Trk neurotrophin receptor family immunoreactivity in rat and human pituitary tissues

Several lines of evidence suggest that neurotrophins may be involved in pituitary function. By immunocytochemical methods we analyzed the cellular distribution of their functional receptors in the pituitary gland. In the rat pituitary gland Trks were differentially distributed. Punctate immunoreactivity for TrkA was observed within the neural lobe, whereas numerous nerve endings were immunostained for TrkB and TrkC in the intermediate lobe. Endocrine cells of the intermediate lobe exhibited intense immunoreactivity for the three Trks, whereas scattered endocrine cells of the anterior lobe displayed a robust immunostaining for TrkC. In addition, TrkA and TrkB immunoreactivity was located in normal and neoplastic endocrine cells from human pituitary adenomas. The differential distribution of Trks in the hypophysis suggests a potential role of different neurotrophins in pituitary functions.     

Association of the p75 neurotrophin receptor with TRAF6

In addition to the Trk tyrosine kinase receptors, neurotrophins also bind to a second receptor, p75, a member of the tumor necrosis factor receptor superfamily. Several signaling pathways have been implicated for p75 in the absence of Trk receptors, including induction of NF-kappaB and c-Jun kinase activities and increased production of ceramide. However, to date, the mechanisms by which the p75 receptor initiates intracellular signal transduction have not been defined. Here we report a specific interaction between p75 and TRAF6 (tumor necrosis factor receptor-associated factor-6) after transient transfection in HEK293T cells. The interaction was ligand-dependent and maximal at 100 ng/ml of nerve growth factor (NGF). Other neurotrophins also promoted the association of TRAF6 with p75 but to a lesser extent. The binding of TRAF6 was localized to the juxtamembrane region of p75 by co-immunoprecipitation and Western blotting. To assess the functional significance of this interaction, we have tested responses in cultured Schwann cells that express p75 and TRAF6. An NGF-mediated increase in the nuclear localization of the p65 subunit of NF-kappaB could be blocked by the introduction of a dominant negative form of TRAF6 in Schwann cells. These results indicate that TRAF6 can potentially function as a signal transducer for NGF actions through the p75 receptor.     

Early events in neurotrophin signalling via Trk and p75 receptors

Biological responses to neurotrophins appear to be mediated by multiple signalling pathways. These emanate from, and are regulated by, the contributions of both Trk and p75 receptors. Early events in Trk signalling are becoming more clearly defined and point to cooperate interaction of both Ras-dependent and Ras-independent pathways. Work over the past year has clarified the steps by which Trk receptor occupation leads to Ras activation and has highlighted the required roles of Ras and extracellular signal regulated kinases in certain neurotrophin responses, including neurite outgrowth. Pharmacologic and mutagenesis studies have additionally supported the importance of the phosphatidylinositol-3' kinase and SNT protein pathways in neurotrophin signalling. Although many findings point to clear involvement for p75 in neurotrophin signalling, the molecular mechanisms by which these occur are just beginning to be identified. Recent studies indicate that p75 dramatically influences Trk activity and ligand interactions, and may mediate signals through the ceramide second-messenger pathway.     

Cloning of the gene for monogalactosyldiacylglycerol synthase and its evolutionary origin

Monogalactosyldiacylglycerol (MGDG) synthase (UDPgalactose:1,2-diacylglycerol 3-beta-D-galactosyltransferase; EC 2.4.1.46) catalyzes formation of MGDG, a major structural lipid of chloroplast. We cloned a cDNA for the synthase from cucumber cDNA library. The full-length cDNA clone was 2142 bp, and it contains a 1575-bp open reading frame encoding 525 aa. The open reading frame consists of the regions for a mature protein (422 aa; Mr of 46,552) and transit peptide to chloroplast (103 aa). Although the molecular weight of mature protein region matched that purified from cucumber cotyledons, it was quite different from those purified from spinach (approximately 20 kDa) reported by other groups. The mature region of the protein was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The expression in E. coli showed that the protein catalyzed MGDG synthesis very efficiently. Therefore, we concluded that the cDNA encodes MGDG synthase in cucumber. In addition, the deduced amino acid sequence of the MGDG synthase cDNA showed homology with MurG of Bacillus subtilis and E. coli, which encode a glycosyltransferase catalyzing the last step of peptidoglycan synthesis in bacteria. This sequence homology implies that the machinery of chloroplast membrane biosynthesis is evolutionarily derived from that of cell wall biosynthesis in bacteria. This is consistent with the endosymbiotic hypothesis of chloroplast formation.     

