Inhibition of basal FGF receptor signaling by dimeric Grb2.

Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this "background" level of receptor phosphorylation is insufficient to effect a downstream response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2. Dimeric Grb2 binds to the C termini of two FGFR2 molecules. This heterotetramer is capable of a low-level receptor transphosphorylation, but C-terminal phosphorylation and recruitment of signaling proteins are sterically hindered. Upon stimulation, FGFR2 phosphorylates tyrosine residues on Grb2, promoting dissociation from the receptor and allowing full activation of downstream signaling. These observations establish a role for Grb2 as an active regulator of RTK signaling.

Elevated receptor for activated C kinase 1 expression is involved in intracellular Ca2+ influx and potentially associated with compromised regulatory T cell function in patients with asthma.

BACKGROUND: Regulatory T cells (T(regs)) are activated during anergy in response to T cell receptor (TCR) activation and functional immune suppression. Anergy of paediatric T(regs) is partially dependent on intracellular calcium mobility; following TCR activation, T(regs) do not exhibit increased intracellular Ca(2+) concentration ([Ca(2+) ](i)). OBJECTIVE: We determined whether [Ca(2+) ](i) in adult T(regs) defined their anergy, if intracellular Ca(2+) movement was linked to regulatory functions, whether [Ca(2+)](i) was indicative of asthma pathology, and the potential molecular mechanism responsible for Ca(2+) movement in T(regs). METHODS: T(regs) were purified by the magnetic bead method, and their regulatory functions were assessed by monitoring carboxyfluorescein succinimidyl ester-labelled responder T cell proliferation. The Ca(2+) response of Fura-2-labelled cells was measured using a video image analysis system. To analyse the functions of T(regs) at the molecular level, we generated Jurkat Tet-On(®) clones with doxycycline (Dox)-induced forkhead box P3 (FOXP3) protein expression. RESULTS: CD4(+) CD25(+) CD127(-/low) T(regs) from participants without asthma did not elicit Ca(2+) influx in response to TCR activation, exhibited little proliferation and suppressed proliferation of CD4(+) CD25(-) T cells. In contrast, under similar conditions, T(regs) from patients with asthma exhibited increased [Ca(2+)](i) and robust proliferation with partial loss of regulatory functions. FOXP3 protein levels in Tet-On(®) clones were high after both 2- and 5-day Dox treatment; however, 5-day cells were comparable with T(regs) from patients with asthma, whereas 2-day cells were similar to T(regs) from participants without asthma. Increasing [Ca(2+)](i) induced a high level of receptor for activated C kinase 1 (RACK1) expression in 5-day cells. CONCLUSIONS AND CLINICAL RELEVANCE: We confirmed that T(regs) in patients with asthma are functionally impaired and that the abnormal regulatory functions of these cells can be analysed by [Ca(2+)](i) following TCR engagement. Furthermore, the impaired functioning of T(regs) evident in patients with asthma may be due to a high level of RACK1.

Precooling via immersion in CO2-enriched water at 25°C decreased core body temperature but did not improve 10-km cycling time trial in the heat.

This study compared the effects of precooling via whole-body immersion in 25°C CO2-enriched water (CO2WI), 25°C unenriched water (WI) or no cooling (CON) on 10-km cycling time trial (TT) performance. After 30 min of precooling (CO2WI, CON, WI) in a randomized, crossover manner, 11 male cyclists/triathletes completed 30-min submaximal cycling (65%VO2peak), followed by 10-km TT in the heat (35°C, 65% relative humidity). Average power output and performance time during TT were similar between conditions (p = 0.387 to 0.833). Decreases in core temperature (Tcore) were greater in CO2WI (-0.54 ± 0.25°C) than in CON (-0.32 ± 0.09°C) and WI (-0.29 ± 0.20°C, p = 0.011 to 0.022). Lower Tcore in CO2WI versus CON was observed at 15th min of exercise (p = 0.050). Skin temperature was lower in CO2WI and WI than in CON during the exercise (p < 0.001 to 0.031). Only CO2WI (1029 ± 305 mL) decreased whole-body sweat loss compared with CON (1304 ± 246 mL, p = 0.029). Muscle oxygenation by near-infrared spectroscopy (NIRS), thermal sensation, and thermal comfort were lower in CO2WI and WI versus CON only during precooling (p < 0.001 to 0.041). NIRS-derived blood volume was significantly lower in CO2WI and WI versus CON during exercise (p < 0.001 to 0.022). Heart rate (p = 0.998) and rating of perceived exertion (p = 0.924) did not differ between conditions throughout the experiment. These results suggested that CO2WI maybe more effective than WI for enhanced core body cooling and minimized sweat losses.

