Regulation of microtubule dynamics by the neuronal growth-associated protein SCG10.

Dynamic assembly and disassembly of microtubules is essential for cell division, cell movements, and intracellular transport. In the developing nervous system, microtubule dynamics play a fundamental role during neurite outgrowth, elongation, and branching, but the molecular mechanisms involved are unknown. SCG10 is a neuron-specific protein that is membrane-associated and highly enriched in growth cones. Here we show that SCG10 binds to microtubules, inhibits their assembly, and can induce microtubule disassembly. We also show that SCG10 overexpression enhances neurite outgrowth in a stably transfected neuronal cell line. These data identify SCG10 as a key regulator of neurite extension through regulation of microtubule instability.

Investigation of the hepatitis C virus replication complex.

Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, altered cellular membranes, and other host factors, is a hallmark of all positive-strand RNA viruses. In the case of HCV, RNA replication takes place in a likely endoplasmic reticulum-derived membrane alteration referred to as the "membranous web." In vitro transcription-translation, membrane extraction and flotation analyses, immunofluorescence microscopy, fluorescent in situ hybridization, and RNA metabolic labeling followed by confocal laser scanning microscopy have yielded insights into the structure and function of the HCV replication complex. We describe these techniques and highlight selected results.

Protective effect of intravitreal administration of tresperimus, an immunosuppressive drug, on experimental autoimmune uveoretinitis.

PURPOSE: To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS: In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS: Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons.

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).

Antigenic characterization of Brazilian isolates of Anaplasma marginale

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.

The effect of in vitro heating on the distribution of nuclear matrix polypeptides in HeLa cells.

The in situ nuclear matrix was obtained from HeLa cells. After permeabilization with nonionic detergent, the resulting structures were incubated for 1 h at 37 degrees C to determine whether or not such an incubation might result in the redistribution of nuclear polypeptides which resisted extraction with buffers of high-ionic strength (1.6 M NaCl or 0.25 M (NH4)2SO4 as well as DNase I digestion. Using indirect immunofluorescence experiments and monoclonal antibodies we show that heating to 37 degrees C changes the distribution of a 160 kDa protein previously shown to be a component of the inner matrix network. On the other hand, a 125 kDa polypeptide was not affected at all by the incubation. Our results clearly indicate that the inclusion of a 37 degrees C incubation (for example during digestion with DNase I) in the protocol to obtain the in situ nuclear matrix can result in the formation of in vitro artifacts.

Influence of epidermal growth factor on the maturation of fetal rat brain cells in aggregate culture. An immunocytochemical study.

Maturation of astrocytes, neurons, and oligodendrocytes was studied in serum-free aggregating cell cultures of fetal rat telencephalon by an immunocytochemical approach. Cell type-specific immunofluorescence staining was examined by using antibodies directed against glial fibrillary acidic protein (GFAP) and vimentin, two astroglial markers; neuron-specific enolase (NSE) and neurofilament (NF), two neuronal markers, and galactocerebroside (GC), an oligodendroglial marker. It was found that the cellular maturation in aggregates is characterized by distinct developmental increases in immunoreactivity for GFAP, vimentin, NSE, NF, and GC, and by a subsequent decrease of vimentin-positive structures in more differentiated cultures. These findings are in agreement with observations in vivo, and they corroborate previous biochemical studies of this histotypic culture system. Treatment of very immature cultures with a low dose of epidermal growth factor (EGF, 5 ng/ml) enhanced the developmental increase in GFAP, NSE, NF and GC immunoreactivity, suggesting an acceleration of neuronal and glial maturation. In addition, EGF was found to alter the cellular organization within the aggregates, presumably by influencing cell migration.

Congenital toxoplasmosis: evaluation of serological methods for the detection of anti-Toxoplasma gondii IgM and IgA antibodies

A study was carried out to evaluate the presence of serological markers for the immunodiagnosis of the vertical transmission of toxoplasmosis. We tested the sensitivity, specificity and predictive values (positive and negative) of different serological methods for the early diagnosis of congenital toxoplasmosis. In a prospective longitudinal study, 50 infants with suspected congenital toxoplasmosis were followed up in the ambulatory care centre of Congenital Infections at University Hospital in Goiânia, Goiás, Brazil, from 1 January 2004-30 September 2005. Microparticle Enzyme Immunoassay (MEIA), Enzyme-Linked Fluorescent Assay (ELFA) and Immune-Fluorescent Antibody Technique (IFAT) were used to detect specific IgM anti-Toxoplasma gondii antibodies and a capture ELISA was used to detect specific IgA antibodies. The results showed that 28/50 infants were infected. During the neonatal period, IgM was detected in 39.3% (11/28) of those infected infants and IgA was detected in 21.4% (6/28). The sensitivity, specificity and predictive values (positive and negative) of each assay were, respectively: MEIA and ELFA: 60.9%, 100%, 100%, 55.0%; IFAT: 59.6%, 91.7%, 93.3%, 53.7%; IgA capture ELISA: 57.1%, 100%, 100%, 51.2%. The presence of specific IgM and IgA antibodies during the neonatal period was not frequent, although it was correlated with the most severe cases of congenital transmission. The results indicate that the absence of congenital disease markers (IgM and IgA) in newborns, even after confirming the absence with several techniques, does not constitute an exclusion criterion for toxoplasmosis.

