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Biomedical subjects

B Anton

Publications and source records attributed to B Anton.

34 records · Page 2Linked to original sources
PubMed1996

Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.

Animals↗
PubMed1996

Immunohistochemical localization of ORL-1 in the central nervous system of the rat.

A novel member of the opioid receptor family (ORL-1) has been cloned from a variety of vertebrates. ORL-1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N-terminus of ORL-1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II-V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1-CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL-1 was observed in telencephalic areas such as caudate-putamen. In the cerebellum, ORL-1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL-1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL-1 receptor in the rat CNS.

Animals↗
PubMed1996

Expression of the lacZ reporter gene in the rat basal forebrain, hippocampus, and nigrostriatal pathway using a nonreplicating herpes simplex vector.

We recently demonstrated the efficacy of a nonreplicating herpes simplex type 1 virus construct, employing the Moloney murine leukemia virus long terminal repeat promoter, in providing long-term expression of the lacZ gene in rat hippocampal neurons. We now report the utility of this construct in expressing the reporter gene in neurons of the basal forebrain and substantia nigra and examine the spread of the virus to other brain regions. Dorsal and ventrolateral hippocampal formation injection of the virus resulted in numerous beta-gal-expressing cells in the stratum pyramidale, stratum oriens, stratum lacunosum-moleculare, and stratum granulosum. Scattered cells of the medial septum/diagonal band were positively stained following direct injection into this region. More intense staining of the basal forebrain was observed following hippocampal injection as a result of retrograde transport of the virus as shown by PCR analysis of viral DNA. Hippocampal injection also resulted in positive cell staining in several other afferent projection nuclei, namely, the supramammillary bodies, dorsal and caudal linear raphe, and perirhinal/entorhinal cortex. Very few cells were labeled around injection sites in the striatum or substantia nigra. However, substantia nigra zona compacta cells were blue following striatal injection, as were pallidal neurons following nigral injection. These data demonstrate the feasibility of using this virus construct to express foreign genes such as neurotrophic factors in basal forebrain and substantia nigra neurons, taking advantage of retrograde transport of the virus to preserve local anatomy.

Animals↗
PubMed1996

Pentylenetetrazol-induced kindling: early involvement of excitatory and inhibitory systems.

Alterations in the brain of rats receiving a single non-convulsive administration pentylenetetrazol (PTZ), 30 mig/kg, i.p. (single PTZ group) were investigated and compared with those detected in fully PTZ kindled rats (chronic PTZ group). In vitro receptor autoradiography experiments showed that both single and chronic PTZ groups presented mu opioid and benzodiazepine (BDZ) receptor binding in specific brain areas. Using an antibody generated against the delta opioid receptor (DOR-1), it was found that DOR-1 like immunoreactivity was reduced in cortex and amygdala in mice following single and chronic PTZ administration. Microdialysis experiments revealed that the administration of PTZ 30 mg/kg, i.p. in freely moving rats without previous experience with the drug, induces a rise in glutamate release, detected in the first and second 10 min dialysates collected from amygdala (138% and 50%, respectively) and frontal cortex (70% and 45%, respectively) as well as aspartate in frontal cortex in the first and second PTZ-dialysates (143% and 80%, respectively). Subsequently, values returned to basal conditions. It may be speculated that decreased BDZ receptor binding results from enhanced release of GABA. On the other hand, the decrease of mu receptor binding and DOR-1 immunoreactivity observed after PTZ administration may be the result of enhanced levels of opioid peptides probably released over the kindling procedure. In conclusion, the present study indicates that PTZ-kindling is associated with an imbalance between excitatory and inhibitory systems which is apparent early in the epileptogenic process.

Amygdala↗
PubMed1995

Characterization of the murine mu opioid receptor gene.

The analgesic and addictive properties of morphine and other opioid drugs are thought to result from their interaction with mu opioid receptors. Using a delta opioid receptor cDNA as a probe, we have isolated a murine mu opioid receptor cDNA clone (mMOR). Stable expression of mMOR in Chinese hamster ovary cells conferred high binding affinity for mu receptor ligands including morphine and [D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin and low affinity for delta and kappa preferring ligands. Treatment of these cell lines with morphine and other mu agonists inhibited forskolin-induced cAMP accumulation, demonstrating a functional coupling of mMOR to the inhibition of adenylate cyclase. The predicted amino acid sequence of mMOR shares approximately 55% overall amino acid identity with the delta receptor and approximately 97% identity with the recently reported rat mu opioid receptor. Expression of the mu receptor in mouse brain as revealed by in situ hybridization parallels the reported pattern of distribution of mu-selective ligand binding sites. Chromosomal localization (to mouse chromosome 10) and Southern analysis are consistent with a single mu opioid receptor gene in the mouse genome, suggesting that the various pharmacologically distinct forms of the mu receptor arise from alternative splicing, post-translational events, or from a highly divergent gene(s).

Amino Acid Sequence↗
PubMed1995

Long-term survival after intensive care unit admission with sepsis.

