Mapping of gene loci for nephronophthisis type 4 and Senior-Løken syndrome, to chromosome 1p36

For nephronophthisis (NPHP), the primary genetic cause of chronic renal failure in young adults, three loci have been mapped. To identify a new locus for NPHP, we here report on total-genome linkage analysis in seven families with NPHP, in whom we had excluded linkage to all three known NPHP loci. LOD scores >1 were obtained at nine loci, which were then fine mapped at 1-cM intervals. Extensive total-genome haplotype analysis revealed homozygosity in one family, in the region of the PCLN1 gene. Subsequent mutational analysis in this gene revealed PCLN1 mutations, thereby allowing exclusion of this family as a phenocopy. Multipoint linkage analysis for the remaining six families with NPHP together yielded a maximum LOD score (Z_max) of 8.9 (at D1S253). We thus identified a new locus, NPHP4, for nephronophthisis. Markers D1S2660 and D1S2642 are flanking NPHP4 at a 2.9-cM critical interval. In one family with NPHP4, extensive genealogical studies were conducted, revealing consanguinity during the 17th century. On the basis of haplotype sharing by descent, we obtained a multipoint Z_max of 5.8 for D1S253 in this kindred alone. In addition, we were able to localize to the NPHP4 locus a new locus for Senior-Løken syndrome, an NPHP variant associated with retinitis pigmentosa.     

Metal binding of metallothionein-3 versus metallothionein-2: lower affinity and higher plasticity

Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc and copper and in cytoprotection against toxic metals and reactive oxygen species. MT-3 plays a specific role in the brain and is down-regulated in Alzheimer's disease. To evaluate differences in metal binding, we conducted direct metal competition experiments with MT-3 and MT-2 using electrospray ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+ and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity and plasticity. Titration with Cd2+ ions demonstrates that metal-binding affinities of individual clusters of MT-2 and MT-3 are decreasing in the following order: four-metal cluster of MT-2>three-metal cluster of MT-2 approximately four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3 and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted cellular conditions, we studied the secondary structures of apo-MT-3 and apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have almost equal helical content (approximately 10%) in aqueous buffer, but that MT-3 had slightly higher tendency to form alpha-helical secondary structure in TFE-water mixtures. Secondary structure predictions also indicated some differences between MT-3 and MT-2, by predicting random coil for common MTs, but 22% alpha-helical structure for MT-3. Combined, all results highlight further differences between MT-3 and common MTs, which may be related with their functional specificities.     

Cell-penetrating peptides: a comparative membrane toxicity study

Cell-penetrating peptides (CPPs) constitute a new class of delivery vectors with high pharmaceutical potential. However, the abilities of these peptides to translocate through cell membranes can be accompanied by toxic effects resulting from membrane perturbation at higher peptide concentrations. Therefore, we investigated membrane toxicity of five peptides with well-documented cell-penetrating properties, pAntp(43-58), pTAT(48-60), pVEC(615-632), model amphipathic peptide (MAP), and transportan 10, on two human cancer cell lines, K562 (erythroleukemia) and MDA-MB-231 (breast cancer), as well as on immortalized aortic endothelial cells. We studied the effects of these five peptides on the leakage of lactate dehydrogenase and on the fluorescence of plasma membrane potentiometric dye bis-oxonol. In all cell lines, pAntp(43-58), pTAT(48-60), and pVEC(615-632) induced either no leakage or low leakage of lactate dehydrogenase, accompanied by modest changes in bis-oxonol fluorescence. MAP and transportan 10 caused significant leakage; in K562 and MDA-MB-231 cells, 40% of total lactate dehydrogenase leaked out during 10 min exposure to 10 microM of transportan 10 and MAP, accompanied by a significant increase in bis-oxonol fluorescence. However, none of the CPPs tested had a hemolytic effect on bovine erythrocytes comparable to mastoparan 7. The toxicity profiles presented in the current study are of importance when selecting CPPs for different applications.     

Characterisation of a new chimeric ligand for galanin receptors: galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide

In this work, we studied a novel chimeric peptide, M242, galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide, and examined its properties in comparison with its parent peptide, M32, galanin(1-13)-neuropeptide Y(25-36)amide, a previously known high-affinity ligand for galanin receptors, and galanin itself. Binding assays performed in Bowes cells known to express human galanin receptor type 1 (hGalR1) and in Chinese hamster ovary cells overexpressing human galanin receptor type 2 (hGalR2) revealed that all three ligands had comparable affinities: at hGalR1<1 nM and at hGalR2<10 nM. However, in rat hippocampal membranes M242 had a 24-fold lower affinity than galanin (9.4 vs. 0.4 nM) and 134-fold lower affinity than M32 (9.4 vs. 0.07 nM). In the same tissue, we also examined the effects of these peptides on adenylate cyclase activity. M32 showed a weak antagonistic behaviour but M242 acted as a potent biphasic regulator of adenylate cyclase. In conclusion, we present and characterise a new peptide M242, which could be a useful tool in studies of galaninergic signalling.     

