AMPA receptor expression in mouse testis and spermatogonial GC-1 cells: A study on its regulation by excitatory amino acids

Excitatory amino acids (EAAs) are found present in the nervous and reproductive systems of animals. Numerous studies have demonstrated a regulatory role for Glutamate (Glu), d -aspartate ( d -Asp) and N-methyl- d -aspartate (NMDA) in the control of spermatogenesis. EAAs are able to stimulate the Glutamate receptors, including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Here in, we assess expression of the main AMPAR subunits, GluA1 and GluA2/3, in the mouse testis and in spermatogonial GC-1 cells. The results showed that both GluA1 and GluA2/3 were localized in mouse testis prevalently in spermatogonia. The subunit GluA2/3 was more highly expressed compared with GluA1 in both the testis and the GC-1 cells. Subsequently, GC-1 cells were incubated with medium containing l -Glu, d -Glu, d -Asp or NMDA to determine GluA1 and GluA2/3 expressions. At 30 minutes and 2 hours of incubation, EAA-treated GC-1 cells showed significantly higher expression levels of both GluA1 and GluA2/3. Furthermore, p-extracellular signal-regulated kinase (ERK), p-Akt, proliferating cell nuclear antigen (PCNA), and Aurora B expressions were assayed in l -Glu-, d -Glu-, and NMDA-treated GC-1 cells. At 30 minutes and 2 hours of incubation, treated GC-1 cells showed significantly higher expression levels of p-ERK and p-Akt. A consequent increase of PCNA and Aurora B expressions was induced by l -Glu and NMDA, but not by d -Glu. Our study demonstrates a direct effect of the EAAs on spermatogonial activity. In addition, the increased protein expression levels of GluA1 and GluA2/3 in EAA-treated GC-1 cells suggest that EAAs could activate ERK and Akt pathways through the AMPAR. Finally, the increased PCNA and Aurora B levels may imply an enhanced proliferative activity.     

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.     

Immobilization-dependent bone collagen breakdown appears to increase with time: evidence for a lack of new bone equilibrium in response to reduced load during prolonged bed rest.

The purpose of this study was to evaluate the effect of prolonged immobilization on bone, in order to investigate how skeletal turnover adapts to bed rest. We examined indices of bone formation and bone resorption in the serum and urine of fifty-four patients (26 males and 28 females) immobilized after an episode of paralytic stroke. The length of immobilization ranged from 30 to 180 days. A significant, time-dependent increase in markers of resorption - urinary pyridinoline (Pyr) and deoxypyridinoline (D-Pyr), serum Type I collagen cross-linked C-telopeptide (ICTP) - was observed in immobilized patients, as compared to free-living healthy subjects. The positive correlation between resorption markers increase and the length of immobilization suggests that the rate of bone resorption did not decrease with time. On the other hand, the levels of markers of bone formation - bone-specific alkaline phosphatase (B-ALP), and the carboxyl-terminal propeptide of Type I procollagen (PICP) - remained within the normal range in all patients, regardless the length of immobilization. Our results would indicate an uncoupling between bone formation and bone resorption during bed rest, and suggest that the bone collagen break-down was not a self-limiting process in immobilized patients, and that a new equilibrium or "steady state" in response to the reduced load was not reached in the skeleton.     

Endocytosis and cancer

Eukaryotic cells use endocytosis to internalise plasma membrane, surface receptors and their ligands, viruses and various extracellular soluble molecules. Endocytosis has been regarded as a long-term mechanism of signal attenuation via receptor clearance from the cell surface. However, additional, and quite unexpected, functions for endocytosis have emerged, which, together with its attenuation function, project a central role for this process in cellular homeostasis and control of proliferation. Subversion of endocytic control is thus predicted to play a causative role in hyperproliferative conditions, first and foremost cancer.     

Diet and Feeding Ecology of Woolly Monkeys in a Western Amazonian Rain Forest

I investigated the diet and feeding ecology of two social groups of woolly monkeys (Lagothrix lagotricha poeppigii) in Yasuní National Park, Ecuador between April 1995 and March 1996. Woolly monkeys in Yasuní were predominantly frugivorous, with fruits comprising ca. 77% of the yearly diet; the next most common food type in the diet was insect and other animal prey. The fruit diet of woolly monkeys in Yasuní is the most diverse yet recorded for any ateline primate, including spider monkeys (Ateles), which are often regarded as ripe fruit specialists: 208 distinct morphospecies of fruits were consumed by woolly monkeys either during the study or during several preceding months of pilot work. Nonetheless, close to one-third of the yearly diet came from just 3 plant genera—Inga, Ficus, and Spondias—and only 20 genera each contributed to 1% of the diet. For one study group, the proportion of ripe fruit in the diet each month was correlated with the habitat-wide availability of this resource, a pattern evidenced by several other ateline species. However, the relationship was not apparent in the second study group. The modal party size for feeding bouts on all food types was a single monkey, and, contrary to reports for other atelines, neither feeding party size nor the total number of feeding minutes that groups spent in food patches was well predicted by patch size. Both results highlight the independent nature of woolly monkey foraging. Given that woolly monkeys and closely-related spider monkeys focus so heavily on ripe fruits, their very different patterns of social organization are intriguing and raise the question of just how their ecological strategies differ. Two important differences appear to be in the use of animal prey and in the phytochemical composition of the ripe fruits that they consume: spider monkeys rarely forage for animal prey, and woolly monkeys seldom consume the lipid-rich fruits that are an important part of spider monkey diets.     

