Biomedical subjects
M Gelman
Publications and source records attributed to M Gelman.
Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms.
In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues ('boxes' 6 and 7), located on either side of Tyr(397), which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr(397), a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr(397) independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr(397) in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr(397). They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.
Identification of an opioid peptide secreted by rat embryonic mixed brain cells as a promoter of macrophage migration.
Conditioned media from embryonic mixed cells from the rat brain were used in a chemotaxis assay to look for potential chemotactic activity which could account for the infiltration of the developing central nervous system (CNS) by macrophage precursors. The most potent chemotactic activity was found in the conditioned medium from E17 mixed brain cells (E17-CM). Based upon checkerboard analysis, this activity was shown to be chemotactic rather than chemokinetic. This chemoattraction was not restricted to brain macrophages (BM) because it was as pronounced on bone marrow-derived macrophages. The implication of a peptide compound in this activity was suggested by its resistance to heat as well as acid treatments, and by its sensitivity to aminopeptidase M digestion. In agreement with the opioid nature of the peptide, not only naloxone, but also the delta opioid receptor antagonist ICI-174 reduced the migration of BM in response to E17-CM by 60%. This migratory activity was no longer effective when pertussis toxin-treated BM were used. When the chemotactic effects of selective opioid agonists were compared to that of E17-CM, DPDPE, the delta agonist, was the most efficient in attracting BM. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that delta as well as other known opioid receptors were expressed in both BM and E17 mixed brain cells. Finally, a Met-enkephalin-like reactivity was found by RIA in the E17-CM. Altogether, these observations suggest that a delta-like opioid peptide released from embryonic mixed brain cells could be responsible for the infiltration of the developing CNS by macrophages precursors.
Ventilator-associated pneumonia in very low-birth-weight infants at the time of nosocomial bloodstream infection and during airway colonization with Pseudomonas aeruginosa.
PURPOSE: To study retrospectively the incidence of ventilator-associated pneumonia (VAP) at the time of Pseudomonas aeruginosa nosocomial bloodstream infection (BSI) and at the time of P aeruginosa airway colonization. MATERIALS AND METHODS: Fifteen very low-birth-weight infants who had P aeruginosa BSI and 33 others who did not but who had P aeruginosa airway-colonization were studied. We correlated clinical data, blood cultures (BCs), and tracheal cultures (TCs) with radiologic findings from radio-graphs taken within 2 days before, the day of, and 1 day after BCs or TCs were first positive for P aeruginosa. Chest radiographs were graded by using semiquantitative scores for bronchopulmonary dysplasia and for pneumonia. RESULTS: Mean birth weight, gestational age, and age when BC or TC became positive were similar for patients with BSI and colonization. At the time of BSI, 2 infants had airway colonization with P aeruginosa; the TCs of the remaining 13 grew P aeruginosa as a new pathogen. Thirteen of 15 patients with BSI, but none of 33 infants with colonization, died within 2 days of positive BC. VAP was diagnosed in 13 of 15 patients with BSI and in 3 of 33 infants with colonization. CONCLUSION: Mechanically ventilated very low-birth-weight infants whose TCs yield P aeruginosa but whose BCs remain negative infrequently have VAP are presumed airway-colonized and are expected to survive. Conversely, VAP is likely to be found when BCs and TCs simultaneously grow P aeruginosa, and high mortality is anticipated.
Brain tumors: complications of cerebral angiography accompanied by intraarterial chemotherapy.