Serosurvey Study of Toscana Virus in Domestic Animals, Granada, Spain

Toscana virus (TOSV) is transmitted by infected sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis in humans. It remains unclear if there are animal reservoirs able to maintain the virus through the cold months of the year, when the vector is not circulating. From May to October of 2006 and 2007, we conducted a serosurvey study on domestic animals from Granada province (southern Spain). TOSV was investigated in 1186 serum samples from horses, goats, pigs, cats, dogs, sheep, and cows by serology (indirect fluorescence assay), viral culture, and RT-polymerase chain reaction. Specific anti-TOSV antibodies were detected in 429 (36.2%) serum samples. The highest seropositivity rates were observed in cats (59.6%) and dogs (48.3%). These results suggest that an important percentage of the domestic animals have been infected by TOSV. Significantly different seroprevalence rates were detected in goats among distinct geographical areas. All viral cultures were negative. TOSV was detected by RT-polymerase chain reaction in only one serum sample from a goat. Thus, the studied animals do not seem to act as reservoirs for TOSV; otherwise, they could be amplifying hosts for the virus.

The neuroretina is a novel mineralocorticoid target: aldosterone up-regulates ion and water channels in Müller glial cells.

Glucocorticoids reduce diabetic macular edema, but the mechanisms underlying glucocorticoid effects are imperfectly elucidated. Glucocorticoids may bind to glucocorticoid (GR) and mineralocorticoid (MR) receptors. We hypothesize that MR activation may influence retinal hydration. The effect of the MR agonist aldosterone (24 h) on ion/water channel expression (real-time PCR, Western blot, immunofluorescence) was investigated on cultured retinal Müller glial cells (RMGs, which contribute to fluid homeostasis in the retina), in Lewis rat retinal explants, and in retinas from aldosterone-injected eyes. We evidenced cell-specific expression of MR, GR, and 11-beta-hydroxysteroid dehydrogenase type II. Aldosterone significantly enhances expression of sodium and potassium channels ENaC-alpha (6.5-fold) and Kir4.1 (1.9-fold) through MR and GR occupancy, whereas aquaporin 4 (AQP4, 2.9-fold) up-regulation is MR-selective. Aldosterone intravitreous injection induces retinal swelling (24% increase compared to sham-injected eyes) and activation of RMGs. It promotes additional localization of Kir4.1 and AQP4 toward apical microvilli of RMGs. Our results highlight the mineralocorticoid-sensitivity of the neuroretina and show that aldosterone controls hydration of the healthy retina through regulation of ion/water channels expression in RMGs. These results provide a rationale for future investigations of abnormal MR signaling in the pathological retina.

Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

The microenvironment hosting a tumor actively participates in regulating tumor cell proliferation, migration, and invasion. Among the extracellular matrix proteins enriched in the stroma of carcinomas are the tenascin family members tenascin-C and tenascin-W. Whereas tenascin-C overexpression in gliomas is known to correlate with poor prognosis, the status of tenascin-W in brain tumors has not been investigated so far. In the present study, we analyzed protein levels of tenascin-W in 38 human gliomas and found expression of tenascin-W in 80% of the tumor samples, whereas no tenascin-W could be detected in control, nontumoral brain tissues. Double immunohistochemical staining of tenascin-W and von Willebrand factor revealed that tenascin-W is localized around blood vessels, exclusively in tumor samples. In vitro, the presence of tenascin-W increased the proportion of elongated human umbilical vein endothelial cells (HUVECs) and augmented the mean speed of cell migration. Furthermore, tenascin-W triggered sprouting of HUVEC spheroids to a similar extent as the proangiogenic factor tenascin-C. In conclusion, our study identifies tenascin-W as a candidate biomarker for brain tumor angiogenesis that could be used as a molecular target for therapy irrespective of the glioma subtype.-Martina, E., Degen, M., Rüegg, C., Merlo, A., Lino, M. M., Chiquet-Ehrismann, R., Brellier, F. Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

In vitro heat exposure induces a redistribution of nuclear matrix proteins in human K562 erythroleukemia cells.

By using both conventional and confocal laser scanning microscopy with three monoclonal antibodies recognizing nuclear matrix proteins we have investigated by means of indirect fluorescence whether an incubation of isolated nuclei at the physiological temperature of 37 degrees C induces a redistribution of nuclear components in human K562 erythroleukemia cells. Upon incubation of isolated nuclei for 45 min at 37 degrees C, we have found that two of the antibodies, directed against proteins of the inner matrix network (M(r) 125 and 160 kDa), gave a fluorescent pattern different from that observed in permeabilized cells. By contrast, the fluorescent pattern did not change if nuclei were kept at 0 degrees C. The difference was more marked in case of the 160-kDa polypeptide. The fluorescent pattern detected by the third antibody, which recognizes the 180-kDa nucleolar isoform of DNA topoisomerase II, was unaffected by heat exposure of isolated nuclei. When isolated nuclear matrices prepared from heat-stabilized nuclei were stained by means of the same three antibodies, it was possible to see that the distribution of the 160-kDa matrix protein no longer corresponded to that observable in permeabilized cells, whereas the fluorescent pattern given by the antibody to the 125-kDa polypeptide resembled that detectable in permeabilized cells. The 180-kDa isoform of topoisomerase II was still present in the matrix nucleolar remnants. We conclude that a 37 degrees C incubation of isolated nuclei induces a redistribution of some nuclear matrix antigens and cannot prevent the rearrangement in the spatial organization of one of these antigens that takes place during matrix isolation in human erythroleukemia cells. The practical relevance of these findings is discussed.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(2+) and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca(2+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.