The hominoid-specific oncogene TBC1D3 activates Ras and modulates epidermal growth factor receptor signaling and trafficking

Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues ( Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006) Genomics 88, 731-736 ). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans.     

Complement inhibition reduces injury in the type 2 diabetic heart following ischemia and reperfusion

Chronic inflammation exacerbates the cardiovascular complications of diabetes. Complement activation plays an important role in the inflammatory response and is known to be involved in ischemia-reperfusion (I/R) injury in the nondiabetic heart. The purpose of this study was to determine if increased complement deposition explains, in part, the increased severity of neutrophil-mediated I/R injury in the type 2 diabetic heart. Nondiabetic Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats underwent 30 min of coronary artery occlusion followed by 120 min of reperfusion. Another group of ZDF rats was treated with the complement inhibitor FUT-175 before reperfusion. Left ventricular (LV) tissue samples were stained for complement deposition and neutrophil accumulation following reperfusion. We found significantly more complement deposition in the ZDF LV compared with the ZLC (P < 0.05), and complement deposition was associated with significantly greater neutrophil accumulation. In whole blood samples taken preischemia and at 120 min reperfusion, neutrophils exhibited significantly more CD11b expression in the ZDF group compared with the ZLC group (P < 0.05). Furthermore, intracellular adhesion molecule (ICAM)-1 expression following I/R was increased significantly in ZDF hearts compared with ZLC hearts (P < 0.001). These results indicate that, in the ZDF heart, increased ICAM-1 and polymorphonuclear neutrophil (PMN) CD11b expression play a role in increasing PMN accumulation following I/R. The infarct size of the ZDF was significantly greater than ZLC (P < 0.05), and treatment with FUT-175 significantly decreased infarct size, complement deposition, and PMN accumulation in the diabetic heart. These findings indicate an exacerbated inflammatory response in the type 2 diabetic heart that contributes to the increased tissue injury observed following ischemia and reperfusion.     

Standardization of food allergen extracts for skin prick test

The aim of the study was to standardize and evaluate technically optimized food allergen extracts for use in skin prick test (SPT). The standardization procedure comprised 36 allergic histories in 32 food allergic patients with 21 healthy, non-atopic individuals serving as controls. The patients had a history of allergic symptoms upon ingestion of either cow’s milk (n=3), hen’s egg (n=9), wheat (n=4), hazelnut (n=14) or cod (n=6). They also had specific IgE in serum to the food in question and a positive SPT with a fresh preparation of the food. The diagnosis had been confirmed by a double-blind, placebo-controlled food challenge, except for the hazelnut-allergic patients. The controls were subjected to an open food challenge with all the foods to ensure tolerance. The standardization was performed by means of titrated SPT in accordance with the guidelines on biological standardization from the Nordic Council on Medicine. Regression analysis of the skin wheal areas was performed for each patient and the median protein concentration of allergen preparation (median C_h10) eliciting a wheal area of the same size as histamine 10 mg/ml was calculated. The median C_h10 was 0.56 mg/ml for milk, 0.88 mg/ml for egg, 5.4 mg/ml for wheat, 2.1 mg/ml for hazelnut and 0.017 mg/ml for the cod extract. The sensitivity of the median C_h10 estimated from the SPT data was 1 for milk, 0.98 for egg, 1 for wheat, 1 for hazelnut and 0.87 for the cod extract. The allergenic activity of the hazelnut extract was further investigated by leukocyte histamine release (HR) and immunoblotting experiments using sera from 27 hazelnut allergic patients. The clinical sensitivity of the optimized hazelnut extract evaluated by HR was 0.78 compared to 0.30 for a commercially available hazelnut extract (Soluprick). Immunoblotting results showed a stronger IgE binding capacity and additional IgE-binding bands of the optimized hazelnut extract compared with the Soluprick extract.     

Prelaying exodus of Cory's Shearwaters (Calonectris diomedea borealis) on Selvagem Grande

Summary We stayed on the Portugese island of Selvagem Grande, 30° N 16° W, 300 km south of Madeira and 200 km north of Tenerife, from 9 May to 3 June 1991 in order to study the prelaying exodus in Cory's Shearwaters (Calonectris diomedea borealis) and its connection with the colony attendance cycles described earlier. Laying took place on 1.5 June ±3.4 days. During the preceding weeks males and females of Cory's Shearwater show very different attendance patterns. Females are absent for 19±4.8 days at minimum. Absences of males are much shorter and also much more variable. They last 7.9±6.0 days (coefficient of variation 76 %, as against 12 % in females). The first incubation stint of the males is badly synchronised with the laying: the delay between laying and the start of the males' first stint may amount up to one week, without compromising the breeding success. Two well marked attendance peaks have been observed, separated by an interval of 18 days, twice the length of that observed in June 1988. We suggest that this anomaly in the daily attendance graph is correlated with the prelaying exodus. The function of the prelaying exodus and its relation with the energy cost of producing the egg and the necessity of storing lipid reserves are discussed.