New horizons in the evolutionary science of the human family.

Familial relationships cannot be properly understood outside of an evolutionary framework. Pseudoscientific and traditional modes of thought have steered us away from an accurate account of ourselves and our kin. Recent theoretical and empirical advances in the evolutionary sciences, such as the theories of inclusive fitness, parental investment, and parent-offspring conflict, have aided our understanding of familial conflict and cooperation. We suggest that a gene's eye perspective of human families can likewise illuminate much of human psychology and behavior by contrasting individual interests with genetic interests. Furthermore, theoretical and empirical work on genetic imprinting and extended phenotypic action-at-a-distance have unveiled the extent to which co-evolutionary arms races and manipulation lie at the heart of familial interactions and psychological disorders. We posit that human cultural trends and morals can ultimately be grounded on an evolutionary foundation: not only do human laws and institutions reflect group-level manifestations of gene-level cooperative adaptations, but also they may reflect gene-level manipulative adaptations. An awareness of evolutionary dynamics can advance human well-being and unveil the hidden mechanisms beneath all human and nonhuman relationships. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Coexpression of mRNA for the full-length neurotrophin receptor trk-C and trk-A in favourable neuroblastoma

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may sometimes regress spontaneously in infants, or progress to a poor clinical outcome despite intensive therapy. Neuroblastomas express neurotrophin receptors and high levels of mRNA for trk-A correlates with favourable outcome, whereas trk-B mRNA is expressed by more unfavourable tumours. Using a sensitive RNase protection assay, mRNA expression for the neurotrophin receptor trk-C was investigated in 50 tumour samples from 45 children at different stages including metastatic and relapsing tumour tissue, out of which 22 were also investigated for trk-A mRNA. Thirty-seven of 43 primary tumours (86%) showed trk-C mRNA with more than 300-fold difference between the highest and the lowest values. A higher trk-C index (trk-C mRNA/GAPDH mRNA) was associated with favourable features such as younger age (P = 0.009-0.003), favourable tumour stage (1, 2 or 4S; P < 0.001) and favourable prognosis (P = 0.044). Better survival probability was shown in children with intermediate or high trk-C index compared with patients with low or undetectable levels (P = 0.031). All localised tumours co-expressed mRNA for trk-A and trk-C receptors. RT-PCR analysis detected mRNA encoding the cytoplasmic trk-C tyrosine kinase region only in favourable neuroblastomas. We conclude that favourable neuroblastoma may express the full-length trk-C receptor while unfavourable tumours, especially those with MYCN amplification, seem to either express no trk-C or truncated trk-C receptors with unknown biological function. Trk-C and possibly its preferred ligand NT-3 may be involved in the biology of favourable neuroblastomas showing apoptosis or differentiation.     

Expression of the trk family of neurotrophin receptors in developing and adult dorsal root ganglion neurons

Expression of trk receptors is a major determinant of neurotrophin responsiveness of sensory neurons. Although it has been apparent for some time that subpopulations of dorsal root and trigeminal ganglion neurons respond in vitro to each of the members of the neurotrophin family, the extent to which functionally distinct subclasses of sensory neurons are dependent on the actions of different neurotrophins for their development and function remains an active area of investigation. One step towards elucidating the role of various neurotrophins in development and function of sensory neurons has been to examine the distribution of trk receptors on sensory neurons. These studies have clearly revealed that members of the trk family are differentially expressed in functionally distinct populations of both developing and mature sensory neurons and, further, have provided evidence consistent with a shift in neurotrophin responsiveness during the development of sensory neurons.     

