Zebrafish smoothened functions in ventral neural tube specification and axon tract formation

Sonic hedgehog (Shh) signaling patterns many vertebrate tissues. shh mutations dramatically affect mouse ventral forebrain and floor plate but produce minor defects in zebrafish. Zebrafish have two mammalian Shh orthologs, sonic hedgehog and tiggy-winkle hedgehog, and another gene, echidna hedgehog, that could have overlapping functions. To examine the role of Hedgehog signaling in zebrafish, we have characterized slow muscle omitted (smu) mutants. We show that smu encodes a zebrafish ortholog of Smoothened that transduces Hedgehog signals. Zebrafish smoothened is expressed maternally and zygotically and supports specification of motoneurons, pituitary cells and ventral forebrain. We propose that smoothened is required for induction of lateral floor plate and a subpopulation of hypothalamic cells and for maintenance of medial floor plate and hypothalamic cells.     

Mechanotransduction through the cytoskeleton

We constructed a model cytoskeleton to investigate the proposal that this interconnected filamentous structure can act as a mechano- and signal transducer. The model cytoskeleton is composed of rigid rods representing actin filaments, which are connected with springs representing cross-linker molecules. The entire mesh is placed in viscous cytoplasm. The model eukaryotic cell is submitted to either shock wave-like or periodic mechanical perturbations at its membrane. We calculated the efficiency of this network to transmit energy to the nuclear wall as a function of cross-linker stiffness, cytoplasmic viscosity, and external stimulation frequency. We found that the cytoskeleton behaves as a tunable band filter: for given linker molecules, energy transmission peaks in a narrow range of stimulation frequencies. Most of the normal modes of the network are spread over the same frequency range. Outside this range, signals are practically unable to reach their destination. Changing the cellular ratios of linker molecules with different elastic characteristics can control the allowable frequency range and, with it, the efficiency of mechanotransduction.     

Involvement of Amino-Acid Side Chains of Membrane Proteins in the Binding of Glutathione to Pig Cerebral Cortical Membranes

Glutathione (GSH), a general antioxidant and detoxifying compound, is the most abundant thiol-containing peptide in the central nervous system. It has been earlier shown to regulate the functions of glutamate receptors and to possess specific binding sites in both neurons and glial cells. The possible involvement of disulfide bonds, cysteinyl, arginyl, lysyl, glutamyl, and aspartyl residues in the binding of tritiated GSH to specific sites in pig cerebral cortical synaptic membranes was now studied after covalent modification of membrane proteins. Treatment of synaptic membranes with the thiol-modifying reagents 5,5-dithio-bis(2-nitrobenzoate) (DTNB) and 4,4-dithiodipyridine (DDP) dramatically enhanced the binding of [³H]GSH in a dose-dependent manner. Dithiothreitol (DTT) alone reduced the binding, but pretreatment of the membranes with DTT potentiated the enhancing effect of DTNB. On the other hand, when the modification with DTNB was followed by treatment with DTT, the enhancement by DTNB was completely reversed. N-ethylmaleimide, a thiol alkylating agent, and phenylisothiocyanate, a thiol- and amino-group modifying compound, reduced the binding, and their effects were additive. The guanidino-modifying agent phenylglyoxal reduced the binding but the carboxyl-modifying reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide had no significant effect. The results indicate that cysteinyl side chains and disulfide bonds are essential in the binding of GSH to membrane proteins and that arginyl and lysyl side chains may also be directly involved in this process.     

Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.     

Polyphasic typing of Cryptosporidium baileyi: a suggested model for characterization of cryptosporidia

The present study was undertaken to characterize the oocyst morphology, host specificity, organ location, virulence, and sequences of the small subunit ribosomal RNA, 70-kDa heat shock protein, and oocyst wall protein genes of Cryptosporidium baileyi, and to compare this strain with other Cryptosporidium species. This study also aims to serve as a model for polyphasic (phenetic and genetic) characterization of Cryptosporidium species and strains. On the basis of these results, further genetic and phenetic characterization of an avian isolate is needed if the difference between the length or width, or both, of oocysts of an isolate and of C. baileyi is > or = 10% or if the difference between the oocyst shape index of the isolate and of C. baileyi is > or = 3% (or both). The isolate is infectious for mammals or lower vertebrates, or the host range is narrow, i.e., infectious only for some bird species; after oral or intratracheal inoculation, the parasites are not located in the cloaca and in the bursa of Fabricius or the respiratory tract; clinical disease or weight gain reduction can be observed after oral inoculation; the genetic distance for the examined gene between C. baileyi and the isolate is similar in magnitude to that observed between most closely related Cryptosporidium species.     

Identification and incidence of fungal strains in chronic rhinosinusitis patients

The fungal revolution taking place in otorhinology inspired us to study the frequency of occurrence of fungi in the nasal mucus of chronic rhinosinusitis (CRS) patients (with or without polyposis) in order to evaluate the incidence of eosinophilic fungal sinusitis in CRS patients. Ninety-six samples were examined from patients with CRS. In 74 cases mucus was collected non-invasively, and in 22 cases during operation. The Gram-stained direct smears of all samples were also evaluated. Bacteria and fungi colonizing in the mucus were detected by culturing method. The control group consisted of 50 healthy volunteers. Typical aerobic pathogenic bacteria could be isolated from 34 patients. Fifty-seven aerobic bacteria were isolated, i.e. 1.6 bacteria/positive patient with a maximum of 3 different bacteria/sample. The most frequently isolated bacteria were Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, and Haemophilus influenzae. Yeasts and moulds could be detected from 79 patients (83%): Candida albicans, Candida spp., Aspergillus spp., Cladosporium spp, and Penicillium spp. were isolated most frequently. Altogether 237 yeasts and moulds were isolated, i.e. 3.0 different fungi/positive patient, with a maximum of 5 different fungi/sample. In the control group aerobic pathogens were not isolated, only apathogenic species. Fungi were isolated from 22 healthy patients (44%). These data indicate that fungi are frequently involved in the aetiology of CRS. IgE-medicated hypersensitivity to fungal allergens could not be proven in our patients.     

Controllability of selection-mutation systems

The well-known Fisher type selection-mutation model is studied from the point of view of mathematical systems theory. Mutation rates are considered as control functions. Based on a general sufficient condition for local controllability of non-linear systems with invariant manifold, a method is proposed to guarantee the controllability of the considered population into a polymorphic equilibrium.