Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain

An understanding of the logic of odor perception requires a functional analysis of odor-evoked patterns of activity in neural assemblies in the brain. We have developed a sensitive imaging system in the Drosophila brain that couples two-photon microscopy with the specific expression of the calcium-sensitive fluorescent protein, G-CaMP. At natural odor concentration, each odor elicits a distinct and sparse spatial pattern of activity in the antennal lobe that is conserved in different flies. Patterns of glomerular activity are similar upon imaging of sensory and projection neurons, suggesting the faithful transmission of sensory input to higher brain centers. Finally, we demonstrate that the response pattern of a given glomerulus is a function of the specificity of a single odorant receptor. The development of this imaging system affords an opportunity to monitor activity in defined neurons throughout the fly brain with high sensitivity and excellent spatial resolution.     

Axonal ephrin-As and odorant receptors: coordinate determination of the olfactory sensory map

Olfactory sensory neurons expressing a given odorant receptor (OR) project with precision to specific glomeruli in the olfactory bulb, generating a topographic map. In this study, we demonstrate that neurons expressing different ORs express different levels of ephrin-A protein on their axons. Moreover, alterations in the level of ephrin-A alter the glomerular map. Deletion of the ephrin-A5 and ephrin-A3 genes posteriorizes the glomerular locations for neurons expressing either the P2 or SR1 receptor, whereas overexpression of ephrin-A5 in P2 neurons results in an anterior shift in their glomeruli. Thus the ephrin-As are differentially expressed in distinct subpopulations of neurons and are likely to participate, along with the ORs, as one of a complement of guidance receptors governing the targeting of like axons to precise locations in the olfactory bulb.     

Evaluation of Candida species' susceptibility to fluconazole with the disk diffusion method

Infections caused by yeasts belonging to the genus Candida have increased dramatically in the last decades, especially in hospital settings. Concomittantly, antimycotic resistance has emerged, as well as the appearance of non-Candida albicans isolates. To standardize in vitro antifungal susceptibility tests, the agar diffusion test was developed using disks impregnated with the antimycotic compound. Electronic recording of the inhibition zone (BIOMIC), furnishes objective values for the minimal inhibitory concentration (MIC). The fluconazole susceptibility patterns were determined for Candida species isolated from 2.139 patients seen in outpatient clinics or in health-care centers in Colombia, Ecuador and Venezuela. Candida albicans was the species most frequently isolated (62%), followed at a distance by Candida parapsilosis (11%), Candida tropicalis (8.5%), Candida glabata (3.5%) and Candida krusei (2.2%). MIC determinations showed that 88.1% of these isolates were susceptible to fluconazole, 5.1% were susceptible-dose-dependant and 6.8% resistant. An important proportion (92.1%) of the C. albicans isolates proved susceptible while resistance predominated in the remaining species. These results indicate that the BIOMIC method is rapid and simple, constituting a suitable tool for the epidemiologic surveillance of resistance in Candida species.     

Analysis of the transforming growth factor-beta1 (TGF-beta1) codon 25 gene polymorphism by LightCycler-analysis in patients with chronic hepatitis C infection

The polymorphism at position 25 of the gene encoding transforming growth factor-beta1 (TGF-beta1), which changes the amino acid sequence of the signal peptide sequence (arginine to proline), is causing a variation in TGF-beta1 production. The homozygous genotype (Arg25Arg) is associated with higher TGF-beta1 production than the heterozygous (Arg25Pro) genotype. Therefore, the possible involvement of this genetic variation in the TGF-beta1 gene for induction and progression of various diseases is under close investigation. At present, several labor-intensive established assays ranging from amplification refractory mutation system (ARMS)-PCR methodologies, sequence specific oligonucleotide probing (SSOP), restriction fragment length polymorphism (RFLP) analysis, 5' nuclease assays, and specialized fingerprinting protocols are applied to analyze the polymorphism in question. We developed a novel approach for analyzing this polymorphism in a LightCycler system and determined the allele frequency distributions between patients with different degrees of hepatic fibrosis induced by chronic hepatitis C virus infection. In patients with severe hepatic fibrosis (METAVIR-score 3-4), the Pro25 allele was twice as frequent compared to patients with mild fibrosis (METAVIR-score 0-2). However, we found no association of necroinflammatory activity and genotype distribution. This suggests that the stage of hepatic fibrosis, rather than the grade (inflammation), is influenced by the presence of proline at codon 25 in patients with chronic hepatitis C.     

Revisiting the Glires concept--phylogenetic analysis of nuclear sequences

The so-called Glires hypothesis postulates a sister-group relationship between Rodentia (e.g., rat and mouse) and Lagomorpha (e.g., rabbit). Recent molecular phylogenetic analyses have yielded incongruent results, and either supported or refuted the Glires grouping. In order to study this inconsistency we have reconstructed phylogenetic trees based on data sets of 20 orthologous nuclear protein coding genes (6441 aa, sites) and 12 mitochondrial protein coding genes (3559 aa sites). The size of the nuclear data set is considerably larger than any comparable data set hitherto used to study the Glires concept. Analysis of the nuclear data strongly supported the phylogenetic tree (frog, chicken, ((rat, mouse), (rabbit, (human, (cattle, dog))))), while the mt data could not conclusively resolve the position of rabbit relative to that of human. This result was supported by all methods. Thus, the Glires hypothesis was rejected by this study.     

