The c-Cbl-associated protein and c-Cbl are two new partners of the SH2-containing inositol polyphosphate 5-phosphatase SHIP2

SHIP2 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains motifs susceptible to mediate protein-protein interaction. Using yeast two-hybrid, GST-pulldown, and coimmunoprecipitation studies, we isolated the CAP cDNA as a specific partner of SHIP2 proline-rich domain and showed by GST-pulldown experiments that the interaction took place with the SH3C of CAP. The interaction was not modulated in COS-7 cells stimulated by EGF neither in CHO cells overexpressing the insulin receptor in the presence or absence of insulin stimulation. We also showed that SHIP2 was able to coimmunoprecipitate with endogenous c-Cbl protein in the absence of CAP and with the insulin receptor in CHO-IR cell extracts. The presence of SHIP2 in a complex around the insulin receptor could account for the very specific increase in insulin sensitivity of SHIP2 knock-out mice.     

Use of cyclic peptide phage display library for the identification of a CD45RC epitope expressed on murine B cells and their precursors

The alternative splicing and variable expression of the exons near to the N-terminus of the leukocyte common antigen (L-CA, CD45) result in distinct extracellular isoforms expressed by cells with different functional and developmental properties. Here we report the tissue reactivity pattern and epitope specificity of a novel rat monoclonal antibody (IBL-8) against a restricted epitope of mouse CD45. We found that this mAb reacts with an epitope displayed by B cells and their precursors (both in newborn spleen and adult bone marrow). Moreover, peripheral CD8-positive T cells were also recognised at an intermediate intensity, whereas the CD4 T cell subset was weakly reactive. The epitope of this mAb was determined with M13 filamentous phages that display cysteine constrained nonapeptides on their coat proteins. The isolated bacteriophages expressing the putative epitope showed an isoform-specific inhibition of the binding of exon-specific mAbs. Deduced amino acid sequence data of these phages indicate that the epitope recognised by the IBL-8 mAb lies at the 136-144 region of the mouse CD45 molecule within its C exon, with a TAFP consensus sequence at its centre.     

Prolonged culture in low glucose induces apoptosis of rat pancreatic beta-cells through induction of c-myc

Prolonged culture in low-glucose concentrations (相似文献   

Confined diffusion without fences of a g-protein-coupled receptor as revealed by single particle tracking

Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.     

On translocation through a membrane channel via an internal binding site: kinetics and voltage dependence

Here we present a model for maltodextrin translocation through maltoporin channels. In a first step, our theoretical analysis does consider the case of a single binding site for a given substrate in a structurally unaffected channel with a possibly different entrance barrier on either side. It is shown how by means of conventional electrical conductance measurements (including current noise analysis) the basic equilibrium and rate constants can be determined as functions of the applied voltage. Then also the net translocation rate of the substrate becomes accessible quantitatively. This most simple model mechanism has been extended to include a voltage-dependent fast conformational change of the channel that prevents the binding process. The so developed approach has been tested with experimental data for a single maltoporin trimer being reconstituted in black lipid membranes when studied in the presence of maltohexaose as the substrate. The experimental results turned out to be clearly incompatible with binding alone. They are, however, very satisfactorily fitted by pertinent theoretical curves if also inhibition of binding by a conformational transition is taken into account. Accordingly, quantitative evaluations of the underlying parameters and eventually of the translocation rate have been carried out successfully. Our analysis reveals a set of parameters necessary for an optimal translocation that nicely corresponds to natural conditions.     

Mammalian meiosis involves DNA double-strand breaks with 3′ overhangs

Meiotic recombination in yeast is initiated at DNA double-strand breaks (DSBs), processed into 3′ single-strand overhangs that are active in homology search, repair and formation of recombinant molecules. Are 3′ overhangs recombination intermediaries in mouse germ cells too? To answer this question we developed a novel approach based on the properties of the Klenow enzyme. We carried out two different, successive in situ Klenow enzyme-based reactions on sectioned preparations of testicular tubules. Signals showing 3′ overhangs were observed during wild-type mouse spermatogenesis, but not in Spo11 ^?/? males, which lack meiotic DSBs. In Atm ^?/? mice, abundant positively stained spermatocytes were present, indicating an accumulation of non-repaired DSBs, suggesting the involvement of ATM in repair of meiotic DSBs. Thus the processing of DSBs into 3′ overhangs is common to meiotic cells in mammals and yeast, and probably in all eukaryotes.     