OBJECTIVE: To evaluate the long-term survival of critically ill patients with sepsis and to assess the factors predictive of long-term survival (> 1 month after admission date). DESIGN: Prospective, cohort study. SETTING: Medical/surgical intensive care unit (ICU) in a multidisciplinary community hospital. PATIENTS: All patients admitted to the ICU from January 1, 1987 to March 31, 1991 who both demonstrated clinical evidence of the systemic inflammatory response syndrome and yielded blood cultures positive for a bacterium or fungus (n = 153). INTERVENTIONS: Random set of procedures normally performed in an ICU setting. MEASUREMENTS AND MAIN RESULTS: Patient characteristics, including age, blood culture results, comorbid conditions, and severity of illness as estimated by the Acute Physiology Score of the Acute Physiology and Chronic Health Evaluation II prognostic system were recorded. Follow-up evaluation utilizing the National Death Index provided survival outcome for all patients 1 yr after hospital discharge. The mortality rate at hospital discharge was 51.0%, and mortality rates at 1 month, 6 months, and 1 yr after admission date were 40.5%, 64.7%, and 71.9%, respectively. A total of 33 patients survived beyond the period of observation. The analyses demonstrated the following findings: a) the survival rate was negatively correlated with the Acute Physiology Score up to 1 month after hospital admission date, but uncorrelated thereafter; b) fungal infections, such as Candida, had the shortest survival prospects of any blood-borne infection; and c) both malignancy and human immunodeficiency virus infection contributed to poorer outcomes, but differed in their patterns of long-term survival. CONCLUSIONS: The most critical period for surveillance of bacteremic patients was in months 2 through 6 after discharge, during which time, the percentage of patients surviving decreased dramatically. The degree of physiologic derangement, as measured by the Acute Physiology Score, was a useful measure of prognosis within the first month after the score was assessed at ICU admission. However, beyond this period, prognostic utility decreased significantly. Healthcare providers should use caution concerning the expected survival of hospitalized patients with human immunodeficiency virus, based on experience with distinct conditions, such as malignancies.

APACHE↗
PubMed1993

Rat-1 fibroblasts engineered with GAD65 and GAD67 cDNAs in retroviral vectors produce and release GABA.

We have used a retroviral cDNA expression system to drive the expression of the different forms of glutamic acid decarboxylase (GAD65, GAD67, or both). Individual clones of engineered Rat-1 cells make the appropriate GAD mRNAs and GAD polypeptides, show GAD enzymatic activity, and make GABA. Clones expressing GAD65 had higher enzymatic activity than those expressing GAD67. As is the case for brain GADs and for GADs produced in engineered bacteria, the enzymatic activity of GAD65 is more responsive to added pyridoxal phosphate than that of GAD67. Immunostaining for both GADs is scattered throughout the cytoplasm. GAD65 immunostaining is less homogeneous than that of GAD67 and also appears to be associated with the surfaces of large vesicle-like structures. Cells expressing GAD65 and GAD67 showed similar immunostaining patterns with anti-GABA antibodies and contained substantial amounts of GABA (ranging from 7 to 18 pmol of GABA/10(6) cells), which was roughly proportional to their levels of GAD activity. GABA is released from the engineered cells into the surrounding medium under resting conditions, suggesting that cells programmed with GAD cDNAs might serve as effective sources of GABA in cell transplantation experiments.

Animals↗
PubMed1992

The effect of human immunodeficiency virus infection on the distribution and outcome of pneumonia in intensive care units.

To determine the frequency and distribution of pneumonia in an intensive care unit (ICU), we retrospectively examined the records of 1,854 consecutive ICU admissions between January 1987 and April 1990. A total of 266 patients met criteria for pneumonia (unilateral or bilateral infiltrate by chest roentgenogram, plus 2 of the following: leukocyte count > 10 x 10(9) per liter, temperature > 38.5 degrees C, or culture of blood or sputum positive for pathogens). Pneumocystis carinii pneumonia in patients infected with the human immunodeficiency virus was the most frequent cause (28%) precipitating an ICU admission in this series of patients. Streptococcus pneumoniae (13%), Staphylococcus aureus (8%), Haemophilus influenzae (4%), and viruses (4%) were also commonly observed. Overall mortality was 20%. An APACHE II score of greater than 24, the need for intubation, and the presence of P carinii were predictive of increased mortality. Age, sex, and length of stay did not predict final results. Patients with P carinii pneumonia who required intubation had an overall mortality of 54%, which was higher than patients without P carinii pneumonia who required intubation (P < .05). Our experience shows the changing spectrum of pneumonia in ICUs. In contrast to reports of a decade ago in which S pneumoniae and Pseudomonas aeruginosa are cited as most common, P carinii is now most prevalent in our ICU. Although our findings reflect the increasing incidence of human immunodeficiency virus infection in San Francisco, California, they may also be pertinent to other areas in the United States where the incidence of this infection continues to increase.

AIDS-Related Opportunistic Infections↗
PubMedDate not supplied

Pediatric respiratory therapy beyond the neonatal ICU.

This article is intended to generate interest in respiratory therapy for older infants and children by pointing out the special requirements and these patients and emphasizing the important role that respiratory therapists can have in their management.

Child, Preschool↗