Genetically modified laboratory animals--what welfare problems do they face?

In this article, we respond to public concern expressed about the welfare of genetically modified (GM) nonhuman animals. As a contribution to the debate on this subject, we attempt in this article to determine in what situations the practice of genetic modification in rodents may generate significant welfare problems. After a brief discussion of the principles of animal welfare, we focus on the problem of animal suffering and review some types of gene modifications likely to cause predictable welfare problems. In this article, we also consider suffering that may be involved in the process of generating GM animals. Finally, we discuss the role of GM animals in attempts to reduce, replace, and refine the use of animals in research.     

Biosynthesis of fungal melanins and their importance for human pathogenic fungi

For more than 40 years fungi have been known to produce pigments known as melanins. Predominantly these have been dihydroxyphenylalanine (DOPA)-melanin and dihydroxynaphthalene (DHN)-melanin. The biochemical and genetical analysis of the biosynthesis pathways have led to the identification of the genes and corresponding enzymes of the pathways. Only recently have both these types of melanin been linked to virulence in some human pathogenic and phytopathogenic fungi. The absence of melanin in human pathogenic and phytopathogenic fungi often leads to a decrease in virulence. In phytopathogenic fungi such as Magnaporthe grisea and Colletotrichum lagenarium, besides other possible functions in pathogenicity, DHN-melanin plays an essential role in generating turgor for plant appressoria to penetrate plant leaves. While the function of melanin in human pathogenic fungi such as Cryptococcus neoformans, Wangiella dermatitidis, Sporothrix schenckii, and Aspergillus fumigatus is less well defined, its role in protecting fungal cells has clearly been shown. Specifically, the ability of both DOPA- and DHN-melanins to quench free radicals is thought to be an important factor in virulence. In addition, in several fungi the production of fungal virulence factors, such as melanin, has been linked to a cAMP-dependent signaling pathway. Many of the components involved in the signaling pathway have been identified.     

TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells

Communication between G protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signalling systems involves cell surface proteolysis of EGF-like precursors. The underlying mechanisms of EGFR signal transactivation pathways, however, are largely unknown. We demonstrate that in squamous cell carcinoma cells, stimulation with the GPCR agonists LPA or carbachol specifically results in metalloprotease cleavage and release of amphiregulin (AR). Moreover, AR gene silencing by siRNA or inhibition of AR biological activity by neutralizing antibodies and heparin prevents GPCR-induced EGFR tyrosine phosphorylation, downstream mitogenic signalling events, cell proliferation, migration and activation of the survival mediator Akt/PKB. Therefore, despite some functional redundancy among EGF family ligands, the present study reveals a distinct and essential role for AR in GPCR-triggered cellular responses. Furthermore, we present evidence that blockade of the metalloprotease-disintegrin tumour necrosis factor-alpha-converting enzyme (TACE) by the tissue inhibitor of metalloprotease-3, a dominant-negative TACE mutant or RNA interference suppresses GPCR-stimulated AR release, EGFR activation and downstream events. Thus, TACE can function as an effector of GPCR-mediated signalling and represents a key element of the cellular receptor cross-talk network.     

Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals

Changes in plasma nitrite concentration in the human forearm circulation have recently been shown to reflect acute changes in endothelial nitric oxide synthase (eNOS)-activity. Whether basal plasma nitrite is a general marker of constitutive NOS-activity in vivo is yet unclear. Due to the rapid metabolism of nitrite in blood and the difficulties in its analytical determination literature data on levels of nitrite in mammals are largely inconsistent. We hypothesized that constitutive NOS-activity in the circulatory system is relatively uniform throughout the mammalian kingdom. If true, this should result in comparable systemic plasma nitrite levels in different species. Using three different analytical approaches we determined plasma nitrite concentration to be in a nanomolar range in a variety of species: humans (305 +/- 23 nmol/l), monkeys (367 +/- 62 nmol/l), minipigs (319 +/- 24 nmol/l), dogs (305 +/- 50 nmol/l), rabbits (502 +/- 21 nmol/l), guinea pigs (412 +/- 44 nmol/l), rats (191 +/- 43 nmol/l), and mice (457 +/- 51 nmol/l). Application of different NOS-inhibitors in humans, minipigs, and dogs decreased NOS-activity and thereby increased vascular resistance. This was accompanied by a significant, up to 80%, decrease in plasma nitrite concentration. A comparison of plasma nitrite concentrations between eNOS(-/-) and NOS-inhibited wild-type mice revealed that 70 +/- 5% of plasma nitrite is derived from eNOS. These results provide evidence for a uniform constitutive vascular NOS-activity across mammalian species.