Fibrinonecrotic enteritis and orchitis associated with Salmonella enterica subsp houtenae infection in a short-tailed boa (Boa constrictor amarali).

Salmonella enterica subsp houtenae has been recovered from a wide variety of species, including reptiles, and has been linked to important clinical manifestations in snakes and lizards. We describe a case of concomitant fibrinonecrotic enteritis and orchitis associated with S. enterica subsp houtenae infection in a short-tailed boa (Boa constrictor amarali). At necropsy, the mucosa of the large intestine was covered by a focally extensive fibrinonecrotic exudate (diphtheritic pseudomembrane). The left testicle was enlarged, firm and diffusely expanded by a thick fibrinous yellow exudate. Polymerase chain reaction and conclusive antigenic testing indicated that the bacteria isolated from the lesions were S. enterica subsp houtenae, and the virulence genes InvA, slyA, stn and spvC were identified. This report reinforces that, although S. enterica subsp houtenae has been isolated from asymptomatic reptiles, it has the potential to cause life-threatening disease in snakes.

[Factors influencing life quality improvement in patients of different age one year after coronary artery bypass surgery].

The study was aimed at examining the factors having an impact on changes in the quality of life (QL) parameters a year after coronary artery bypass surgery (CABG) in different age groups. 408 patients aged 31-79 years had their QL assessed by means of the SF-36 questionnaire before and one year after elective CABG. Additionally, the patients were tested for the presence of type D personality before and one year after CABG by means of the DS-14 questionnaire. The groups were comparable in terms of previous carotid endarterectomy, peripheral arterial and aortic reconstruction number (p > 0.05). One year after CABG the improved QL (p < 0.01) was reported whereas type D patients had significantly poorer psychological test results (p < 0.01). One year after CABG type D personality and high Euroscore had an independent impact on the QL improvement while previous carotid endartectomies resulted in a significantly improved psychological health component. Thus, along with the detection and treatment of non-coronary lesions the personality type assessment and focused behavioral interventions can contribute to the QL improvement in long-term CABG period.

[Quality of life of patients after coronary bypass surgery: effect of age and personality type D].

AIM: To study effect of age and presence of personality type D on quality of life (QL) of patients in one year after coronary bypass surgery (CBS). MATERIAL AND METHODS: Assessment of QL before and after surgery was carried out in 408 patients. Two groups were formed: patients younger than 60 years (n=233) and older than 60 years (n=175). Quality of life and presence of personality type D was assessed by questionnaires SF-36 and DS-14, respectively. RESULTS: In a year after CBS improvement of physical and psychological components of QL (p<0.05) was noted in both groups. Personality type D was more frequent among patients older than 60 years (p<0.01). Patients with personality type D had worse parameters of irrespective of age (p<0.01). Personality type D correlated directly with age QL (p=0.003) and inversely with parameters of QL (p<0.01). Presence of personality type D was an independent predictor of low level of physical (together with index of atherogenecity prior to surgery) and psychological components of QL at multiple logistic regression analysis. CONCLUSION: Assessment of personality traits of patients before CBS can be helpful for targeted measures aimed at improvement of QL what is especially important for older age groups.

Expansion microscopy reveals subdomains in C. elegans germ granules.

Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid-liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule in C. elegans, our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with WT animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, Mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. This study will serve as an important tool in our understanding of germ granule biology and the biological role of liquid-liquid phase separation.

Differential labelling of human sub-cellular compartments with fluorescent dye esters and expansion microscopy.

Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human HeLa cell line. When combined with the super-resolution technique expansion microscopy (ExM) involving swellable acrylamide hydrogels, fluorescent esters reveal nanoscale features including cytoplasmic membrane-bound compartments and nucleolar densities. We observe differential labelling patterns linked to the biochemical properties of the conjugated dye. Alterations in staining density and compartment specificity were seen depending on the timepoint of application in the ExM protocol. Additional complexity in labelling patterns was detected arising from inter-ester interactions. Our findings raise a number of considerations for the use of fluorescent esters. We demonstrate esters as a useful addition to the repertoire of stains of the cellular proteome, whether applied either on their own to visualise overall cellular morphology, or as counterstains providing ultrastructural context alongside specific target markers like antibodies.

[Quality of life in patients of different age groups with ischemic heart disease: effect of multifocal atherosclerosis].