PURPOSE: To evaluate the rate of complications associated with diagnostic cerebral angiography accompanied by intraarterial chemotherapy for the treatment of primary and metastatic brain tumors. MATERIALS AND METHODS: Three hundred ninety-two consecutive transfemoral cerebral angiographic procedures accompanied by intraarterial chemotherapy were performed in 48 patients (28 men, 20 women), and complications were evaluated. RESULTS: The most common local complications were groin hematomas, which occurred in 10 (2.6%) of the 392 procedures and none of which required therapy. Two carotid arterial dissections (0.5%) were reported in two patients who were asymptomatic and did not require further treatment. Both improved at follow-up examinations. Only one patient required surgery for a delayed popliteal embolus. Systemic transient complications occurred five times (1.3%). There were seven (1.8%) transient neurologic events, which were paresis and visual disturbances. Six (1.5%) transient seizure events were recorded. There were no permanent neurologic complications. CONCLUSION: Intraarterial chemotherapy for brain tumors is a safe procedure with a low complication rate.
Focal air trapping in patients with HIV infection: CT evaluation and correlation with pulmonary function test results.
OBJECTIVE: HIV-positive individuals commonly have symptoms of airway disease. We evaluated thin-section CT scans of HIV-infected individuals during inspiration and expiration for evidence of focal air trapping. We also correlated imaging findings with pulmonary function test results. SUBJECTS AND METHODS: Fifty-nine subjects, 48 of whom were HIV-positive and 11 of whom were HIV-negative, underwent thin-section CT of the thorax during inspiration and expiration. All subjects also underwent pulmonary function tests. Two radiologists, who were unaware of the subjects' HIV status and smoking history and of the results of pulmonary function tests, evaluated the CT scans for the presence and severity of focal air trapping. RESULTS: Expiratory CT revealed focal air trapping in 33 subjects: 30 were HIV-positive and three were HIV-negative (p = .0338). The mean values of forced expiratory volume in 1 sec (FEV1), forced mid expiratory flow, and diffusion capacity (DL(CO)) were significantly lower for subjects with focal air trapping (mean = 88.85, 84.52, and 80.80, respectively) than for those with normal findings on CT (mean = 100.84, 99.24, and 95.82, respectively; p = .001, p = .021, and p = .003, respectively). We found no significant differences in smoking history between HIV-positive and HIV-negative subjects. Severe air trapping on expiratory CT scans was seen in three subjects: All three had HIV infection, low CD4 counts, and abnormally decreased FEV1 and DL(CO) values. CONCLUSION: Focal air trapping was a common finding on thoracic CT scans obtained during expiration in HIV-positive subjects. In addition, focal air trapping was associated with significantly lower FEV1, forced mid expiratory flow, and DL(CO) values than those found for subjects in whom CT revealed no focal air trapping. These results suggest that small airways disease may accompany a decline in pulmonary function in HIV-positive individuals.
A 21-kDa polypeptide belonging to a new family of proteins is expressed in the Golgi apparatus of neural and germ cells.
We have isolated a full-length murine clone corresponding to the rat neuronal p1A75 partial cDNA (Sutcliffe, J. G., Milner, R. J., Shinnick, T. M., and Bloom, F. E. (1983) Cell 33, 671-682). It encodes a 185-residue polypeptide that displays 56% identity with p19, a protein selectively expressed in the Golgi apparatus of neural cells (Sabéran-Djoneidi, D., Marey-Semper, I., Picart, R., Studler, J.-M., Tougard, C., Glowinski, J., and Lévi-Strauss, M. (1995) J. Biol. Chem. 270, 1888-1893). An antibody directed against the recombinant polypeptide allowed us to demonstrate the existence of the natural 21-kDa protein (p21) in brain and its prominent juxtanuclear Golgi-like localization in cultured neurons. Ultrastructural observation of cultured neurons and analysis of transfected COS cells revealed a specific labeling of the Golgi apparatus, suggesting, as for p19, the presence of a Golgi targeting signal in its primary sequence. Surprisingly, p21, which is much more strongly expressed in the olfactory epithelium than p19, is also present in the Golgi complex of spermatocytes and in the flagellar middle piece of late spermatids.
Identification of CSF-1 as a brain macrophage migratory activity produced by astrocytes.
