Responses of petrosal ganglion neurons in vitro to hypoxic stimuli and putative transmitters.
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Acetylcholine↗
Biomedical subjects
J Arroyo
Publications and source records attributed to J Arroyo.
Applications of flow cytometry to clinical microbiology.
Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.
Flow Cytometry↗
Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates.
A chimeric yellow fever (YF)-dengue type 2 (dengue-2) virus (ChimeriVax-D2) was constructed using a recombinant cDNA infectious clone of a YF vaccine strain (YF 17D) as a backbone into which we inserted the premembrane (prM) and envelope (E) genes of dengue-2 virus (strain PUO-218 from a case of dengue fever in Bangkok, Thailand). The chimeric virus was recovered from the supernatant of Vero cells transfected with RNA transcripts and amplified once in these cells to yield a titer of 6.3 log(10) PFU/ml. The ChimeriVax-D2 was not neurovirulent for 4-week-old outbred mice inoculated intracerebrally. This virus was evaluated in rhesus monkeys for its safety (induction of viremia) and protective efficacy (induction of anti-dengue-2 neutralizing antibodies and protection against challenge). In one experiment, groups of non-YF-immune monkeys received graded doses of ChimeriVax-D2; a control group received only the vaccine diluents. All monkeys (except the control group) developed a brief viremia and showed no signs of illness. Sixty-two days postimmunization, animals were challenged with 5.0 log(10) focus forming units (FFU) of a wild-type dengue-2 virus. No viremia (<1.7 log(10) FFU/ml) was detected in any vaccinated group, whereas all animals in the placebo control group developed viremia. All vaccinated monkeys developed neutralizing antibodies in a dose-dependent response. In another experiment, viremia and production of neutralizing antibodies were determined in YF-immune monkeys that received either ChimeriVax-D2 or a wild-type dengue-2 virus. Low viremia was detected in ChimeriVax-D2-inoculated monkeys, whereas all dengue-2-immunized animals became viremic. All of these animals were protected against challenge with a wild-type dengue-2 virus, whereas all YF-immune monkeys and nonimmune controls became viremic upon challenge. Genetic stability of ChimeriVax-D2 was assessed by continuous in vitro passage in VeroPM cells. The titer of ChimeriVax-D2, the attenuated phenotype for 4-week-old mice, and the sequence of the inserted prME genes were unchanged after 18 passages in Vero cells. The high replication efficiency, attenuation phenotype in mice and monkeys, immunogenicity and protective efficacy, and genomic stability of ChimeriVax-D2 justify it as a novel vaccine candidate to be evaluated in humans.
Amino Acid Substitution↗
Chimeric yellow fever virus 17D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys.
ChimeriVax-JE is a live, attenuated recombinant virus prepared by replacing the genes encoding two structural proteins (prM and E) of yellow fever 17D virus with the corresponding genes of an attenuated strain of Japanese encephalitis virus (JE), SA14-14-2 (T. J. Chambers et al., J. Virol. 73:3095-3101, 1999). Since the prM and E proteins contain antigens conferring protective humoral and cellular immunity, the immune response to vaccination is directed principally at JE. The prM-E genome sequence of the ChimeriVax-JE in diploid fetal rhesus lung cells (FRhL, a substrate acceptable for human vaccines) was identical to that of JE SA14-14-2 vaccine and differed from sequences of virulent wild-type strains (SA14 and Nakayama) at six amino acid residues in the envelope gene (E107, E138, E176, E279, E315, and E439). ChimeriVax-JE was fully attenuated for weaned mice inoculated by the intracerebral (i.c.) route, whereas commercial yellow fever 17D vaccine (YF-Vax) caused lethal encephalitis with a 50% lethal dose of 1.67 log(10) PFU. Groups of four rhesus monkeys were inoculated by the subcutaneous route with 2.0, 3.0, 4.0, and 5. 0 log(10) PFU of ChimeriVax-JE. All 16 monkeys developed low viremias (mean peak viremia, 1.7 to 2.1 log(10) PFU/ml; mean duration, 1.8 to 2.3 days). Neutralizing antibodies appeared between days 6 and 10; by day 30, neutralizing antibody responses were similar across dose groups. Neutralizing antibody titers to the homologous (vaccine) strain were higher than to the heterologous wild-type JE strains. All immunized monkeys and sham-immunized controls were challenged i.c. on day 54 with 5.2 log(10) PFU of wild-type JE. None of the immunized monkeys developed viremia or illness and had mild residual brain lesions, whereas controls developed viremia, clinical encephalitis, and severe histopathologic lesions. Immunized monkeys developed significant (>/=4-fold) increases in serum and cerebrospinal fluid neutralizing antibodies after i.c. challenge. In a standardized test for neurovirulence, ChimeriVax-JE and YF-Vax were compared in groups of 10 monkeys inoculated i.c. and analyzed histopathologically on day 30. Lesion scores in brains and spinal cord were significantly higher for monkeys inoculated with YF-Vax. ChimeriVax-JE meets preclinical safety and efficacy requirements for a human vaccine; it appears safer than yellow fever 17D vaccine but has a similar profile of immunogenicity and protective efficacy.