The aim of the research was to study the quality of life for the patients of different age groups with coronary artery disease and to evaluate the factors affecting its decline. The present study included 709 patients aged 31 to 79 years examined before elective coronary artery bypass graft surgery. All patients underwent coronary angiography, echocardiography, ultrasonography of the aorta, brachiocephalic, and peripheral arteries. With age, significantly increased incidence of multifocal atherosclerosis (p = 0.01 for the trend). To study the quality of life (QL) a non-specific questionnaire SF-36 was used, according to which the reduction of QL equally in all the groups (p > 0.05) was detected. Multivariate analysis showed that the level of QL was influenced by diabetes, myocardial infarction and the presence of multifocal atherosclerosis (p < 0.05). Thus, in order to improve the QL in aged patients with multifocal atherosclerosis is reasonable to conduct of reconstructive operations at different arterial basins.

[Prevalence of multifocal atherosclerosis in different age groups].

Aim of the study was to assess prevalence of lesions in several arterial beds in patients with atherosclerosis of various localization in the clinic of cardiovascular surgery. We examined 1018 patients (825 men and 193 women, aged 31-78 years, mean age 59+/-12 years) in the period of preparation to elective surgical interventions on coronary arteries or other arterial beds. All patients were divided into 4 age groups: group 1 - younger than 60 years (n=542), group 2 - 60-64 years (n=215), group 3 - 65-69 years (n=141), group 4-70 years and older (n=120). All patients were subjected to coronary angiography and Doppler ultrasound investigation (USI) of extracranial arteries. USI of arteries of lower extremities and angiography of peripheral arteries were carried out if indicated. Presence of 50% or greater stenosis was considered a criterion of involvement of an arterial vascular bed. Lesions in 2 or more beds were found in 321 patients (31.5%). Stenoses in 2 and 3 arterial beds were revealed in 24 and 3.5%, respectively, of patients in group 1, and in 31.8 and 10%, respectively, of patients in group 4 (p=0.008). Purposeful diagnostics of multifocal atherosclerosis in patients of the given category apparently should not be limited by older age groups.

Verification of the Usefulness of an Assessment and Risk Control Sheet that Promotes Management of Cancer Drug Therapy.

Background: Proper management of adverse events is crucial for the safe and effective implementation of anticancer drug treatment. Showa University Hospital uses our interview sheet (assessment and risk control [ARC] sheet) for the accurate evaluation of adverse events. On the day of anticancer drug treatment, a nurse conducts a face-to-face interview. As a feature of the ARC sheet, by separately describing the symptoms the day before treatment and the day of treatment and sharing the information on the medical record, it is possible to clearly determine the status of adverse events. In this study, we hypothesized that the usefulness and points for improvement of the ARC sheet would be clarified by using and evaluating a patient questionnaire. Methods: This study included 174 patients (144 at Showa University Hospital (Hatanodai Hospital) and 30 at Showa University Koto Toyosu Hospital (Toyosu Hospital) who underwent pre-examination interviews by nurses and received cancer chemotherapy at the outpatient center of Hatanodai and Toyosu Hospital. In the questionnaire survey, the ARC sheet's content and quality, respondents' satisfaction, structural strengths, and points for improvement were evaluated on a five-point scale. Results: The patient questionnaire received responses from 160 participants, including the ARC sheet use group (132 people) and the non-use group (28 people). Unlike the ARC sheet non-use group, the ARC sheet use group recognized that the sheet was useful to understand the adverse events of aphthous ulcers (p = 0.017) and dysgeusia (p = 0.006). In the satisfaction survey questionnaire, there was a high sense of security in the pre-examination interviews by nurses using the ARC sheet. Conclusions: The ARC sheet is considered an effective tool for comprehensively evaluating adverse events. Pre-examination interviews by nurses using ARC sheets accurately determined the adverse events experienced by patients with anxiety and tension due to confrontation with physicians.

Indirect recruitment of the signalling adaptor Shc to the fibroblast growth factor receptor 2 (FGFR2).