Abi1 is essential for the formation and activation of a WAVE2 signalling complex

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.     

Abl-dependent tyrosine phosphorylation of Sos-1 mediates growth-factor-induced Rac activation

The non-receptor tyrosine kinase Abl participates in receptor tyrosine kinase (RTK)-induced actin cytoskeleton remodelling, a signalling pathway in which the function of Rac is pivotal. More importantly, the activity of Rac is indispensable for the leukaemogenic ability of the BCR-Abl oncoprotein. Thus, Rac might function downstream of Abl and be activated by it. Here, we elucidate the molecular mechanisms through which Abl signals to Rac in RTK-activated pathways. We show that Sos-1, a dual guanine nucleotide-exchange factor (GEF), is phosphorylated on tyrosine, after activation of RTKs, in an Abl-dependent manner. Sos-1 and Abl interact in vivo, and Abl-induced tyrosine phosphorylation of Sos-1 is sufficient to elicit its Rac-GEF activity in vitro. Genetic or pharmacological interference with Abl (and the related kinase Arg) resulted in a marked decrease in Rac activation induced by physiological doses of growth factors. Thus, our data identify the molecular connections of a pathway RTKs-Abl-Sos-1-Rac that is involved in signal transduction and actin remodelling.     

Pattern and process in Amazon tree turnover, 1976-2001

Previous work has shown that tree turnover, tree biomass and large liana densities have increased in mature tropical forest plots in the late twentieth century. These results point to a concerted shift in forest ecological processes that may already be having significant impacts on terrestrial carbon stocks, fluxes and biodiversity. However, the findings have proved controversial, partly because a rather limited number of permanent plots have been monitored for rather short periods. The aim of this paper is to characterize regional-scale patterns of 'tree turnover' (the rate with which trees die and recruit into a population) by using improved datasets now available for Amazonia that span the past 25 years. Specifically, we assess whether concerted changes in turnover are occurring, and if so whether they are general throughout the Amazon or restricted to one region or environmental zone. In addition, we ask whether they are driven by changes in recruitment, mortality or both. We find that: (i) trees 10 cm or more in diameter recruit and die twice as fast on the richer soils of southern and western Amazonia than on the poorer soils of eastern and central Amazonia; (ii) turnover rates have increased throughout Amazonia over the past two decades; (iii) mortality and recruitment rates have both increased significantly in every region and environmental zone, with the exception of mortality in eastern Amazonia; (iv) recruitment rates have consistently exceeded mortality rates; (v) absolute increases in recruitment and mortality rates are greatest in western Amazonian sites; and (vi) mortality appears to be lagging recruitment at regional scales. These spatial patterns and temporal trends are not caused by obvious artefacts in the data or the analyses. The trends cannot be directly driven by a mortality driver (such as increased drought or fragmentation-related death) because the biomass in these forests has simultaneously increased. Our findings therefore indicate that long-acting and widespread environmental changes are stimulating the growth and productivity of Amazon forests.     

Interaction between two ubiquitin-protein isopeptide ligases of different classes,CBLC and AIP4/ITCH

In metazoans, CBL proteins are RING finger type ubiquitin-protein isopeptide (E3) ligases involved in the down-regulation of epidermal growth factor tyrosine kinase receptors (EGFR). Among the three CBL proteins described in humans, CBLC (CBL3) remains poorly studied. By screening in parallel a human and a Caenorhabditis elegans library using the two-hybrid procedure in yeast, we found a novel interaction between Hsa-CBLC and Hsa-AIP4 or its C. elegans counterpart Cel-WWP1. Hsa-AIP4 and Cel-WWP1 are also ubiquitin E3 ligases. They contain a HECT (homologous to E6-AP C terminus) catalytic domain and four WW domains known to bind proline-rich regions. We confirmed the interaction between Hsa-CBLC and Hsa-AIP4 by a combination of glutathione S-transferase pull-down, co-immunoprecipitation, and colocalization experiments. We show that these two E3 ligases are involved in EGFR signaling because both become phosphorylated on tyrosine following epidermal growth factor stimulation. In addition, we observed that CBLC increases the ubiquitination of EGFR, and that coexpressing the WW domains of AIP4 exerts a dominant negative effect on EGFR ubiquitination. Finally, coexpressing CBLC and AIP4 induces a down-regulation of EGFR signaling. In conclusion, our data demonstrate that two E3 ligases of different classes can interact and cooperate to down-regulate EGFR signaling.     

The Eps15 homology (EH) domain

The Eps15 homology (EH) domain was originally identified as a motif present in three copies at the NH2-termini of Eps15 and of the related molecule Eps15R. Both of these molecules are substrates for the tyrosine kinase activity of the epidermal growth factor receptor and hence the name 'Eps15 homology' or EH domain [Wong et al. (1994) Oncogene 9, 1591-1597; Wong et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9530-9534; Fazioli et al. (1993) Mol. Cell. Biol. 13, 5814-5828] was derived. The motif was subsequently found in several proteins from yeast to nematode, thus establishing its evolutionary conservation. Initial studies with filter-binding assays and phage-displayed libraries demonstrated its protein:protein interaction abilities and identified specific ligands. Subsequently, structural analyses established the molecular bases of recognition between EH domains and cognate peptides. To date, several EH-containing and EH-binding proteins have been identified, which establish in the cell a network of protein:protein interactions, defined as the EH network. This network coordinates cellular functions connected with endocytosis, actin remodeling and intracellular transduction of signals.