In vivo measurements by differential pulse voltammetry of extra-cellular 5-hydroxyindoleacetic acid in the rat brain

The use of differential pulse voltammetry, performed with electrochemically treated carbon fiber electrodes, enables us to detect in vitro or in vivo, in the striatum of anaesthetized rats, an oxidation peak (3) at a potential of + 300 mV. Electrolytic or 5-7-dihydroxytryptamine lesions of the medial forebrain bundle are followed by a decrease of 59 and 62% respectively of this peak. Biochemical measurements are significantly correlated with the measured peak (3) and decreases. Thus, peak (3) increases obtained after injection of L-tryptophan and/or Reserpine, as well as the decreases observed after injection of Clorgyline or 3-hydroxybenzylhydrazine, confirm that peak (3) is dependent upon 5-hydroxyindoleacetic acid concentration. The detection of a peak (3) in the cerebrospinal fluid and its increase after injection of Probenecid reinforce this conclusion.     

Functional recuperation of the serotoninergic innervation in the rat locus coeruleus

1. 5,6-dihydroxytryptamine (5,6-DHT) or a lesion of the raphe centralis superior (RCS) cause significant decreases in the serotonin (5-HT) content and significant increases in the tyrosine hydroxylase activity in the locus coeruleus (LC) of the rat. This suggests that noradrenaline (NA) synthesis is controlled by serotonin-containing neurons in the raphe system via their terminals in the LC. 2. Radioautography after intraventricular infusion of tritiated serotonin (3H-5-HT) and biochemical determinations of endogenous 5-HT content showed an almost complete disappearance of serotoninergic axonal varicosities and content in the LC region 10-15 days after intraventricular administration of 75 micrograms of 5,6-DHT. Two to 4 months after neurotoxin administration, 5-HT fibers had regrown in the LC but, contrary to the normal innervation pattern, the majority of them invaded the medial most portion of the nucleus and the adjacent subependymal region. The LC region regained almost all of its endogenous 5-HT content in the same time period. 3. Functional recuperation of these 5-HT fibers was demonstrated by the fact that the RCS had, after regeneration, the same functional control on NA synthesis as in the normal animal.     

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities—I

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the αγ isozyme. The most important increase in 14-3-2 antigen and γγ enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is—during embryonic development—the decrease in the specific activity of the cerebral enolase isozyme αα. This could be explained by the replacement—in neuroblasts—of αα enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (γγ) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase.Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).     

Comparison of Switching and Biofilm Formation between MTL-Homozygous Strains of Candida albicans and Candida dubliniensis

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO₂ induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO₂, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved.     

Molecular Characterization of Sewage-Borne Pathogens and Detection of Sewage Markers in an Urban Stream in Caracas,Venezuela