Intraparenchymal migration of macrophages occurs in the CNS during development or as a consequence of tissue injuries. In the present study, we have shown, by using an in vitro chemotaxis assay, that cultured rat astrocytes obtained from the developing cerebral cortex and striatum produce soluble factors, which attract purified brain macrophages. The effect of astrocyte-derived factors on macrophages was strongly reduced in the presence of antibodies neutralizing colony-stimulating factor 1 (CSF-1, also called M-CSF), and recombinant CSF-1 was found to act as a chemotactic agent on brain macrophages. Synthesis of CSF-1 by cultured astrocytes was confirmed by northern detection of CSF-1 transcripts. In contrast, the CSF-1 gene was not expressed by cultured neurons from the cerebral cortex and striatum or by the brain macrophage population responsive to CSF-1 gradient. ELISA detection of CSF-1 in tissue extracts revealed the occurrence of this cytokine in the rat cerebral cortex during postnatal development and in adults. Altogether, our results demonstrate that astrocytes, through CSF-1 secretion, can trigger the polarized migration of brain macrophages and suggest a new mechanism which could regulate the locomotion of these cells in the cerebral cortex during ontogenesis or following lesions.
Alternatively spliced focal adhesion kinase in rat brain with increased autophosphorylation activity.
pp125 focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase transducing signals initiated by integrin engagement and G protein-coupled receptors, is highly expressed in brain. FAK from brain had a higher molecular weight and an increased autophosphorylation activity, than from other tissues. In addition to a 9-base insertion in the 3'-coding region, which defines FAK+, rat striatal FAK mRNAs contained several additional short exons, coding for peptides of 28, 6, and 7 residues, respectively (termed boxes 28, 6, and 7), surrounding the autophosphorylated Tyr-397. In transfected COS 7 cells, the presence of boxes 6 and 7 conferred an increased overall tyrosine phosphorylation, a higher phosphorylation of Tyr-397 assessed with a phosphorylation state-specific antibody, and a more active autophosphorylation in immune precipitates. The presence of box 28 did not alter further these parameters. Two-dimensional phosphopeptide maps of hippocampal FAK were identical to those of FAK+6,7. The presence of the various exons did not alter the interaction of FAK with c-Src, n-Src, or Fyn. Thus, several splice isoforms of FAK are preferentially expressed in rat brain, some of which have an increased autophosphorylation activity, suggesting that FAK may have specific properties in neurons.
Neurons promote macrophage proliferation by producing transforming growth factor-beta2.
The infiltration of bone marrow-derived macrophages into the CNS contributes to growth and reactions of microglia during development or after brain injury. The proliferation of microglial cells is stimulated by colony-stimulating factor 1 (CSF-1), an astrocyte-produced growth factor that acts on mononuclear phagocytes. In the present study, we have shown, using an in vitro model system, that rodent neurons obtained from the developing cerebral cortex produce a soluble factor that strongly enhances the proliferation of macrophages cultured in the presence of CSF-1. Both macrophages isolated from the developing brain and those from the adult bone marrow were stimulated. Kinetic analyses of [3H]thymidine incorporation into macrophages indicated that their response to the neuron-derived factor involved a shortening of the cycle of proliferating cells. The effect of neurons on macrophages was blocked in the presence of antibodies neutralizing transforming growth factor-beta2 (TGF-beta2), whereas recombinant TGF-beta2 stimulated macrophage proliferation in the presence of CSF-1. Neuronal secretion of TGF-beta2 was confirmed by reverse transcription-PCR detection of TGF-beta2 transcripts and immunodetection of the protein within neurons and in their culture medium. In situ hybridization and immunohistochemical experiments showed neuronal expression of TGF-beta2 in sections of cerebral cortex obtained from 6-d-old rats, an age at which extensive developmental recruitment of macrophages occurs in this cerebral region. Altogether, our results provide direct evidence that neurons have the capacity to promote brain macrophage proliferation and demonstrate the role of TGF-beta2 in this neuronal function.
Regulation of a neuronal form of focal adhesion kinase by anandamide.