Animals↗
A novel family of cell wall-related proteins regulated differently during the yeast life cycle.
The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial beta-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Delta crh2Delta strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1-green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle.
Alleles↗
Discovery of distyly in Narcissus (Amaryllidaceae).
There has been disagreement concerning the nature of sexual polymorphisms in Narcissus, a genus of insect-pollinated geophytes native to the Mediterranean. The existence of trimorphic heterostyly in the genus has recently been confirmed, but the occurrence of distyly remains enigmatic. All sexually dimorphic species previously investigated possess two distinct style lengths but anthers of similar height. Stigma-height dimorphism does not qualify as true distyly because of the absence of a reciprocal correspondence in stigma and anther position in the floral morphs. Such reciprocal herkogamy is generally regarded as the defining feature of heterostyly. Here we report on distyly in N. albimarginatus (section Apodanthae), a rare species confined to a single mountain in northwestern Morocco. A population composed of equal numbers of long- and short-styled plants exhibited reciprocal herkogamy with lower anthers of the long-styled morph and upper anthers of the short-styled morph corresponding in height to stigmas of short- and long-styled plants, respectively. The presence of both stigma-height dimorphism and distyly in Narcissus is of general significance to theoretical models of the evolution of heterostyly.
Journal Article↗
Responses to hypoxia of petrosal ganglia in vitro.
NaCN is a classical stimulus used to elicit discharges from carotid body chemoreceptors. The effect is assumed to be mediated by glomus (type I) cells, which release an excitatory transmitter for the excitation of carotid nerve endings. Since the sensory perikarya of the glossopharyngeal nerve (from which the carotid nerve branches) are located in the petrosal ganglion, we tested whether application of this drug to the petrosal ganglion superfused in vitro elicits antidromic discharges in the carotid nerve. NaCN did indeed cause an intense and prolonged burst of nerve impulses in the carotid nerve, while provoking a less intense and much briefer burst of discharges in the glossopharyngeal branch. Carotid nerve responses to NaCN were reduced and shortened by prior or following application of dopamine to the ganglion. Sodium azide applied to the petrosal ganglion evoked a less intense and much briefer burst of impulses in the carotid nerve. Ganglionar application of 2,4-dinitrophenol did not induce discharges in the carotid nerve. Switching the superfusion of the ganglion from a normoxic to a hypoxic solution did not evoke discharges in the carotid nerve. Therefore, the perikarya of carotid nerve neurons are sensitive to NaCN, but are not excited by reducing the pO(2) of the superfusing solution.
2,4-Dinitrophenol↗
Dopamine modulates carotid nerve responses induced by acetylcholine on the cat petrosal ganglion in vitro.