Between- and within-host species selection on cytoplasmic incompatibility-inducing Wolbachia in haplodiploids

Abstract The most common effect of the endosymbiont Wolbachia is cytoplasmic incompatibility (CI), a form of postzygotic reproductive isolation that occurs in crosses where the male is infected by at least one Wolbachia strain that the female lacks. We revisited two puzzling features of Wolbachia biology: how Wolbachia can invade a new species and spread among populations, and how the association, once established in a host species, can evolve, with emphasis on the possible process of infection loss. These questions are particularly relevant in haplodiploid species, where males develop from unfertilized eggs, and females from fertilized eggs. When CI occurs in such species, fertilized eggs either die (female mortality type: FM), or develop into males (male development type: MD), raising one more question: how transition among CI types is possible. We reached the following conclusions: (1) the FM type is a better invader and should be retained preferentially after a new host is captured; (2) given the assumptions of the models, FM and MD types are selected on neither the bacterial side nor the host side; (3) selective pressures acting on both partners are more or less congruent in the FM type, but divergent in the MD type; (4) host and symbiont evolution can drive infection to extinction for all CI types, but the MD type is more susceptible to the phenomenon; and (5) under realistic conditions, transition from MD to FM type is possible. Finally, all these results suggest that the FM type should be more frequent than the MD type, which is consistent with the results obtained so far in haplodiploids.     

Mitochondria-derived glutamate at the interplay between branched-chain amino acid and glucose-induced insulin secretion

In pancreatic beta-cells, glutamate has been proposed to mediate insulin secretion as a glucose-derived factor, although it is also considered for its sole catabolic function. Hence, changes in cellular glutamate levels are a matter of debate. Here, we investigated the effects of glucose and the glutamate precursor glutamine on kinetics of glutamate levels together with insulin secretion in INS-1E beta-cells. Preincubation at low (1 mM) glucose resulted in reduced cellular glutamate levels, which were doubled by exposure to glutamine. In glutamine-deprived cells, 5 mM glucose restored glutamate concentrations. Incubation at 15 mM glucose increased cellular glutamate, along with stimulation of insulin secretion, following both glutamine-free and glutamine-rich preincubations. Nuclear magnetic resonance (NMR) spectroscopy of INS-1E cells exposed to 15 mM D-[1-(13)C]glucose revealed glutamate as the major glucose metabolic product. Branched-chain amino acids, such as leucine, reduced cellular glutamate levels at low and intermediate glucose. This study demonstrates that glucose stimulates glutamate generation, whereas branched-chain amino acids promote competitive glutamate expenditure.     

<Emphasis Type="Italic">Candida</Emphasis> — Agent of the diaper dermatitis?

Occurrence of Candida spp. was determined in a population of 60 infants, 1-15-month-old, with diaper dermatitis, admitted to a neonatal intensive care unit in Hospital Saca (Kosice, Slovakia). Specimens were obtained from the perianal, pubic, inguinal, or gluteal areas that showed signs of secondary infection as manifested by erythema, oozing, vesiculopustular lesions, and pus formation. The most frequently isolated species was C. albicans (41), followed by C. parapsilosis (8), C. tropicalis (4), C. pulcherrima (4), C. guilliermondii (2), and C. zeylanoides (1). Other organisms present in the mixed culture from the diaper area were Staphylococcus aureus (6), Escherichia coli (3), and 2 strains of each group B and D streptococci, and Proteus mirabilis. Infants diapered exclusively in disposable diapers showed less rash than those diapered exclusively or sometimes in cloth diapers.     

Characterization of extradiol dioxygenases from a polychlorinated biphenyl-degrading strain that possess higher specificities for chlorinated metabolites

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBDLB400; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2',6'-dichloro-2,3-dihydroxybiphenyl (2',6'-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBDP6-I and DHBDP6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBDLB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBDP6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3'-Cl DHB approximately DHB approximately 4'-Cl DHB > 2'-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBDP6-I were among the best for DHBDP6-III (4-Cl DHB > 5-Cl DHB approximately 6-Cl DHB approximately 3'-Cl DHB > DHB > 2'-Cl DHB approximately 4'-Cl DHB; DHBDP6-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBDP6-I and 2'-Cl DHB for DHBDP6-III. As observed in DHBDLB400, chloro substituents influenced the reactivity of the dioxygenases with O2. For example, the apparent specificities of DHBDP6-I and DHBDP6-III for O2 in the presence of 2'-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBDP6-I and DHBDP6-III shared the relative inability of DHBDLB400 to cleave 2',6'-diCl DHB (apparent catalytic constants of 0.088 +/- 0.004 and 0.069 +/- 0.002 s(-1), respectively). However, these isozymes had remarkably different apparent K(m) values for this compound (0.007 +/- 0.001, 0.14 +/- 0.01, and 3.9 +/- 0.4 micro M for DHBDLB400, DHBDP6-I, and DHBDP6-III, respectively). The markedly different reactivities of DHBDP6-I and DHBDP6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.