The adaptor protein Shc (Src homology and collagen-containing protein) plays an important role in the activation of signalling pathways downstream of RTKs (receptor tyrosine kinases) regulating diverse cellular functions, such as differentiation, adhesion, migration and mitogenesis. Despite being phosphorylated downstream of members of the FGFR (fibroblast growth factor receptor) family, a direct interaction of Shc with this receptor family has not been described to date. Various studies have suggested potential binding sites for the Shc PTB domain (phosphotyrosine-binding domain) and/or the SH2 (Src homology 2) domain on FGFR1, but no interaction of full-length Shc with these sites has been reported in vivo. In the present study, we investigated the importance of the SH2 domain and the PTB domain in recruitment of Shc to FGFR2(IIIc) to characterize the interaction of these two proteins. Confocal microscopy revealed extensive co-localization of Shc with FGFR2. The PTB domain was identified as the critical component of Shc which mediates membrane localization. Results from FLIM (fluorescence lifetime imaging microscopy) revealed that the interaction between Shc and FGFR2 is indirect, suggesting that the adaptor protein forms part of a signalling complex containing the receptor. We identified the non-RTK Src as a protein which potentially mediates the formation of such a ternary complex. Although an interaction between Src and Shc has been described previously, in the present study we implicate the Shc SH2 domain as a novel mediator of this association. The recruitment of Shc to FGFR2 via an indirect mechanism provides new insight into the regulation of protein assembly and activation of various signalling pathways downstream of this RTK.

The rapamycin-binding domain governs substrate selectivity by the mammalian target of rapamycin.

The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70(S6K). Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70(S6K), were phosphorylated. Rapamycin-FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells.

Socioeconomic Impact of Pediatric Sleep Disorders.

Pediatric disorders tend to affect the immediate support unit, adults and children. High costs for direct consumption of medical care are offset by early diagnosis and treatment of pediatric sleep disorders. Pediatric sleep disorders are underdiagnosed and undertreated. Attention-deficit/hyperactivity disorder may result from insufficient or fragmented sleep. Delaying school start time resulted in decreased car crashes in teen drivers and improved mood.

The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription.

Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense.

[Role of Mg2+-dependent alkaline endodnaase of blood serum in tumorigenesis in A/smail mice].

An enzymoassay of levels of Mg2+-dependent alkaline endoDNAase of protein spectrum of blood serum of noninbred albino mice by SDS-electrophoresis in 10% PAAG established a fairly high heterogeneity of the enzyme. The variety of alkaline endoDNAases must be due to the limited proteolysis of their high-molecular precursor by specific proteases as described in the literature. No alkaline endoDNAase activity was identified by analysis of 10-150 kDa protein spectrum in A/smail line mice without ascites hepatoma. It was detected (pH 8.3) in the 25-45 kDa range on day 10 after tumor transplantation. Considering the gravity of disease (pre-lethal stage), on day 10, it was suggested that the level be accounted for by decay of diseased cells. The lack or extremely low level of endoDNAase activity in original blood serum expressed a mechanism of enhancing sensitivity to tumor cell effects. In mice bearing ascites hepatoma, the enzyme levels (pH 8.3) were much higher on day 10. DNAase activity (pH 7.5) was not induced in response to the heterogenous DNA. Our data point to possible defects in the induction of Mg2+-dependent alkaline endoDNAases (pH 8.3) and DNAses (pH 7.5) as well as their role in raising sensitivity to transplantable ascites hepatoma.

[Apoptosis of phagocytes as one of the probable mechanisms of the pathogenetic action of Yersinia pestis "mouse" toxin].

Apoptosis was evaluated by characteristic morphological changes of cells in preparations stained with histological dyes and in live preparations, as well as by DNA degradation, colorimetrically detected with the use of the diphenylamine reagent. "Mouse toxin" (MT) was found to have a pronounced apoptogenic action with respect to the phagocytic cells of mice, but not guinea pigs. Macrophages were affected by this action stronger than neutrophils, and in both cases this effect was dose dependent. As the dose of MT decreased to 0.01 microg/ml, the proportion of cells dying as the result of apoptosis increased, the necrotic type of damage was almost absent. On the contrary, as MT concentration rose to 1.0 microg/ml and over, the proportion of phagocytes dying due to necrosis increased with a decrease in the number of cells in which the process of apoptosis started. The results of the study are indicative of the fact that the mechanisms programming the death of cells under the action of MT on macrophages and neutrophils took part in the process, which, in its turn, determined their role in the pathogenesis of plague.

[Influence of neutrophilokines on the synthesis of lymphokines in the process of the formation of immunity to plague].

The evaluation of the complex of neutrophilokines whose synthesis was induced by Yersinia pestis vaccine strain EV on the production of lymphokines in the process of the formation of primary and secondary immunity to plague is presented. As revealed in this study, neutrophilokines regulate the synthesis of IL-2 by T helpers of type 1, IL-4 and IL-5 by T helpers of type 2, IL-1 by B lymphocytes, as well as the expression of receptors IL-2 by immunocompetent cells. The helper effect of neutrophilokines is more pronounced in the secondary immune response.