Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors

Beta-arrestins mediate agonist-dependent desensitization and internalization of G protein-coupled receptors. Previously, we have shown that phosphorylation of beta-arrestin1 by ERKs at Ser-412 regulates its association with clathrin and its function in promoting clathrin-mediated internalization of the receptor. In this paper we report that beta-arrestin2 is also phosphorylated, predominantly at residues Thr-383 and Ser-361. Isoproterenol stimulation of the beta(2)-adrenergic receptor promotes dephosphorylation of beta-arrestin2. Mutation of beta-arrestin2 phosphorylation sites to aspartic acid decreases the association of beta-arrestin2 with clathrin, thereby reducing its ability to promote internalization of the beta(2)-adrenergic receptor. Its ability to bind and desensitize the beta(2)-adrenergic receptor is, however, unaltered. These results suggest that, analogous to beta-arrestin1, phosphorylation/dephosphorylation of beta-arrestin2 regulates clathrin-mediated internalization of the beta(2)-adrenergic receptor. In contrast to beta-arrestin1, which is phosphorylated by ERK1 and ERK2, phosphorylation of beta-arrestin2 at Thr-383 is shown to be mediated by casein kinase II. Recently, it has been reported that phosphorylation of visual arrestin at Ser-366 prevents its binding to clathrin. Thus it appears that the function of all arrestin family members in mediating internalization of G protein-coupled receptors is regulated by distinct phosphorylation/dephosphorylation mechanisms.     

Dynamics of green fluorescent protein mutant2 in solution, on spin-coated glasses, and encapsulated in wet silica gels

Single-molecule experiments are performed by investigating spectroscopic properties of molecules either diffusing in and out of the observation volume or fixed in space by different immobilization procedures. To evaluate the effect of immobilization methods on the structural and dynamic properties of proteins, a highly fluorescent mutant of the green fluorescent protein, GFPmut2, was spectroscopically characterized in bulk solutions, dispersed on etched glasses, and encapsulated in wet, nanoporous silica gels. The emission spectrum, the fluorescence lifetimes, the anisotropy, and the rotational correlation time of GFPmut2, encapsulated in silica gels, are very similar to those obtained in solution. This finding indicates that the gel matrix does not alter the protein conformation and dynamics. In contrast, the fluorescence lifetimes of GFPmut2 on glasses are two-to fourfold higher and the fluorescence anisotropy decays yield almost no phase shifts. This indicates that the interaction of the protein with the bare glass surface induces a significant structural perturbation and severely restricts the rotational motion. Single molecules of GFPmut2 on glasses or in silica gels, identified by confocal image analysis, show a significant stability to illumination with bleaching times of the order of 90 and 60 sec, respectively. Overall, these data indicate that silica gels represent an ideal matrix for following biologically relevant events at a single molecule level.     

Temperature influence on embryonic development of Anopheles albitarsis and Anopheles aquasalis

Temperature influence on the embryonic development of Anopheles aquasalis and An. albitarsis was investigated. At 26 degrees C, 75% and 60% of respectively An. aquasalis and An. albitarsis eggs hatched, with one peak of eclosion, between the 2nd and 3rd day after oviposition. At 20 +/- 2 degrees C, around 66-70% of An. aquasalis eggs hatched, with one eclosion peak, on the 5th day. On the other hand, An. albitarsis eclosion at 21+/- 2 degrees C decreased to 10-22%, with two eclosion peaks, on the 4th-5th day and on the 9th-12th day. These data indicate a stronger temperature influence over An.albitarsis than over An. aquasalis embryos.     

Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover

To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.     

Low levels of STRP variability are not universal in American Indians

Data related to 15 short tandem repeat polymorphisms (STRPs) are reported for five Brazilian Indian populations, and a set of them compared with results previously reported for Asian, neo-Brazilian, North American, Iberian, and African populations. The low variability observed for these markers among the Suruí Indians is confirmed, but the other populations show variability levels that are similar to those found elsewhere. Previous suggestions of population bottlenecks in the prehistorical colonization of the New World were not confirmed. On the other hand, STRPs again showed to be good markers for the establishment of population relationships.     