Splitting schizophrenia: periodic catatonia-susceptibility locus on chromosome 15q15

The nature of subtypes in schizophrenia and the meaning of heterogeneity in schizophrenia have been considered a principal controversy in psychiatric research. We addressed these issues in periodic catatonia, a clinical entity derived from Leonhard's classification of schizophrenias, in a genomewide linkage scan. Periodic catatonia is characterized by qualitative psychomotor disturbances during acute psychotic outbursts and by long-term outcome. On the basis of our previous findings of a lifetime morbidity risk of 26.9% of periodic catatonia in first-degree relatives, we conducted a genome scan in 12 multiplex pedigrees with 135 individuals, using 356 markers with an average spacing of 11 cM. In nonparametric multipoint linkage analyses (by GENEHUNTER-PLUS), significant evidence for linkage was obtained on chromosome 15q15 (P = 2.6 x 10(-5); nonparametric LOD score [LOD*] 3.57). A further locus on chromosome 22q13 with suggestive evidence for linkage (P = 1.8 x 10(-3); LOD* 1.85) was detected, which indicated genetic heterogeneity. Parametric linkage analysis under an autosomal dominant model (affecteds-only analysis) provided independent confirmation of nonparametric linkage results, with maximum LOD scores 2.75 (recombination fraction [theta].04; two-point analysis) and 2.89 (theta =.029; four-point analysis), at the chromosome 15q candidate region. Splitting the complex group of schizophrenias on the basis of clinical observation and genetic analysis, we identified periodic catatonia as a valid nosological entity. Our findings provide evidence that periodic catatonia is associated with a major disease locus, which maps to chromosome 15q15.     

Investigation of the electrostatic charge of basidiospores of the Phellinus igniarius group

The polarity and magnitude of primary electric charges carried by basidiospores in the airborne state were investigated in living fungal fruiting bodies under natural forest conditions using a portable experimental device designed by the author. The operating principle was the falling of spores in the homogeneous horizontal electric field. The vertical and horizontal components of the trajectories of the spores were determined according to their deposition sites on electrodes (vertical metal plates). Altogether 33 samples of spores were examined for polarity, 10 of these samples (with 10⁴–10⁶ spores per sample) also were used to calculate the mean spore charge-to-mass quotient and the mean spore charge. The detection limits of spore charge-to-mass quotient varied in the range from (4.9±2.3)×10^?5 to (1.36±0.33)×10^?4 C kg^?1. Basidiospores (subglobose, smooth, diameter of 4–6 μm) of the closely related (sibling) species Phellinus alni, P. nigricans, P. populicola and P. tremulae (Hymenochaetales, Basidiomycota) carried positive electrical charges that have mean values from 48 to 305 elementary charges. The intraspecies variation of the spore charge could depend on the natural variation in spore size.     

A Novel Whole-Cell Mechanism for Long-Term Memory Enhancement

Olfactory-discrimination learning was shown to induce a profound long-lasting enhancement in the strength of excitatory and inhibitory synapses of pyramidal neurons in the piriform cortex. Notably, such enhancement was mostly pronounced in a sub-group of neurons, entailing about a quarter of the cell population. Here we first show that the prominent enhancement in the subset of cells is due to a process in which all excitatory synapses doubled their strength and that this increase was mediated by a single process in which the AMPA channel conductance was doubled. Moreover, using a neuronal-network model, we show how such a multiplicative whole-cell synaptic strengthening in a sub-group of cells that form a memory pattern, sub-serves a profound selective enhancement of this memory. Network modeling further predicts that synaptic inhibition should be modified by complex learning in a manner that much resembles synaptic excitation. Indeed, in a subset of neurons all GABA_A-receptors mediated inhibitory synapses also doubled their strength after learning. Like synaptic excitation, Synaptic inhibition is also enhanced by two-fold increase of the single channel conductance. These findings suggest that crucial learning induces a multiplicative increase in strength of all excitatory and inhibitory synapses in a subset of cells, and that such an increase can serve as a long-term whole-cell mechanism to profoundly enhance an existing Hebbian-type memory. This mechanism does not act as synaptic plasticity mechanism that underlies memory formation but rather enhances the response of already existing memory. This mechanism is cell-specific rather than synapse-specific; it modifies the channel conductance rather than the number of channels and thus has the potential to be readily induced and un-induced by whole-cell transduction mechanisms.     

Crosstalk between L-type calcium channels and ZnT-1, a new player in rate-dependent cardiac electrical remodeling

Crosstalk between two membrane transport systems is an established mechanism underlying regulation. In this study, we investigated the interaction between ZnT-1, a putative plasma membrane zinc transporter, and L-type voltage-dependent calcium channels (LTCC). In the atrium of the myocardium decreased activity of the LTCC is a dominant feature of patients with atrial fibrillation. The trigger for this inhibition has been attributed to the rapid firing rates and consequent calcium overload in the atrial cardiomyocytes. However, the underlying mechanism of LTCC inhibition is still to be elucidated. Here, we showed that the expression of ZnT-1 inhibits the activity of L-type channels during electrical remodeling induced by rapid pacing. (i) Direct manipulations of ZnT-1 expression in cultured cardiomyocytes either by ZnT-1 overexpression or by ZnT-1 silencing with siRNA, decreased or enhanced, respectively, the barium influx through the LTCC. (ii) Co-expression of ZnT-1 with LTCC in Xenopus oocytes decreased whole cell barium current through LTCC. (iii) Rapid pacing of cultured cardiomyocytes (4 h, 100 ms cycle) increased ZnT-1 protein expression and inhibited the voltage-dependent divalent cation influx through the LTCC. Moreover, silencing ZnT-1 with siRNA prevented the rapid pacing induced inhibition of the LTCC (iv) Atrial pacing of anesthetized adult rats (4 h, 50 ms cycle) led to a significant increase in atrial ZnT-1 protein expression in parallel with the typical decrease of the refractory period in the atria. Taken together, these findings demonstrate that crosstalk between ZnT-1 and the L-type calcium channels may underlie atrial response to rapid pacing, suggesting that ZnT-1 is a significant participant in rate-dependent cardiac electrical remodeling.