Molecular characterization of two sewage-borne pathogens identified hepatitis A virus (HAV) subgenotype IA and Giardia duodenalis assemblages A and B as predominant genotypes circulating in an urban area of Venezuela. This study reveals epidemiological features of human pathogens of worldwide distribution and the efficacy of molecular methods for accurate assessment of sewage pollution.Multiple microbial pathogens may frequently be found in surface waters that receive uncontrolled municipal sewage discharges. The range and diversity of sewage-borne pathogens in surface waters are geographically specific and strongly dependent on the burden of infectious diseases in the population, the seasonal patterns of infectious diseases in the community, and the availability of sewage treatment processing (5, 7). The metropolitan city of Caracas, the capital and largest city of Venezuela, is located in northern South America near the Caribbean coast. Water pollution is a big issue in Caracas, like in many other cities in South America, where most of the human sewage (∼97%) from overpopulated urbanized areas is discharged without any treatment into nearby rivers and coastal environments. Despite these facts, the seriousness of sewage-related health issues is not at the forefront of public concern in this country.Giardia is the protozoan parasite most frequently detected in human fecal samples submitted to diagnostic laboratories from major cities in Venezuela. The frequency of giardiasis reported in the population varies between 21% and 45% but may increase up to 75% among school age children (8). Notwithstanding, the epidemiology of giardiasis in Venezuela remains unknown, and no previous studies have documented the distribution of species and genotype assemblages associated with human infections.Hepatitis A virus (HAV), the etiological agent of hepatitis A in humans, has distinguishable epidemiological patterns of distribution and endemicity closely related to socioeconomic development (9, 12). Water- and food-borne outbreaks of HAV have been well documented worldwide (6, 19). Seroepidemiological studies conducted in selected populations in Venezuela have demonstrated high endemicity of hepatitis A infection among low socioeconomic population strata, with seroprevalences between 48% and 98% (13). Nevertheless, studies on HAV genotype circulation in major urban areas of the country are scarce, as is research on predominant exposure routes and potential transmission patterns through the environment.The analysis of nucleic acid sequences of sewage-borne pathogens may provide relevant information on predominant species and genotypes of human-pathogenic viruses and parasites circulating in specific geographical areas (11, 12, 15). The molecular approach may be of relevance for countries lacking reliable disease surveillance programs and proper understanding of the potential transmission of specific human pathogens through the environment. In this research, Giardia cysts and HAV recovered from an urban stream were characterized by multiple molecular methods along with nucleotide sequence analysis to identify predominant genotypes circulating in a major urban area of Venezuela''s capital. The strength and efficacy of multiple molecular methods for accurate assessment of human sewage pollution and risks of exposure to sewage-borne pathogens were also investigated.Dry season sampling (October through March) was conducted in a heavily polluted urban stream (>10⁶ fecal coliforms/100 ml) that flows in a southeast direction through the metropolitan city of Caracas (16). Water sample volumes of 100 ml were collected in three sterile centrifuge tubes two to four times per month. Giardia cysts were concentrated by centrifugation (100 ml at 1,500 × g for 15 min) followed by DNA extraction by the freeze-thaw method in the presence of Chelex-100 (3) for sucrose-purified cysts. Human-pathogenic assemblage occurrence was determined by nested PCR amplification and sequence analysis of the triosephosphate isomerase (tpi) gene (18). Multiple sequence alignments were performed with ClustalW (21), and phylogenetic analyses were conducted using MEGA4 software (20). The genetic diversity of Giardia isolates was inferred by the neighbor-joining method (17) using a bootstrap test of 1,000 replicates. Giardia cysts counts were obtained by fluorescence microscopy using BTF EasyStain monoclonal antibody stain (BTF Precise Microbiology, Inc.) and 4′6-diamidino-2-phenylindole (DAPI). The recovery efficiency of cysts was determined in five experiments using ColorSeed C&G spike suspensions as internal quality controls (14).HAV particles were concentrated from 35 ml by ultracentrifugation and elution with 0.25 N glycine buffer following procedures previously described (16). Viral RNA was extracted from sample concentrates with Trizol (Invitrogen, Inc., Carlsbad, CA) following the manufacturer''s instructions. General detection of HAV was based on amplification of the 5′ nontranslated region (5′ NTR), while analysis of genetic diversity involved sequencing and phylogenetic analysis of the VP1 amino terminus and the full VP1 gene (2, 10). Sequence alignment was conducted with the DNAman software 5.2.2 (Lynnon BioSoft, Quebec, Canada) followed by phylogenetic analysis.Molecular detection of sewage pollution was accomplished by PCR amplification of Bacteroidales human-specific 16S rRNA genes, Bacteroides thetaiotamicron 16S rRNA genes, and the nifH gene of Methanobrevibacter smithii using primers and PCR conditions originally described by Field et al. (4), Carson et al. (1), and Ufnar et al. (22), respectively.Giardia duodenalis tpi nucleotide sequences amplified directly from urban stream waters were included after phylogenetic analysis into two well-defined clusters of assemblages A and B. These results were supported by high bootstrap values, as indicated in Fig. ​Fig.1.1. The level of cysts recovered from these samples ranged from 10,400 to 62,000 cysts/liter; however, mean percent recoveries varied from 20% to 50%, which suggests that the urban stream may harbor and receive much higher loads of cysts.Open in a separate windowFIG. 1.Phylogenetic tree of G. duodenalis assemblages A (VPW2, VPW3, and VPW7) and B (VPW1, VPW4, VPW5, and VPW6) from urban stream samples forming two clusters in a neighbor-joining analysis of tpi nucleotide sequences. Only bootstrap values >80% are shown in the tree.Three genomic regions used for detection and characterization of HAV revealed the predominance of HAV strains belonging to subgenotype IA, the most frequent genotype associated with human disease worldwide (9). A neighbor-joining tree constructed from the alignment of nucleotide sequences from urban stream samples and sequences of HAV strains from case patients (unpublished data) was used to investigate the relationship between genotypes present in environmental and clinical samples. The comparative analysis indicated a high degree of identity (98 to 99%) between nucleotide sequences from the urban stream and the strains from sporadic HAV cases. The phylogenetic analysis grouped all of these sequences into two unique clades within subgenotype IA, strongly supported by significant bootstrap values (Fig. 2A, B, and C).Open in a separate windowFIG. 2.Phylogenetic analysis of the HAV 5′ NTR (A; 284 nucleotides [nt]), VP1 amino terminus (B; 172 nt), and complete VP1 (C; 820 nt) regions. Nucleotide sequences of HAV reference strains are designated by their GenBank accession number, including the name of the country of origin, except for Venezuelan isolates, which are shown in bold. S, isolates derived from human sporadic cases; W, urban stream isolates. Phylogenetic analysis was performed by neighbor joining, and phylogenetic distances were calculated by the Kimura two-parameter test. Bootstrap values ≥90% are shown in the trees. Letters in bold indicate the subtype.Three reliable published assays for detection of human-specific markers of fecal pollution identified and confirmed the predominant point source of water pollution. Sequence analysis of three randomly selected PCR products from each marker revealed ≥99% sequence identity with published sequences (GenBank) derived from different geographical areas, thus indicating the validity and specificity of the molecular markers as reliable indicators of human sewage pollution in Venezuela.The results of this research demonstrate that the molecular assays applied for detection and characterization of sewage-borne pathogens in surface waters may have practical applications for epidemiological investigations on distribution of predominant human-specific genotypes circulating in urban populations. Previous studies identified the most predominant waterborne gastroenteritis viruses circulating in Metropolitan Caracas (16).The molecular-based monitoring approach for rapid and precise identification of sewage-borne pathogens and sewage markers in surface waters has important implications for sewage-related health issues that require special attention in Venezuela and South America. Deficient sewerage coverage and lack of municipal wastewater treatment, commonly associated with informal settlements around densely populated urban areas, are responsible for many of the environmental degradation and public health problems that occur in these countries. The precise identification of human pathogens in the environment offers an appropriate and alternative approach for initial assessment of risks of exposure to waterborne pathogens. Current bacterial indicators of fecal pollution (fecal coliforms, Escherichia coli, and enterococci) do not allow identification of the relative sources of impacts (i.e., sewage, urban runoff, and agricultural waste) on surface waters. Thus, the molecular detection of sewage-borne pathogens and sewage markers in surface waters may be more effective than the bacterial indicator approach for forecasting pathogen distribution and for managing and reducing risks associated with inappropriate sewage disposal into natural waters in Venezuela and South America.     