We have recently reported that application of acetylcholine (ACh) or nicotine to the petrosal ganglion-the sensory ganglion of the glossopharyngeal nerve-elicits a burst of discharges in the carotid nerve branch, innervating the carotid body and sinus, but not in the glossopharyngeal branch, innervating the tongue and pharynx. Thus, the perikarya of sensory neurons for the carotid bifurcation exhibit selective cholinosensitivity. Since dopamine (DA) modulates carotid nerve chemosensory activity, we searched for the presence of DA sensitivity at the perikarya of these neurons in the cat petrosal ganglion superfused in vitro. Applications of DA in doses of up to 5 mg to the ganglion did not modify the rate of spontaneous discharges in the carotid nerve. However, if DA was applied 30 s before ACh injections, ACh-evoked reactions were modified: low doses of DA enhanced the subsequent responses to ACh, while high doses of DA depressed the responses to ACh. This depressant effect of DA on ACh responses was partially antagonized by adding spiroperone to the superfusate. Our results show that the response to ACh of petrosal ganglion neurons projecting through the carotid nerve is modulated by DA acting on D(2) receptors located in the somata of these neurons. Thus, dopaminergic modulation of cholinosensitivity could be shared also by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.
Acetylcholine↗
Immunogenicity, genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-Japanese encephalitis virus (ChimeriVax-JE) as a live, attenuated vaccine candidate against Japanese encephalitis.
Yellow fever (YF) 17D vaccine virus, having a 60-year history of safe and effective use, is an ideal vector to deliver heterologous genes from other medically important flaviviruses. A chimeric YF/Japanese encephalitis (JE) virus (ChimeriVax-JE virus) was constructed by insertion of the premembrane and envelope (prME) genes of an attenuated human vaccine strain (SA14-14-2) of Japanese encephalitis (JE) virus between core and nonstructural (NS) genes of a YF 17D infectious clone. The virus grew to high titers in cell cultures and was not neurovirulent for 3- to 4-week-old mice at doses </=6 log10 plaque forming units (pfu) inoculated by the intracerebral (IC) route. In contrast, commercial YF 17D vaccine was highly neurovirulent for weanling mice by the same route. Mice inoculated subcutaneously with one dose of >/=10(3) pfu of ChimeriVax-JE virus were solidly protected against intraperitoneal challenge with a virulent JE virus. Genetic stability of the chimera was assessed by sequential passages in cell cultures or in mouse brain. All attenuating residues and the avirulent phenotype were preserved after 18 passages in cell cultures or 6 passages in mouse brains.
Animals↗
Recombinant, chimaeric live, attenuated vaccine (ChimeriVax) incorporating the envelope genes of Japanese encephalitis (SA14-14-2) virus and the capsid and nonstructural genes of yellow fever (17D) virus is safe, immunogenic and protective in non-human primates.
Yellow fever 17D virus, a safe and effective live, attenuated vaccine, was used as a vector for genes encoding the protective antigenic determinants of a heterologous member of the genus Flavivirus, Japanese encephalitis (JE) virus, the leading cause of acute viral central nervous system infection and death throughout Asia. The viral envelope (prM and E) genes of a full-length cDNA clone of YF 17D virus were replaced with the corresponding genes of JE SA14-14-2, a strain licensed as a live, attenuated vaccine in China. Full-length RNA transcripts of the YF/JE chimaera were used to transfect Vero cells. The progeny virus (named 'ChimeriVax-JE'), was used to define safety after intracerebral (i.c.) inoculation of rhesus monkeys. Monkeys (N = 3) inoculated with a high dose (6.6 log10 pfu) developed a brief viremia, showed no signs of illness, developed high titers of anti-JE neutralizing antibody, and had minimal brain and spinal cord lesion scores according to criteria specified in the WHO monkey neurovirulence test. A control group of 3 monkeys that received a lower dose (4.2 log10 pfu) of commercial YF 17D vaccine had slightly higher lesion scores. To develop a lethal monkey model of JE for vaccine protection tests, we inoculated groups of monkeys i.c. or intranasally (i.n.) with a JE virus strain found to be highly neurovirulent and neuroinvasive for mice. Monkeys inoculated i.c., but not i.n., developed severe encephalitis after an incubation period of 8-13 days. The ChimeriVax-JE virus was passed in a cell line acceptable for human use (diploid fetal rhesus lung) and 4.3 or 5.3 log10 pfu were inoculated into groups of 3 monkeys by the subcutaneous route. All 6 animals developed brief viremias (peak titer < 2.0 log10 pfu/ml) and subsequently had anti-JE but no yellow fever neutralizing antibodies. On day 64, the monkeys were challenged i.c. with 5.5 log10 pfu of virulent JE virus. The immunized animals had no detectable viremia post-challenge, whereas 4 unimmunized controls became viremic. Only 1 of 6 (17%) vaccinated monkeys but 4 of 4 (100%) unvaccinated controls developed encephalitis. Histopathological examination 30 days after challenge confirmed that the protected, immunized animals had no or minimal evidence of encephalitis. These data demonstrated the ability of the ChimeriVax-JE to induce a rapid humoral immune response and to protect against a very severe, direct intracerebral virus challenge. Target areas of neuronal damage and inflammation in monkeys infected IC with wild-type JE, the chimaeric virus and YF 17D were similar, indicating that the histopathological scoring system used for the WHO yellow fever monkey neurovirulence test will be applicable to control testing of chimaeric seed viruses and vaccines.