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Vorbrutzeitliches Verlassen der Kolonie beim Gelbschnabelsturmtaucher (Calonectris diomedea borealis) auf Selvagem Grande

Zusammenfassung Zwischen 9. Mai und 3. Juni 1991 untersuchten wir auf der portugisischen Insel Selvagem Grande, 30° N 16° W, 300 km südlich von Madeira und 200 km nördlich von Teneriffa gelegen, das Verlassen der Kolonie vor der Eiablage des Gelbschnabelsturmtauchers (Calonectris diomedea borealis) und die Beziehung zur Anwesenheit in der Kolonie. Gelegt wurde am 1,5 Juni ±3,4 Tage. In den Monaten vor der Eiablage zeigten Männchen und Weibchen sehr unterschiedliche Muster der Anwesenheit in der Kolonie. Während die Weibchen im Minimum durchschnittlich 19±4,8 Tage abwesend waren, war diese Abwesenheit bei den Männchen mit 7,9±6,0 Tagen wesentlich kürzer und variabler (Variationskoeffizient 76 % gegenüber 12 % bei den Weibchen). Die erste Brutbeteiligung des Männchens war kaum mit der Eiablage synchronisiert, doch war dadurch der Bruterfolg nicht beeinträchtigt. Die Anwesenheit in der Kolonie zeigte zwei Gipfel, die 18 Tage auseinander lagen. In 1988 betrug dieser zeitliche Abstand nur etwa die Hälfte. Es wird vermutet, dass dieser Unterschied mit dem Ausmaß des vorbrutzeitlichen Verlassens der Kolonie korreliert. Die Funktion dieses vorbrutzeitlichen Exodus und seine Beziehung zu den energetischen Kosten der Eiproduktion und der Bildung von Lipidreserven werden diskutiert.

     

Successional Changes in an Evolving Anaerobic Chlorophenol-Degrading Community Used To Infer Relationships between Population Structure and System-Level Processes

The response of a complex methanogenic sediment community to 2-chlorophenol (2-CP) was evaluated by monitoring the concentrations of this model contaminant and important metabolic intermediates and products and by using rRNA-targeted probes to track several microbial populations. Key relationships between the evolving population structure, formation of metabolic intermediates, and contaminant mineralization were identified. The nature of these relationships was intrinsically linked to the metabolism of benzoate, an intermediate that transiently accumulated during the mineralization of 2-CP. Before the onset of benzoate fermentation, reductive dehalogenation of 2-CP competed with methanogenesis for endogenous reducing equivalents. This suppressed H₂ levels, methane production, and archaeal small-subunit (SSU)-rRNA concentrations in the sediment community. The concentrations of bacterial SSU rRNA, including SSU rRNA derived from “Desulfovibrionaceae” populations, tracked with 2-CP levels, presumably reflecting changes in the activity of dehalogenating organisms. After the onset of benzoate fermentation, the abundance of Syntrophus-like SSU rRNA increased, presumably because these syntrophic organisms fermented benzoate to methanogenic substrates. Consequently, although the parent substrate 2-CP served as an electron acceptor, cleavage of its aromatic nucleus also influenced the sediment community by releasing the electron donors H₂ and acetate. Increased methane production and archaeal SSU-rRNA levels, which tracked with the Syntrophus-like SSU-rRNA concentrations, revealed that methanogenic populations in particular benefited from the input of reducing equivalents derived from 2-CP.     

Direct Profiling of Environmental Microbial Populations by Thermal Dissociation Analysis of Native rRNAs Hybridized to Oligonucleotide Microarrays

Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.     

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.     

Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses.

The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (T(d)) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5' terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T(d) and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T(d) and signal intensity, and it decreased the variability of the signal. Although T(d)s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T(d)s than those with mismatches in the first or second position. The trained NNs predicted the T(d) with high accuracies (R(2) = 0.93). However, the NNs predicted the signal intensity only moderately accurately (R(2) = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T(d)s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5' terminus plays a greater role in determining the T(d) and signal intensity of duplexes than the type of mismatch.     

The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria.

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationships among selected species of mycobacteria and related organisms. The phylogeny inferred reflects the traditional classification, with major branches of the phylogenetic tree in general correspondence to the four Runyon groups and with numerical classification analyses. All the mycobacterial species compared, with the exception of M. chitae, are closely related (average similarity values greater than 95%). The slow growers form a coherent line of descent, distinct from the rapid growers, within which the overt pathogens are clustered. The distant relationship between M. chitae and the remaining mycobacteria suggests that this organism is incorrectly classified with the mycobacteria. M. paratuberculosis 18 was indistinguishable from M. avium-M. intracellulare-M. scrofulaceum serovar 1 by this analysis.     

Development of germ cells in chick ovarian medulla]

Ovaries from chick embryos and chicken have been investigated with a view to the evolution of germ cells in the medullary. They can enter meiosis and reach pachytene. They seem to be eliminated by the way of the lacunas. Some observations can be utilized in a discussion about the initiation of the meiosis and the evolution of the ovocytes.