Anandamide is an endogenous ligand for central cannabinoid receptors and is released after neuronal depolarization. Anandamide increased protein tyrosine phosphorylation in rat hippocampal slices and neurons in culture. The action of anandamide resulted from the inhibition of adenylyl cyclase and cyclic adenosine 3', 5'-monophosphate-dependent protein kinase. One of the proteins phosphorylated in response to anandamide was an isoform of pp125-focal adhesion kinase (FAK+) expressed preferentially in neurons. Focal adhesion kinase is a tyrosine kinase involved in the interactions between the integrins and actin-based cytoskeleton. Thus, anandamide may exert neurotrophic effects and play a role in synaptic plasticity.
Production of monocyte chemotactic protein-1 by rat brain macrophages.
In the present study, we show that cultured rat brain macrophages release a soluble factor that stimulates the migration of bone marrow-derived macrophages, as determined by an in vitro chemotaxis assay. A checkerboard analysis indicated that most of this effect resulted from a polarized migration of the cells (chemotactic phenomenon), rather than in an increase in cell motility (chemokinesis). This activity was significantly decreased by an immune serum directed against the rat monocyte chemoattractant protein-1 (chemokine MCP-1). Northern blot analysis demonstrated expression of the MCP-1 gene in cultured brain macrophages, but its absence in unstimulated bone marrow-derived macrophages. Up-regulation of MCP-1 expression was observed when lipopolysaccharide was added to cultured brain macrophages, a peak occurring after a 6 h period of stimulation. Also, inflammatory cytokines such as interleukin (IL)-1 beta, colony stimulating factor-1, tumour necrosis factor-alpha and IL-6 individually increased the basal level of MCP-1 mRNA. Subsequently, we demonstrated the in vivo production of MCP-1 in the adult rat brain following injury induced by a local injection of kainic acid. MCP-1 synthesis was localized in both astrocytes and brain macrophages. These results suggest that the activation of resting microglial cells into brain macrophages and their subsequent secretion of chemokines could contribute to the mechanism(s), leading to the infiltration of the CNS by blood-derived monocytes, as observed in several pathologies.
A selective toxicity toward cultured mesencephalic dopaminergic neurons is induced by the synergistic effects of energetic metabolism impairment and NMDA receptor activation.
Numerous observations strongly support the hypothesis that dopaminergic neurons could be particularly vulnerable to an impairment of their energetic metabolism. In order to demonstrate the existence of such a selective vulnerability, the toxic effects of rotenone, an inhibitor of complex I of the respiratory chain, and of glutamate, which is very likely involved in the neurotoxicity induced by an energetic stress, were analyzed on cultured mouse mesencephalic neurons. Toxicity toward dopaminergic and GABAergic neurons was compared by measuring the residual uptakes of dopamine and GABA. Exposure to 5 nM rotenone for 6 hr or to a low concentration of glutamate (100 microM) for 1 hr did not lead to a high selective toxic effect on dopaminergic neurons. In contrast, dopaminergic neurons were three times less resistant to the sequential exposure to rotenone and glutamate than GABAergic neurons. A particular resistance of mesencephalic GABAergic neurons to the synergistic toxic effects of rotenone and glutamate was ruled out since two other neuronal types, the striatal cholinergic and GABAergic neurons, displayed the same weak vulnerability as the mesencephalic GABAergic neurons. This selective toxic effect of glutamate on rotenone-pretreated dopaminergic neurons was blocked by either AMPA or NMDA receptor antagonists and mimicked by combined treatment with AMPA and NMDA, or by NMDA alone when the medium was deprived of Mg2+ ions. Moreover, this NMDA-selective neurotoxicity was critically dependent on the presence of a physiological extracellular sodium concentration, since the use of choline chloride instead of sodium chloride had a protective effect on dopaminergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)
Depolarization and neurotransmitters increase neuronal protein tyrosine phosphorylation.
