Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases

Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.     

Time course of nerve-fiber regeneration in the noise-damaged mammalian cochlea

The time course of events which are essential for nerve-fiber regeneration in the mammalian cochlea was determined using a group of chinchillas that had been exposed for 3.5 hr to an octave band of noise with a center frequency of 4 kHz and a sound pressure level of 108 dB. The animals recovered from 40 min (0 days) to 100 days at which times their inner ears were fixed and the organs of Corti prepared for phase-contrast and bright-field microscopy as plastic-embedded flat preparations. Selected areas identified in the flat preparations were semi-thick and thin sectioned at radial or tangential angles for examination by bright-field and transmission electron microscopy. The following time-ordered events appeared critical for nerve-fiber regeneration: (1) The area of the basilar membrane in which regeneration had a possibility of occurring showed signs of severe injury. Outer hair cells degenerated first followed by outer pillars, inner pillars, inner hair cells and other supporting cells; (2) Myelinated nerve fibers in the osseous spiral lamina became fragmented, starting at the distal ends of the fibers. This degeneration gradually extended back to Rosenthal's canal; (3) Fibrous processes, originating from Schwann-like cells in the osseous spiral lamina, extended laterally on the basilar membrane; (4) Schwann cells lined up medial to the habenulae perforata in the areas of severest damage, apparently ready to migrate through the habenulae onto the basilar membrane; (5) Schwann-cell nuclei appeared on the basilar membrane beneath the developing layer of squamous epithelium which was in the process of replacing the degenerated portion of the organ of Corti; (6) Regenerated nerve fibers with thin myelin sheaths or a simple investment of Schwann cell cytoplasm appeared in areas of total loss of the organ of Corti; and (7) The myelin sheaths on the regenerated nerve fibers gradually became thicker.     

Polysomnographic sleep is not clinically impaired in Vietnam combat veterans with chronic posttraumatic stress disorder

BACKGROUND: Because sleep is typically disturbed in posttraumatic stress disorder (PTSD), this study was undertaken to evaluate a group of Vietnam combat veterans with the disorder using clinical polysomnographic techniques. METHODS: Eighteen Vietnam combat veterans with PTSD and 10 healthy non-combat-exposed Vietnam era veterans participated in 2 nights of polysomnographic study and a multiple sleep latency test. RESULTS: No significant differences between subjects and controls were noted except for greater sleep onset latency to stage 2 (p < .03), and lower arousals/hour from stages 3 & 4 (p < .04) on night 2, and lower subjectively estimated total sleep time on night 1 (p < .005) in the case of PTSD subjects. Otherwise, results from the second night served to replicate those from the first, and no significant differences appeared on 2 successive nights for any polysomnographic variable. No daytime hypersomnolence was detected. CONCLUSIONS: Polysomnographically recorded sleep was notably better than expected in the presence of clinically significant PTSD with typical histories of disrupted sleep. In these subjects, there is no clinically significant sleep disorder or typical pattern of sleep disturbance detectable by standard polysomnography.     

Aging and high-frequency hearing sensitivity

As part of a large population-based study of hearing and aging, ultra high-frequency (9-20 kHz) threshold measures are reported for 3396 participants grouped by age (48-59 years, n = 1233; 60-69 years, n = 1031; 70-79 years, n = 851; 80-92 years, n = 281). Ultra high-frequency (UHF) thresholds were higher for older age groups. The percentage of unmeasurable responses also was significantly higher for older age groups and for higher frequencies in the UHF range. The observed age effects remained significant after adjusting for gender. In general, UHF thresholds were significantly higher for men compared to those for women at lower UHF frequencies (9-14 kHz), but were not significantly different by gender for the highest UHF frequencies (16, 18, and 20 kHz). After accounting for hearing loss at traditional audiometric frequencies (250-8000 Hz), the age effect still remained; even for comparable degrees of sensorineural hearing loss, participants in older age groups evidenced higher UHF thresholds.     