Animals↗
In vitro laser welding of amniotic membranes.
OBJECTIVE: To test in vitro the feasibility of welding amniotic membranes using Nd:YAG laser energy. STUDY DESIGN: Fresh fetal membranes from term pregnancies were washed and cut into 1 cm2 pieces. Pooled cryoprecipitate (CPT), 50% albumin (Alb), or polytetrafluoroethilene (e-PTFE) were used as solder medium. The optimal settings of the laser were determined. Results were assessed quantitatively and semi-quantitatively using Pearson Chi-square analysis. RESULTS: Laser welding of amniotic membranes was successful in 82.6% of experiments with e-PTFE and in 10.7% of experiments with CPT (P < 0.001). The strength of the welding was also significantly better with e-PTFE (P < 0.001). Optimal results were obtained using 1-7 Watts and 0.1-1 seconds. Laser welding was unsuccessful in 100% of experiments with Alb. CONCLUSIONS: Laser welding of fetal membranes can be accomplished with e-PTFE and to a lesser degree with the CPT using Nd:YAG energy under low wattage-high exposure settings. Further studies are underway to test other grafting or soldering materials.
Extraembryonic Membranes↗
Treatment of iatrogenic previable premature rupture of membranes with intra-amniotic injection of platelets and cryoprecipitate (amniopatch): preliminary experience.
OBJECTIVE: Our aim was to describe the treatment of iatrogenic previable premature rupture of membranes with the intra-amniotic injection of platelets and cryoprecipitate (amniopatch). STUDY DESIGN: Patients with iatrogenic previable premature rupture of membranes and without evidence of intra-amniotic infection underwent transabdominal intra-amniotic injection of platelets and cryoprecipitate through a 22-gauge needle. The study was approved by the Institutional Review Board of St Joseph's Hospital in Tampa, Florida, and all patients gave written informed consent. RESULTS: Seven patients with iatrogenic preterm premature rupture of membranes underwent placement of an amniopatch. Membrane sealing was verifiable in 6 of 7 patients. Three patients had iatrogenic preterm premature rupture of membranes after operative fetoscopy, 3 cases were after genetic amniocentesis, and 1 was after diagnostic fetoscopy. Three pregnancies progressed well, with restoration of the amniotic fluid volume and no further leakage. Two patients had unexplained fetal death despite successful sealing. One case of bladder outlet obstruction had no further leakage, but oligohydramnios persisted and did not allow unequivocal documentation of sealing. One patient miscarried from twin-twin transfusion, but the amniotic cavity was sealed. CONCLUSIONS: Iatrogenic preterm premature rupture of membranes can be treated effectively with an amniopatch. The technique is simple and does not require knowledge of the exact location of the defect. Unexpected fetal death from the procedure may be attributable to vasoactive effects of platelets or indigo carmine. Although the appropriate dose of platelets and cryoprecipitate needs to be established, the amniopatch may mean that iatrogenic preterm premature rupture of membranes no longer needs to be considered a devastating complication of pregnancy.
Adult↗
Prognostic value of early computerized tomography scanning following craniotomy for traumatic hematoma.