In rat hippocampal slices and in neurons in primary culture, K(+)-induced depolarization increased markedly and rapidly tyrosine phosphorylation of a 110-kDa protein (pp110) and, to a lesser degree, of a 120-kDa protein (pp120), in a calcium-dependent fashion. Glutamate, 1-aminocyclopentane-trans-1,3-dicarboxylic acid (an agonist of metabotropic glutamate receptors), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (an agonist of ionotropic glutamate receptors) stimulated also tyrosine phosphorylation of pp110 and pp120. These effects were not observed in astrocytes in primary culture. In hippocampal slices tyrosine phosphorylation of pp110 and pp120 was stimulated by Ca(2+)-ionophores and by phorbol esters and antagonized by a chelator of intracellular Ca2+ and by drugs that inhibit protein kinase C. Stimulation of muscarinic and alpha 1-adrenergic receptors increased also tyrosine phosphorylation of pp110 and pp120. These results demonstrate that membrane depolarization and stimulation of neurotransmitter receptors activate a tyrosine phosphorylation pathway in neurons. This pathway involves an increase in intracellular Ca2+ concentrations and the activation of protein kinase C. It may provide a biochemical basis for some neurotrophic effects of electrical activity and neurotransmitters and may contribute to the role of tyrosine phosphorylation in long-term potentiation.
Candida albicans spondylodiscitis and vertebral osteomyelitis in patients with intravenous heroin drug addiction. Report of 3 new cases.
We describe 3 patients with a history of intravenous (iv) heroin addiction presenting with indolent, persisting lumbar pain. Clinical findings and initial investigations were unremarkable. Gallium scintigraphy revealed septic spondylodiscitis, and cultures of material obtained by biopsy were positive for Candida albicans in all 3 cases. Two patients were treated with iv amphotericin B and the other with fluconazole with excellent results. Surgical treatment was needed only in one patient because of neurological involvement.
The high sensitivity to rotenone of striatal dopamine uptake suggests the existence of a constitutive metabolic deficiency in dopaminergic neurons from the substantia nigra.
The toxicity of the 1-methyl-4-phenylpyridinium ion (MPP+), an inhibitor of complex I of the respiratory chain, on nigrostriatal dopaminergic neurons contrasts with its relative inefficiency towards other catecholaminergic cell populations in spite of their ability to accumulate this neurotoxin through their high-affinity uptake system. A constitutive metabolic deficiency of the nigrostriatal dopaminergic neurons could account for their particular vulnerability to MPP+. In order to substantiate this hypothesis, we compared the inhibitory effects of rotenone, an inhibitor of mitochondrial oxidative phosphorylation, on the uptake of dopamine, serotonin, noradrenaline and GABA in mouse striatal synaptosomes, and of dopamine, serotonin and GABA in cultured mesencephalic neurons. In both preparations, the uptake of dopamine was much more affected than that of other neurotransmitters by rotenone. This result was confirmed using two other unrelated inhibitors of oxidative phosphorylation. Moreover, dopamine uptake in synaptosomes from the dorsolateral striatum was more sensitive to rotenone than uptake in synaptosomes from the nucleus accumbens. This indicates that intrinsic metabolic properties of the nigrostriatal dopaminergic neurons may explain the strong inhibition by rotenone of striatal dopamine uptake. Altogether, these results suggest that a constitutive metabolic deficiency could account, at least in part, for the selective vulnerability of the nigrostriatal dopaminergic pathway to the action of the neurotoxin MPP+.
[Antitubercular agents: pharmacogenetic factors in the development of adverse effects].
We studied the distribution of the acetylator phenotype in 47 patients aged 15 to 77 years receiving isonyazid as antituberculous therapy. 62% were fast and 32% slow acetylators. Ethnical, socio-economic and biologic factors were not related to acetylator type. The incidence of liver alterations, mainly elevated transaminase levels, was higher than reported in the literature and was not shown to be influenced by acetylator type. Adverse reactions to isonyacid were not related to drug serum levels.