Increased growth promoting but not mast cell degranulation potential of a covalent dimer of c-Kit ligand

The native form of soluble c-kit ligand (KL) is a noncovalent dimer. We have isolated a soluble, disulfide-linked dimer of murine KL (KL-CD) by expressing KL in Escherichia coli and refolding the denatured protein under conditions that promote the formation of both noncovalent dimers (KL-NC) and KL-CD. KL-CD exhibits a 10- to 15-fold increase in the ability to stimulate the growth of both the human megakaryocytic cell line MO7e and murine bone marrow-derived mast cells relative to KL-NC. Colony-forming assays of murine bone marrow progenitor cells also reflected this increased potency. However, KL-CD and KL-NC are equally able to prime mast cells for enhanced IgE-dependent degranulation in vitro and activate mast cells in vivo. Improving the growth-promoting activity of KL without changing its mast cell activation potential suggests that KL-CD or a related molecule could be administered in the clinic at doses that stimulate hematopoietic recovery while avoiding significant mast cell activation.     

Cognition and metacognition at extreme altitudes on Mount Everest.

The FACTRETRIEVAL2 test battery, which assesses both retrieval of general information from memory and metacognition about that retrieval, was administered to people before and after a recent expedition to Mount Everest and at extreme altitudes above 6,400 m (higher than any mountain in North America or Europe). Major findings were as follows: First, the same extreme altitudes known to impair learning did not affect accuracy or latency of retrieval, and this robustness of retrieval occurred for both recall and forced-choice recognition. Second, extreme altitude did affect metacognition: Climbers showed a decline in their feeling of knowing both while at extreme altitude and after returning to Kathmandu (i.e., both an effect and an aftereffect of extreme altitude). Third, extreme altitude had different effects than alcohol intoxication (previously assessed by T. O. Nelson et al [see PA, Vol 73:29460]). Alcohol intoxication affected retrieval without affecting metacognition, whereas extreme altitude affected metacognition without affecting retrieval; this different pattern for extreme altitude versus alcohol intoxication implies that (a) hypoxia does not always yield the same outcome as alcohol intoxication and (b) neither retrieval nor metacognition is strictly more sensitive than the other for detecting changes in independent variables. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

EPR and electron nuclear double resonance (ENDOR) studies show nitrite binding to the type 2 copper centers of the dissimilatory nitrite reductase of Alcaligenes xylosoxidans (NCIMB 11015)

EPR and 1H, 14,15N ENDOR spectra are described for the type 1 and type 2 Cu(II) centers of dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans. The study was carried out on preparations of NiR containing both type 1 and type 2 Cu sites, and also on preparations of lower activity which contained essentially only type 1 Cu centers. This has enabled ENDOR studies of type 1 and type 2 sites to be carried out largely independently of each other, by appropriate choice of the excitation field. Spectra were recorded both in the absence and presence of nitrite, allowing a clear determination of which of the two types of Cu center constitutes the substrate binding site. The EPR results show large changes in the type 2 site gparallel (which decreases by 0.065) and CuAparallel (which increases by 2.0 mT) while the type 1 site EPR is not affected. In addition, both 1H and 14N ENDOR of the type 2 Cu site undergo considerable changes on addition of nitrite whereas the type 1 Cu site ENDOR is unaffected. Our results clearly demonstrate that nitrite binds to the type 2 copper and that this process significantly perturbs the ligation of this copper by the protein histidine residues. No 15N ENDOR resonances were observed from 15N nitrite. The accessibility of the copper sites to solvent has been studied using 2H2O. The results indicate that nitrite binds to the type 2 Cu by displacing a proton, probably on a water molecule bound to the copper atom.     

Determination of osteoporosis in patients with fractured femoral neck using the Singh index: a Jerusalem study

The Singh index was used to measure the degree of osteoporosis in patients with fracture of the neck of the femur and to compare its prevalence with a random population sample in Jerusalem. The frequency of osteoporosis was 22.7% among women in the fracture series as compared with 1% in the control group. These figures are much lower than those in comparable series. The Singh index was not as sensitive an indicator of spinal osteoporosis as a direct measurement of vertebral bone density and could not be correlated with the incidence of hip fractures.