Transgenic mice expressing the intracellular domain of the p75 neurotrophin receptor undergo neuronal apoptosis

We have asked whether p75(NTR) may play a role in neuronal apoptosis by producing transgenic mice that express the p75(NTR) intracellular domain within peripheral and central neurons. These animals showed profound reductions in numbers of sympathetic and peripheral sensory neurons as well as cell loss in the neocortex, where there is normally little or no p75(NTR) expression. Developmental loss of facial motor neurons was not observed, but induced expression of the p75(NTR) intracellular domain within adult animals led to increased motor neuron death after axotomy. Biochemical analyses suggest that these effects were not attributable to a p75(NTR)-dependent reduction in trk activation but instead indicate that the p75(NTR) intracellular domain may act as a constitutive activator of signaling cascades that regulate apoptosis in both peripheral and central neurons.     

Regulation of neurotrophin receptor expression by retinoic acid in mouse sympathetic neuroblasts

We have studied the effect of retinoic acid on the expression of the neurotrophin receptors trkA, trkC, and p75 by neuroblasts and neurons at different axial levels along the embryonic mouse paravertebral sympathetic chain. In dissociated cultures of sympathetic neuroblasts, retinoic acid inhibited the developmental increase in trkA mRNA expression and the developmental decrease in trkC mRNA expression that normally occurs in these cells but did not affect p75 mRNA expression. At higher concentrations, retinoic acid also increased the proliferation of sympathetic neuroblasts. After sympathetic neuroblasts became postmitotic, retinoic acid no longer affected receptor expression. Studies with retinoic acid receptor agonists and antagonists indicated that the effects of retinoic acid on neurotrophin receptor expression were mediated mainly by alpha retinoic acid receptors, not beta or gamma receptors. The observation that alpha-antagonists increased trkA mRNA expression in intact sympathetic ganglion explants suggests that endogenous retinoic acid is a physiological regulator of trkA receptor expression.     

Neurotrophins regulate sequential changes in neurotrophin receptor expression by sympathetic neuroblasts

We have examined the mechanisms controlling the induction of the two NGF receptors, trkA and p75, in proliferating neuroblasts immuno-isolated from thoracolumbar embryonic sympathetic ganglia. Contrary to prior studies, we find that induction of p75 follows rather than precedes that of trkA; this late induction is consistent with the fact that p75 functions at relatively late stages of sympathetic development. trkA induction is apparently not controlled by a cell-intrinsic mechanism. Rather, this receptor is induced by environmental signals including NT-3, which also acts as an interim survival factor for these neuroblasts. trkA induction by NT-3 is consequent to its promotion of mitotic arrest, as anti-mitotic drugs also efficiently induce trkA. p75 expression is induced in trkA-expressing cells by NGF. Thus, the development of sympathetic neurons involves sequential actions of different neurotrophins, which also regulate the expression of their own and each other's receptors.     

The distribution of neurotrophin receptor TrkC-like immunoreactive fibers and varicosities in the rhesus monkey brain

The distribution of immunoreactivity for the neurotrophin receptor tyrosine kinase TrkC was examined in the brain of the adult rhesus monkey. TrkC-like immunoreactivity was widespread and consisted primarily of varicose fibers. The most dense populations of fibers were in the basal forebrain (in the cholinergic cell groups Ch1, Ch2 and Ch4), in the raphé complex throughout its rostrocaudal extent, and in the locus coeruleus. Other fibers were present in the thalamus, hypothalamus, central gray matter of the midbrain, dorsal midline of the brainstem and the cerebral cortex. The only neuronal cell bodies with consistent labeling were located in the lateral hypothalamus. Purkinje cells in the cerebellum showed variable labeling. Specific labeling of varicosities and cell bodies was abolished by omission of the primary antiserum or by preabsorption with the TrkC peptide antigen. We conclude that TrkC-like immunoreactivity can be detected in a wide variety of subcortical locations in the adult rhesus monkey. Labeling was particularly prominent in the vicinity of the major cholinergic, serotonergic and adrenergic nuclei, known from other studies to be vulnerable in the ageing brain. This suggests that the ligand for TrkC, neurotrophin-3, may persist as a survival factor for critical neurons into adulthood.     

Neurotrophins inhibit major histocompatibility class II inducibility of microglia: involvement of the p75 neurotrophin receptor

Major histocompatibility complex (MHC) molecules are rare in the healthy brain tissue, but are heavily expressed on microglial cells after inflammatory or neurodegenerative processes. We studied the conditions leading to the induction of MHC class II molecules in microglia by using explant cultures of neonatal rat hippocampus, a model of interacting neuronal networks. Interferon-gamma (IFN-gamma)-dependent MHC class II inducibility in microglia cells was very low, but strongly increased in the hippocampal slices after the blockade of neuronal activity by neurotoxins [tetrodotoxin (TTX), omega-conotoxin] or glutamate antagonists. None of these agents acted directly on isolated microglia cells. We found that neurotrophins modulate microglial MHC class II expression. MHC class II inducibility was enhanced by neutralization of neurotrophins produced locally within the cultured tissues and was inhibited by the addition of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT3). NGF and, to a lower extent, NT3 acted directly on isolated microglia via the p75 neurotrophin receptor and inhibited MHC class II inducibility as shown by blockade of the p75 neurotrophin receptor with antibodies. Our data suggest that neurotrophins secreted by electrically active neurons control the antigen-presenting potential of microglia cells, and indicate that this effect is mediated partly via the p75 neurotrophin receptor.     

NGF induces apoptosis in a human neuroblastoma cell line expressing the neurotrophin receptor p75NTR

Galactocerebroside (GalC) and sulfatide are major constituent lipids in vertebrate myelin. Their precise immunolocalization in electron microscopy so far has been hampered by the fact that lipids are not immobilized by chemical fixation and thus get extracted during dehydration with organic solvents. Here, we examined the suitability of cryotechniques for the preservation and immunohistochemical localization of myelin glycolipids in rat brain at the ultrastructural level. Native cerebral cortex tissue, obtained by fine-needle biopsy, was cryoimmobilized by high-pressure freezing and dehydrated by freeze-substitution before embedding in Epon. This procedure resulted in an excellent preservation of brain ultrastructure. Concomitantly, immunogold labeling of ultrathin sections with the well-defined monoclonal antibodies (mAbs) O1, O4, and R-mAb, which were shown to react with GalC and/or sulfatide and some structurally related glycolipids, revealed a good conservation of relevant epitopes. These data suggest that in adult rat cerebral cortex, the most relevant antigens recognized by R-mAb, O1, and O4, namely GalC and sulfatide, are exclusively expressed in myelin structures. Because these mAbs are common markers for the identification of developing oligodendrocytes, this "postembedding glycolipid-labeling technique" holds great potential for studying oligodendroglial differentiation in normal and pathological conditions at the ultrastructural level.     

The IgG Fc receptor family

IgG immune complexes are of central importance in the humoral immune system and strongly implicated in the pathogenesis of hematologic and rheumatic autoimmune disorders. Cross-linking of receptors for the Fc domain of IgG antibodies (FcgammaRs) triggers a wide variety of effector functions including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. In this way, FcgammaR provide an essential feedback between the humoral and cellular immune response. In the past, significant advances have been made in the molecular dissection of FcgammaR function using cellular transfection systems. Current approaches designed to target and change individual FcgammaR genes in mice have given further insight into their specific contributions to systemic processes, also indicating them to be important immunoregulatory receptors involved in various disease states of allergy, autoimmunity, and inflammation. Future work on targeting FcgammaR binding sites in combination with humanized FcgammaR mouse models will lead to novel therapeutic strategies in the treatment of IgG-mediated human disease in which FcgammaR activation plays an integral part.     

Testing the validity of an instrument for assessing family of origin history

Family of origin history information is often gathered for research and clinical purposes in psychology, but there has been relatively little examination of the validity of the data obtained. This report describes several tests of the validity of the recently developed Family Background Questionnaire, a relatively behaviorally specific instrument designed to comprehensively assess characteristics of one's family of origin. Using a sample of 678 nonclinical and clinical participants, it was found that a history of incest, parental chemical dependency, clinical status, socioeconomic status, and birth order all predicted Family Background Questionnaire scores as hypothesized and that 38% of the variance in Total Scale scores was accounted for by these variables.     

Early ambulatory experience in the undergraduate education of family physicians

A number of benefits have been claimed for early ambulatory experience in the training of family physicians, although few practical examples have been reported. This paper describes an approach to the education of first and second year medical students interested in family medicine which places heavy emphasis on community-based ambulatory care. During the first year, an elective introductory preceptorship permits students to participate in the office practice of a family physician in a limited role. Seminars are offered concurrently to provide integrating principles and perspective. In the second year, a nine-month-long continuity clerkship is offered in which students gain intimate contact with a small panel of families and practice the skills of primary care in the offices of family physicians. Clinical experience is accompanied by weekly seminars and integrated with elements of the required curriculum. Selected evaluation data are presented, which attest to the achievement of course objectives and provide support for the claim that this approach is beneficial to students seeking careers in primary care.     

Expression of the low affinity neurotrophin receptor, P75NGFR, in the rat forebrain, following unilateral bulbectomy

It has been hypothesized that the main olfactory bulb, with its relatively rich source of neurotrophins, may provide trophic support for neurons that project to the bulb. We monitored expression of the common, low affinity receptor for neurotrophins, p75NGFR, in the olfactory bulb and basal forebrain of unilaterally bulbectomized and sham-treated rats, 1-16 weeks post-surgery, using the monoclonal antibody MAb192. An induction of p75NGFR-immunoreactivity was observed in both the glomerular and olfactory nerve layers of the right, contralateral main olfactory bulb of lesioned animals. The naturally occurring regeneration taking place in the olfactory neuroepithelium is known to be altered by olfactory bulbectomy, with subsequent changes in the sensory input to the remaining bulb. These changes in expression of p75NGFR in the olfactory bulb support the hypothesis we have developed in previous papers, that changes in the extent of the peripheral input from the olfactory neuroepithelium to the main olfactory bulb regulate p75NGFR expression in both the glomerular and the olfactory nerve layers. Expression of p75NGFR in the basal forebrain of bulbectomized animals was found to be no different than sham-treated controls and does not support the hypothesis that the olfactory bulb provides trophic support to this region of the central nervous system.     

Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF)

Nerve growth factor (NGF) is the prototype of a family of neurotrophins that support important neuronal programs such as differentiation and survival of a subset of sympathetic, sensory, and brain neurons. NGF binds to two classes of cell surface receptors: p75LANR and p140TrkA. NGF binding to p140TrkA initiates the neuronal signaling pathway through activation of the tyrosine kinase activity, which subsequently results in a rapid signal transduction through a phosphorylation cascade. To examine this crucial signaling step in more detail, the TrkA extracellular domain polypeptide (TrkA-RED) was overexpressed in Sf21 insect cells and purified to homogeneity. The recombinant TrkA-RED is a 70 kDa acidic glycoprotein with a pI of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, Kd, of 2.9 nM. Thus, the recombinant TrkA-RED is functionally competent and can be used to elucidate the interaction of NGF and TrkA receptor. Circular dichroism difference spectra indicated that, upon association of NGF with TrkA-RED, a minor conformational change occurred to form a complex with decreased ordered secondary structure. Interaction between NGF and TrkA-RED was also demonstrated by size exclusion chromatography, light scattering, and chemical crosslinking with evidence for formation of a higher molecular weight complex consistent with a (TrkA-RED)2-(NGF dimer) complex. Association and dissociation rates of 5.6 x 10(5) M(-1) s(-1) and 1.6 x 10(-3) s(-1), respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF-induced receptor dimerization concomitant with a small conformational change.