Increased secretion of leukemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signaling and airway contractility

Airway smooth muscle cells (ASMC) play a major role in airway inflammation, hyperresponsiveness, and obstruction in asthma. However, very little is known regarding the relation between inflammatory mediators and cytokines and immature ASMC. The aim of this study was to evaluate 1) the secretion of leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine) by ASMC; 2) intracellular calcium concentration ([Ca(2+)](i)) signaling; and 3) the effect of LIF on mast cell chemotaxis and rat airway contractility. Immature and adult human ASMC were cultured. ELISA and real-time PCR were performed to assess LIF protein secretion and mRNA production, [methyl-(3)H]thymidine incorporation to quantify ASMC DNA synthesis, a Boyden chamber to evaluate the effect of LIF on mast cell chemotaxis, microspectroflurimetry using indo-1 (at baseline and after stimulation bradykinin, U-46619, histamine, and acetylcholine, in the presence or absence of LIF or TNF-alpha) for [Ca(2+)](i) signaling, and isolated rat pup tracheae to determine the effect of LIF on airway contractility to ACh. TNF-alpha-stimulated immature ASMC produce more LIF mRNA and protein than adult ASMC, although this cytokine induces a moderate increase in DNA synthesis (+20%) in adult ASMC only. Human recombinant LIF exerts no chemotactic effect on human mast cells. In immature ASMC, ACh-induced [Ca(2+)](i) response was enhanced twofold after incubation with LIF, whereas TNF-alpha increased the [Ca(2+)](i) to U-46619 threefold. In TNF-alpha-exposed adult ASMC, [Ca(2+)](i) responses to ACh were of greater magnitude (sixfold increase) than in immature ASMC. Human recombinant LIF increased contractility to ACh by 50% in immature, isolated rat tracheae. Stimulated immature human ASMC greatly secrete LIF, thus potentially contributing to neuroimmune airway inflammation and subsequent remodeling. Increased LIF secretion enhances airway reactivity and [Ca(2+)](i) signaling.     

GMDD: a database of GMO detection methods

Background  

Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed.