An unusual cause of abdominal pain: spontaneous bilateral adrenal hemorrhage.

Bilateral adrenal hemorrhage (BAH) is a rare condition that can lead to acute adrenal insufficiency and death if not recognized and treated promptly. We report the case of a 30-year-old male who presented to the emergency department with acute abdominal pain, nausea, and vomiting. On emergency room admission, the first abdominal CT revealed normal adrenal glands without enlargement, but with the development of hypotension and hypoglycemia, a second CT performed four days later showed enlargement due to hemorrhage in both adrenals. The diagnosis of BAH associated with acute adrenal insufficiency was retained. Prompt treatment with intravenous and oral corticosteroids resulted in successful conservative management. We describe the clinical, biological, radiological and etiological features of this condition based on a review of the literature.

Binding of brain-derived neurotrophic factor to the nerve growth factor receptor.

The neurotrophic proteins BDNF and NGF are related in their primary structures, and both have high- and low-affinity receptors on their responsive neurons. In this study, we investigate the extent to which these receptors can discriminate between BDNF and NGF. We found that a 1000-fold excess of the heterologous ligand is needed to reduce binding to the high-affinity receptor by 50%, but that the same concentrations of BDNF and NGF similarly reduce the binding of either ligand to the low-affinity receptor. Results obtained with cells transfected with the low-affinity NGF receptor gene indicate that these cells bind BDNF, in addition to NGF, whereas cells before transfection do not. These data indicate that the low-affinity NGF receptor is also a low-affinity BDNF receptor and that whatever is conferring high-affinity binding and biological response also considerably reinforces the ability of the low-affinity receptor to discriminate between NGF and BDNF.

Altered balance of glutamatergic/GABAergic synaptic input and associated changes in dendrite morphology after BDNF expression in BDNF-deficient hippocampal neurons.

Cultured neurons from bdnf-/- mice display reduced densities of synaptic terminals, although in vivo these deficits are small or absent. Here we aimed at clarifying the local responses to postsynaptic brain-derived neurotrophic factor (BDNF). To this end, solitary enhanced green fluorescent protein (EGFP)-labeled hippocampal neurons from bdnf-/- mice were compared with bdnf-/- neurons after transfection with BDNF, bdnf-/- neurons after transient exposure to exogenous BDNF, and bdnf+/+ neurons in wild-type cultures. Synapse development was evaluated on the basis of presynaptic immunofluorescence and whole-cell patch-clamp recording of miniature postsynaptic currents. It was found that neurons expressing BDNF::EGFP for at least 16 h attracted a larger number of synaptic terminals than BDNF-deficient control neurons. Transfected BDNF formed clusters in the vicinity of glutamatergic terminals and produced a stronger upregulation of synaptic terminal numbers than high levels of ambient BDNF. Glutamatergic and GABAergic synapses reacted differently to postsynaptic BDNF: glutamatergic input increased, whereas GABAergic input decreased. BDNF::EGFP-expressing neurons also differed from BDNF-deficient neurons in their dendrite morphology: they exhibited weaker dendrite elongation and stronger dendrite initiation. The upregulation of glutamatergic synaptic input and the BDNF-induced downregulation of GABAergic synaptic terminal numbers by postsynaptic BDNF depended on tyrosine receptor kinase B activity, as deduced from the blocking effects of K252a. The suppression of dendrite elongation was also prevented by block of tyrosine receptor kinase B but required, in addition, glutamate receptor activity. Dendritic length decreased with the number of glutamatergic contacts. These results illuminate the role of BDNF as a retrograde synaptic regulator of synapse development and the dependence of dendrite elongation on glutamatergic input.

Neurotrophin receptors.

The neurotrophins are members of a family of four related proteins that allow the survival and differentiation of specific sub-sets of embryonic vertebrate neurons. On neurons, two types of neurotrophin receptors can be distinguished on the basis of their dissociation constants: low affinity receptors (Kd 10(-9) M) and high affinity receptors (Kd 10(-11) M). Several genes coding for neurotrophin receptors have been cloned and the expression in fibroblasts of the recombinant membrane proteins allows comparisons to be made between the binding properties of the neurotrophins on such cell lines and neurons. As a result, it appears that much of the low affinity binding sites detected on neurons for all neurotrophins can be attributed to a single molecular entity, the low affinity neurotrophin (or NGF) receptor. This receptor binds all known neurotrophins with similar affinity but different binding kinetics. Its role in neurotrophic signal transduction remains to be established. In addition to this receptor, three members of the trk-subfamily of tyrosine kinase receptors have recently been identified as receptors for the neurotrophins. These receptors (whose intrinsic tyrosine kinase activity can be stimulated by the various neurotrophins) bind the neurotrophins with higher affinity and higher ligand specificity when compared with the low affinity receptor. However, the observation has been made that some of the recombinant trk-receptors on cell lines bind more than one neurotrophin (though typically with lower affinity than their own ligands).(ABSTRACT TRUNCATED AT 250 WORDS)

Final steps of the maturation of Omp F, a major protein from the outer membrane of Escherichia coli.

Pulse-labelling experiments with E. coli cells allowed us to follow the incorporation of de novo proteins into the outer membrane of the cell envelope. Labelled membrane samples containing increasingly different levels of newly synthesized Omp F protein were subjected to chemical cross-linking with a bifunctional cleavable reagent in order to investigate the process of trimer formation of the protein. From the results obtained, we conclude that the formation of functional Omp F trimers is substantially delayed to, and can be distinguished from, the incorporation of Omp F monomers to the outer membrane.

An unusual linkage between polysaccharides and some major proteins from the outer membrane of Escherichia coli.

A small population of OmpA, a major protein from the outer membrane of Escherichia coli, was found covalently associated with either lipopolysaccharide or O-antigen polysaccharide. Radioactive oligosaccharide was elicited linked to OmpA after treatment of the membranes with periodate that hydrolyzed large sugars. Association of saccharides to OmpA could be enhanced by treatment of the outer membrane with NaBH4.

Role of neurotrophins in the control of neural development: neurotrophin-3 promotes both neuron differentiation and survival of cultured chick retinal cells.

The effects of neurotrophins brain-derived neurotrophic factor and neurotrophin-3 on cultured dissociated cells from chick retina were studied at several embryonic ages from day 4 to day 13. Precursor cells from days 4-7 retinas proliferated in vitro and, after 20 h in culture, a proportion of them underwent spontaneous differentiation, as judged by both [3H]thymidine uptake and acquisition of neuronal morphology and neuron-specific markers. Brain-derived neurotrophic factor did not affect neuronal differentiation, although this factor supports survival of differentiated retinal ganglion cells [Rodríguez-Tébar et al. (1989) Devl Biol. 136, 296-303]. However, in cultures from young undifferentiated retinas, neurotrophin-3 produced up to a 2.5-fold increase in the number of [3H]thymidine-positive neurons, i.e. those that in vitro replicated their DNA. Moreover, in older retinas, neurotrophin-3, like brain-derived neurotrophic factor, supported the survival of differentiated retinal ganglion cells over a short developmental period. This effect was negligible at embryonic day 5, maximal at day 9, decreased at day 11 and was absent at embryonic day 13. Neurotrophin-3 also supported the survival of a population of amacrine neurons. This effect was modest at embryonic day 9, and increased at days 11 and 13. Our results show that, whereas the action of brain-derived neurotrophic factor is restricted to differentiated neurons, neurotrophin-3 exerts two distinct successive actions on retinal cells in vitro: first, this factor promotes either differentiation of neuroepithelial cells or maturation of recently differentiated neurons, and later in development, this factor supports the survival of differentiated retinal ganglion and amacrine cells but only during a discrete post-differentiation period.

Developmentally regulated vitronectin influences cell differentiation, neuron survival and process outgrowth in the developing chicken retina.

Vitronectin is a multifunctional protein involved in the regulation of the immune system and blood coagulation. Here we report that the expression of vitronectin is developmentally regulated in the embryonic retina of the chicken. Vitronectin immunoreactivity was detected in chicken retinas from embryonic day 5, encompassing the cell bodies of most neuroepithelial cells. At this developmental stage, alpha v integrin subunit expression was distributed across the retina, suggesting a ligand/receptor interaction. Expression of both vitronectin and alpha v increased during development and reached a maximum at embryonic day 9, a time when most differentiated neurons grow processes and initiate synapse formation. At this age, vitronectin immunoreactivity appeared to be located predominantly in the fiber and inner plexiform layers of the differentiated stratified retina. alpha v immunoreactivity and mRNA expression was seen associated with all layers formed by differentiated neurons, being most abundant in the ganglion cell and inner nuclear layers. Later in development, levels of vitronectin decreased and immunoreactivity appeared exclusively associated with the fiber layer. In accordance with this pattern of expression, vitronectin as a substrate sustained both proliferation and differentiation of cultured neuroepithelial cells from embryonic day 5 retinas. At later stages, vitronectin supported survival and neurite outgrowth of most differentiated neurons. Our data suggest that vitronectin is a ubiquitous component of the retinal extracellular matrix, serving as a substrate for developmental processes such as proliferation, differentiation of neuron progenitors, cell survival, and axonal and dendritic growth of differentiated neurons.

An abnormal response of retinoblastoma cells (Y-79) to neurotrophins.

PURPOSE: To clarify the expression of neurotrophins and their receptors in retinoblastoma (Rb) cells, to elucidate their potential role in the proliferation of neuroectodermal tumor cells, and to establish conditions for Rb cell differentiation. METHODS: The Rb-derived cell line Y-79 was grown in serum-free suspension or monolayer culture. Proliferating and differentiated cells were isolated and submitted to semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, immunostaining, and flow cytometry. The proliferation rate of the cells was estimated by 5-bromo-2'-deoxyuridine (BrdU) incorporation, and the effects of neurotrophins and laminin on BrdU-incorporation, process outgrowth, or immunostaining were determined. RESULTS: In contrast to previously studied normal retinal precursor cells, Y-79 cells not only express nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and p75, but also the corresponding high affinity receptors TrkA, TrkB, and TrkC. Proliferation was stimulated by exogenous and endogenous neurotrophin receptor ligands. Inhibition of protein kinase phosphorylation with K252a blocked proliferation and promoted differentiation. The effect of K252a on differentiation was enhanced by the addition of soluble laminin. After 9 days of combined treatment, the fraction of differentiated cells amounted to 30%, differentiation being characterized by improved attachment, neurite outgrowth, expression of NF-68, and a loss of glial fibrillary acidic protein (GFAP) and parvalbumin immunoreactivity. These changes were accompanied by a downregulation of TrkB and TrkC, but not TrkA or p75. Differentiated cells were isolated and further grown in the absence of K252a. However, despite the high level of TrkA expression in differentiated cells, the addition of NGF had no effect on their survival. CONCLUSIONS: A mitogenic action of neurotrophins could contribute to retinal tumor growth.

Neurotrophins and other growth factors in the generation of retinal neurons.

The generation of neurons in the vertebrate retina, as in other areas of the developing nervous system, largely depends on extracellular signals. Of the known signaling molecules, neurotrophins play decisive, defined, and distinct roles. The three neurotrophins identified in the chick, namely, neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF), are expressed in either the pigment epithelium (NT-3 and BDNF) or in the neural retina (NGF) at the onset of neuron birth. In addition, trkC and trkB, receptors for NT-3 and BDNF, respectively, together with p75, the low-affinity neurotrophin receptor, are expressed in the retina at the same developmental period. The role of these three neurotrophins in the differentiation of neurons in the chick retina has been elucidated by a combination of in vitro and in vivo experiments. Thus, NT-3 promotes the conversion of neuroepithelial cells into neurons, whereas BDNF and NGF control the programmed cell death (apoptosis) that affects early postmitotic neuroblasts. BDNF, acting via its trkB receptor, is a survival factor for these cells, whereas NGF, binding to p75 receptor, acts as a killing factor, thereby controlling the provisional number of newly generated neurons.

Expression of depolarizing voltage- and transmitter-activated currents in neuronal precursor cells from the rat brain is preceded by a proton-activated sodium current.

The early expression of amiloride-sensitive proton-activated sodium currents (INa(H] was demonstrated using the giga-seal whole-cell voltage clamp technique in cells from the primordial tectum of E12 rat embryos. Less than 10% of these cells stained for tetanus toxin receptors after 2 h in vitro. However, after 10 h in vitro all cells with neuronal geometry were tetanus toxin-positive and capable of generating voltage-activated Na currents (INa(V] and high-voltage activated Ca2+-currents (ICa(HV]. INa(H) was expressed roughly in parallel with INa(V) and ICa(HV), but exceeded the former currents in amplitude by 50-100 times, reaching 600 pA and more. In 25% of the cells tested within the first 5 h in vitro INa(H) was, in fact, the only cationic inward current resolved. Responses to quisqualate and kainate appeared only after 3 days in vitro, and responses to N-methyl-D-aspartate/glycine were seen only after 4 days in vitro. These results suggest that the channels carrying INa(H) are present at the earliest stages of neuronal development.

Structural modification of Escherichia coli peptidoglycan induced by bicyclomycin.

We have studied the modification of Escherichia coli peptidoglycan induced by bicyclomycin. For this purpose liquid chromatography for peptidoglycan analysis has been used. The main alteration found was an increase of diaminopimelyl-diaminopimelyl bridge containing subunits. Our results show that bicyclomycin impairs the normal breakage of that interpeptidic bond, whose cleavage is needed for the normal remodeling of peptidoglycan and cell growth. Based on the analysis of the possible structure of diaminopimelyl-diaminopimelyl bond and bicyclomycin, we propose a hypothesis on the mechanism of action of bicyclomycin.

Thermochemistry of the interaction between peptides and vancomycin or ristocetin.

The thermodynamics of the interaction between the glycopeptide antibiotics vancomycin and ristocetin and bacterial peptidoglycan peptide analogs have been studied by means of a microcalorimetric titration technique. From results of the calorimetric measurements, changes in Gibbs energies, enthalpies, entropies and heat capacities for the binding reactions have been calculated. The derived thermodynamic data have been discussed on the basis of stereochemical data available for the interaction of acetyl-D-alanyl-D-alanine with each of the two antibiotics. The significance of entropic factors connected with conformational changes of the antibiotics is stressed.

Retinoic acid induces NGF-dependent survival response and high-affinity NGF receptors in immature chick sympathetic neurons.

An important step in the development of peripheral sensory and sympathetic neurons is the onset of the survival response and dependence on the presence of nerve growth factor (NGF) or other neurotrophic factors. We have recently observed that immature sympathetic neurons from 7-day-old chick embryos are unable to become NGF-responsive in vitro and we have now used these cells to identify molecules that induce NGF-dependent neuronal survival. We found that retinoic acid (RA) induces the ability of these cells to survive in the presence of NGF. At RA concentrations of 10(-9)-10(-8)M virtually all neurons survived in the presence of NGF. RA was found to also induce the biologically active, high-affinity NGF receptor: high-affinity receptors were undetectable on dissociated E7 sympathetic neurons and were observed in vitro only in RA-treated neurons. These findings suggest that the induction of high-affinity NGF receptors may be sufficient to activate the survival response in sympathetic neurons and imply an important role for RA during neuron differentiation in the peripheral nervous system.

Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo.

Brain-derived neurotrophic factor (BDNF), a protein known to support the survival of embryonic sensory neurons and retinal ganglion cells, was derivatized with 125I-Bolton-Hunter reagent and obtained in a biologically active, radioactive form (125I-BDNF). Using dorsal root ganglion neurons from chick embryos at 9 d of development, the basic physicochemical parameters of the binding of 125I-BDNF with its receptors were established. Two different classes of receptors were found, with dissociation constants of 1.7 x 10(-11) M (high-affinity receptors) and 1.3 x 10(-9) M (low-affinity receptors). Unlabeled BDNF competed with 125I-BDNF for binding to the high-affinity receptors with an inhibition constant essentially identical to the dissociation constant of the labeled protein: 1.2 x 10(-11) M. The association and dissociation rates from both types of receptors were also determined, and the dissociation constants calculated from these kinetic experiments were found to correspond to the results obtained from steady-state binding. The number of high-affinity receptors (a few hundred per cell soma) was 15 times lower than that of low-affinity receptors. No high-affinity receptors were found on sympathetic neurons, known not to respond to BDNF, although specific binding of 125I-BDNF to these cells was detected at a high concentration of the radioligand. These results are discussed and compared with those obtained with nerve growth factor on the same neuronal populations.

Penicillin-binding proteins and peptidoglycan peptide-interacting proteins.

Peptidoglycan peptide-interacting proteins (PPIPs) which include the penicillin-binding proteins (PBPs) are bacterial enzymes which interact with beta-lactams (including penicillins and cephalosporins) and/or acyl-D-ala-D-ala analogues of a non-beta-lactam structure. The PBPs and other PPIPs of Escherichia coli are reviewed.

Neurotrophins: structural relatedness and receptor interactions.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are structurally related proteins that allow the survival of specific populations of embryonic vertebrate neurons. The primary structure of these neurotrophins, deduced from their nucleotide sequences, indicates that all three are synthesized in the form of precursor proteins presumably allowing for appropriate folding, including the formation of disulphide bridges, cleavage and secretion. While no information is yet available on the 3-dimensional structures of the neurotrophins, results from binding studies using the three neurotrophins as ligands indicate that their receptors do recognize similarities, as well as differences, between them. High-affinity receptors, that presumably mediate the biological response, as well as low-affinity receptors are present on neurons responsive to the neurotrophins. Whereas a large excess of heterologous ligand is needed to reduce binding of a particular neurotrophin to its high-affinity receptor, the same concentration of homologous or heterologous ligand similarly reduce the binding of any of the three neurotrophins to the low-affinity receptor. For all three, the low-affinity receptor appears to be the already characterized NGF low-affinity receptor that seems to be an integral part of the high-affinity receptor complexes. These results suggest that the regulation of neuronal survival by target cells can, in part, be explained by the release from these cells of limiting quantities of the structurally related neurotrophins, each being recognized by a specific high-affinity receptor complex located on the nerve terminals of the responsive neurons.

Location of some proteins involved in peptidoglycan synthesis and cell division in the inner and outer membranes of Escherichia coli.

Inner and outer membranes of Escherichia coli were separated by isopycnic centrifugation in sucrose gradients and analyzed for the presence of penicillin-binding proteins. All penicillin-binding proteins--except penicillin-binding protein 3, which is found almost exclusively in the cytoplasmic membrane and is involved in septum formation--are also found in gradient fractions corresponding to the outer membrane. Our results support the hypothesis that approximately half of the total amount of penicillin-binding proteins may be sacculus-located proteins linked to the outer membrane, probably through peptidoglycan bridges.