Heterozygous submicroscopic inversions involving olfactory receptor-gene clusters mediate the recurrent t(4;8)(p16;p23) translocation

The t(4;8)(p16;p23) translocation, in either the balanced form or the unbalanced form, has been reported several times. Taking into consideration the fact that this translocation may be undetected in routine cytogenetics, we find that it may be the most frequent translocation after t(11q;22q), which is the most common reciprocal translocation in humans. Case subjects with der(4) have the Wolf-Hirschhorn syndrome, whereas case subjects with der(8) show a milder spectrum of dysmorphic features. Two pairs of the many olfactory receptor (OR)-gene clusters are located close to each other, on both 4p16 and 8p23. Previously, we demonstrated that an inversion polymorphism of the OR region at 8p23 plays a crucial role in the generation of chromosomal imbalances through unusual meiotic exchanges. These findings prompted us to investigate whether OR-related inversion polymorphisms at 4p16 and 8p23 might also be involved in the origin of the t(4;8)(p16;p23) translocation. In seven case subjects (five of whom both represented de novo cases and were of maternal origin), including individuals with unbalanced and balanced translocations, we demonstrated that the breakpoints fell within the 4p and 8p OR-gene clusters. FISH experiments with appropriate bacterial-artificial-chromosome probes detected heterozygous submicroscopic inversions of both 4p and 8p regions in all the five mothers of the de novo case subjects. Heterozygous inversions on 4p16 and 8p23 were detected in 12.5% and 26% of control subjects, respectively, whereas 2.5% of them were scored as doubly heterozygous. These novel data emphasize the importance of segmental duplications and large-scale genomic polymorphisms in the evolution and pathology of the human genome.     

The formation of synapses in the central nervous system

Interneuronal synapses are specialized contact zones formed between the transmitting pole of one neuron, usually an axon, and the receptive pole of another nerve cell, usually a dendritic process or the soma. The formation of these synaptic contacts is the result of cellular events related to neurite elongation, the establishment of polarity, axon guidance, and target recognition. A series of morphological rearrangements takes place once synaptic targets establish their initial contact. These changes include the clustering of synaptic vesicles in the presynaptic element and the formation of a specialized area capable of signal transduction at the postsynaptic target. The present review discusses the role of different synaptic proteins in the cellular events leading to the formation of synapses among neurons in the central nervous system.     

Interactions between Zn and Cu in LEC rats, an animal model of Wilson's disease

The effect of oral Zn treatment was studied in the liver and kidneys of 26 male Long-Evans Cinnamon (LEC) rats (mutant animals, 5 weeks old) in relation to both the interaction between Zn and Cu and the localisation and concentration of metallothionein (MT). Rats receiving 80 mg zinc acetate daily by gavage and control rats receiving no treatment were killed after 1 or 2 weeks. By immunohistochemical and analytical chemical techniques we revealed that treated rats had higher levels of MT in the hepatic and renal cells compared to untreated ones. Tissue Zn concentrations were significantly higher in treated rats compared to untreated whereas Cu concentrations decreased in the liver and kidneys as indicated by analytical chemical analyses. MT levels also decreased with treatment period. A histochemical procedure, obtained using autofluorescence of Cu-metallothioneins, confirms these findings: after 2 weeks, the signal decreased in both the liver and kidney sections. This gives a greater understanding of the mechanism of Cu metabolism in the two tissues considered. These results suggest that Zn acts both to compete for absorption on the luminal side of the intestinal epithelium and to induce the synthesis of MT.     

NMR spectroscopic and theoretical study of the complexation of the inhibitor allosamidin in the binding pocket of the plant chitinase hevamine

Based on NMR spectroscopic information about the allosamidin-hevamine complex, ab initio MO calculations of the ring current effect of the aromatic moieties of Trp255, Tyr183 and Tyr6 of hevamine were carried out to investigate the role of these amino acid residues in binding interactions with allosamidin in solution. In addition, the intermolecular steric compression effect on the 13C chemical shifts of the allosamizoline carbon atoms and the hydrogen bonding to Glu127 was identified. It can be inferred that the binding forces are strongest in the allosamizoline moiety of allosamidin.