circulating TNF-α, IL-1, IL-6, and endotoxin were also measured. In the SAP group, interstitial congestion and edema, inflammatory cell infiltration, and interstitial hemorrhage occurred in ileum and pancreas tissues. The levels of HMGB1, TNF-α, IL-1, IL-6 and endotoxin were significantly up-regulated in the SAP group compared with those in the sham-operated group, and the 7-day survival rate was 0%. In the SAP+G and SAP+S groups, the inflammatory response of the morphological structures was alleviated, the levels of HMGB1, TNF-α, IL-1, IL-6, and endotoxin were significantly decreased compared with those in the SAP group, and the survival rate was increased. Moreover, in the SAP+G+S group, all histological scores were significantly improved and the survival rate was significantly higher compared with the SAP group. In conclusion, HMGB1 might participate in pancreas and ileum injury in SAP. Growth hormone and somatostatin might play a therapeutic role in the inflammatory response of SAP.

The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells

We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

Molecular characterization and evaluation of antimicrobial susceptibility of enteropathogenic E. coli (EPEC) isolated from minas soft cheese

Foodborne disease caused by microorganisms is a problem of public health. Minas soft cheese is a national product manufactured using simple technology; it has high level of acceptance in the country making its production an important economic activity. Many microorganisms may be present in foods including the bacterium Escherichia coli (E. coli). Overall, E. coli is a harmless commensal bacterium; however, some strains may have a pathogenic potential. Several outbreaks of foodborne diseases associated with consumption of contaminated cheese have been reported, and the presence of pathogenic strains of E. coli has increased. The objective of this study was to isolate, evaluate the antimicrobial susceptibility, and characterize, by Multiplex PCR, the pathogenic E. coli strains isolated from Minas cheese commercialized in Rio de Janeiro. Thirty samples were analyzed and five strains of E. coli (EPEC) were identified. The assessment of antimicrobial susceptibility revealed 40% of the isolates resistant to ampicillin and 40% with intermediate resistance to ampicillin-sulbactam combination. These findings are a warning signal to health authorities since Minas cheese is a ready to eat food product, and therefore should not pose health risks to the population.