Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85 Dex2 ) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85 Dex2 animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85 Dex2 mice.

The sexual mating of the pathogenic yeast Cryptococcus neoformans is important for pathogenesis studies because the fungal virulence is linked to the ␣ mating type (MAT␣). We characterized C. neoformans mating pheromones (MF␣ 1 and MFa1) from 122 strains to understand intervariety hybridization or mating and intervariety virulence. MF␣ 1 in three C. neoformans varieties showed (a) specific nucleotide polymorphisms, (b) different copy numbers and chromosomal localizations, and (c) unique deduced amino acids in two geographic populations of C. neoformans var. gattii. MF␣ 1 of different varieties cross-hybridized in Southern hybridizations. Their phylogenetic analyses showed purifying selection (neutral evolution). These observations suggested that MAT␣ strains from any of the three C. neoformans varieties could mate or hybridize in nature with MATa strains of C. neoformans var. neoformans. A few serotype A/D diploid strains provided evidence for mating or hybridization, while a majority of A/D strains tested positive for haploid MF␣ 1 identical to that of C. neoformans var. grubii. MF␣ 1 sequence and copy numbers in diploids were identical to those of C. neoformans var. grubii, while their MFa1 sequences were identical to those of C. neoformans var. neoformans; thus, these strains were hybrids. The mice survival curves and histological lesions revealed A/D diploids to be highly pathogenic, with pathogenicity levels similar to that of the C. neoformans var. grubii type strain and unlike the low pathogenicity levels of C. neoformans var. neoformans strains. In contrast to MF␣ 1 in three varieties, MFa1 amplicons and hybridization signals could be obtained only from two C. neoformans var. neoformans reference strains and eight A/D diploids. This suggested that a yet undiscovered MFa pheromone(s) in C. neoformans var. gattii and C. neoformans var. grubii is unrelated to, highly divergent from, or rarer than that in C. neoformans var. neoformans. These observations could form the basis for future studies on the role of intervariety mating in C. neoformans biology and virulence.Cryptococcus neoformans causes cryptococcal meningoencephalitis in healthy and immunocompromised individuals. The fungus has two mating types (mating type ␣ [MAT␣] and MATa), three varieties (C. neoformans var. neoformans, C. neoformans var. gattii, and C. neoformans var. grubii), and five serotypes (A, B, C, D, and A/D). C. neoformans var. grubii is most common in cryptococcosis among immunocompromised patients; the isolates are usually serotype A, MAT␣. This variety apparently reproduces asexually in nature, as indicated by the rarity of fertile MATa isolates (9,19,29). Sexual mating is seen in the laboratory both in C. neoformans var. neoformans (serotype D) and C. neoformans var. gattii (serotype B or C). The latter two varieties also show laboratory cross-hybridization; thus, their teleomorphs are classified as Filobasidiella neoformans var. neoformans and F. neoformans var. bacillispora (30). Currently, C. neoformans sexual mating is being ...

The ryanodine-sensitive Ca2+ release channel (RyaCRC) of the sarcoplasmic reticulum plays a key role in the intracellular Ca2+ handling in cardiomyocytes. Altered expression of the RyaCRC has been supposed to contribute to abnormal cellular Ca2+ handling and to myocardial dysfunction in dilated and ischemic cardiomyopathy. In the present study the 3H-ryanodine binding site in human myocardial homogenates was characterized and the density of the RyaCRC (which corresponds to the cardiac ryanodine receptor) was determined in nonfailing and in failing human myocardium. Homogenates were prepared from nonfailing left ventricular myocardium from the hearts of 5 organ donors (NF) and from failing myocardium from 14 explanted hearts of transplant recipients with end-stage heart failure resulting from dilated (DCM, n = 5) or ischemic (ICM, n = 9) cardiomyopathy. Radioligand saturation binding experiments revealed a specific, high-affinity 3H-ryanodine binding site (Kd-values: NF: 0.65 +/- 0.11 nmol/l, DCM: 0.66 +/- 0.09 nmol/l, ICM: 0.88 +/- 0.18 nmol/l; n.s.) in all preparations. Specific 3H-ryanodine binding depended on the free Ca2+ concentration in the assay. It was maximal at 3-100 micro mol/l Ca2+. The binding was inhibited by the RyaCRC antagonists ruthenium red (Ki-value: 0.32 [0.18-0.56] micromol/l, n = 5) and Mg2+ (Ki-value: 2.95 [1.23-7.11] mmol/l, n = 5). The RyaCRC density was 103.5 +/- 11.9 fmol/mg protein in nonfailing myocardium. There was no significant change in the RyaCRC density in dilated or ischemic cardiomyopathy (112.4 +/- 17.1 and 122.7 +/- 13.9 fmol/mg protein) compared to nonfailing control myocardium. In summary, 3H-ryanodine binds specifically and with high-affinity to the RyaCRC in human myocardium. There is no change in the RyaCRC density in failing myocardium of patients with DCM or ICM in comparison to non-failing controls.

The excitatory neurotransmitter glutamate (100 μM) induces intracellular calcium transients in cultured hippocampal astrocytes that can be imaged using the calcium indicator (Fluo3AM) and time-lapse microscopy. In response to glutamate (Fig. 1A), cultured astrocytes exhibit distinct patterns of intracellular Ca2+ oscillations and long-distance intercellular waves. Two distinct types of intercellular Ca2+ waves are attributed to excitation by different agonists of the glutamate receptor subtypes. A long-distance regenerative intercellular wave is induced by the ionotropic glutamate receptor, kainate (Fig. 1B). This is a true wave lasting for 50-125 sec with a constant velocity of 10-20 μm/sec. This wave requires extracellular Ca2+ and Na+ and is driven by the Na+/Ca2+ exchanger. A fast Ca2+ wave which travels at speeds from 10 to 200 μm/sec is dependent upon the metabotropic glutamate receptor, is inducible by t-ACPD and is dependent upon cytoplasmic release of Ca2+ regulated by IP3 (Fig. 1C). This wave is not dependent upon extracellular Ca2+ and is stopped by MCPG, a specific inhibitor of IP3-mediated intracellular Ca2+ release.