OBJECT: Patients with head injuries traditionally were categorized on the basis of whether their lesions appeared to be diffuse, focal, or mass lesions on admission computerized tomography (CT) scanning. In the classification of Marshall, et al., the presence of a hematoma (evacuated or not evacuated) is more significant than any diffuse injury (DI). The CT scan appearance after evacuation of a mass lesion has not been analyzed previously in relation to outcome. The authors have investigated the importance of: 1) neurological assessment at hospital admission; 2) the status of the basal cisterns and associated intracranial lesions on the admission CT scan; and 3) the degree of DI on the early CT scan obtained after craniotomy to identify patients at risk for development of raised intracranial pressure (ICP) and lowered cerebral perfusion pressure (CPP) and to discover the influence of the postoperative CT appearance of the lesion on patient outcome. METHODS: The authors prospectively studied 82 patients with isolated, severe closed head injury (Glasgow Coma Scale [GCS] score < or = 8), all of whom had intracranial hematoma. Both ICP and CPP were continuously monitored, and a CT scan was obtained within 2 to 12 hours after craniotomy. The CT images were categorized according to the classification of Marshall, et al. The mortality rate during the hospital stay was 37%, and 50% of the patients achieved a favorable outcome. Compression of the basal cistern on the admission (preoperative) CT scan was associated with raised ICP and a CPP of less than 70 mm Hg but not with any other features or with poor patient outcome. In 53 patients the postoperative CT scan revealed DIs III or IV and 29 patients had DIs I or II. The percentages of time during the hospital stay in which ICP was higher than 20 mm Hg and CPP was lower than 70 mm Hg as well as unfavorable outcome were higher in the group of patients in whom DI III or IV was present (p < 0.001). Raised ICP, CPP lower than 70 mm Hg, DI III or IV, and unfavorable outcome were more frequently observed in patients who presented with a motor (m)GCS score of 3 or less, bilateral unreactive pupils, associated intracranial injuries, and hypotension (p < 0.001). When logistic regression analysis was performed, an mGCS score of 3 or less (p = 0.0013, odds ratio [OR] 10.8), bilateral unreactive pupils (p = 0.0047, OR 31.8), and DI III or IV observed on CT scanning after surgery (p = 0.015, OR 8.9) were independently associated with poor outcome. CONCLUSIONS: Features on CT scans obtained shortly after craniotomy constitute an independent predictor of outcome in patients with traumatic hematoma. Patients in whom DI III or IV appears on postoperative CT scanning, who often present with an mGCS score of 3 or less and nonreactive pupils, are at high risk for the development of raised ICP and lowered CPP.
Adolescent↗
The deletion of six ORFs of unknown function from Saccharomyces cerevisiae chromosome VII reveals two essential genes: YGR195w and YGR198w.
We have deleted six different ORFs of unknown function located on the right arm of Saccharomyces cerevisiae chromosome VII; namely, YGR187c/HGH1, YGR189c, YGR194c, YGR195w, YGR196c and YGR198w. No basic phenotypes could be attributed to the strains deleted in any of genes YGR187c/HGH1, YGR189c, YGR194c and YGR196c. These deletants did not show mating, sporulation or growth defects under any of the conditions tested. However, spores bearing deletions in either the YGR195w or YGR198w genes were unable to develop into macroscopical colonies. The YGR195w gene product shows significant homology with bacterial ribonuclease PH, an enzyme hitherto undescribed in yeasts, and its deletion causes a loss of viability after one to three rounds of cell division. Overexpression of this gene, using a tetracycline-regulatable promoter system, did not cause any effect on the cells. Contrary to what has been reported for prokaryotic homologs, this enzyme could play an essential role in yeast cell biology. The product encoded by the other essential ORF, YGR198w, shows no significant homology with any protein of known function in the databases. Spores bearing the deletion usually germinate and give rise to microcolonies of 50-100 non-viable cells.
Chromosomes, Fungal↗
The YGR194c (XKS1) gene encodes the xylulokinase from the budding yeast Saccharomyces cerevisiae.
We report the finding of a Saccharomyces cerevisiae gene necessary for growth in culture media with D-xylulose as the sole carbon source. This gene corresponds to the YGR194c open reading frame that we have previously described, and it is renamed now XKS1. Data bank comparisons of the protein encoded by the XKS1 gene showed significant homology with different xylulokinases, indicating a possible role in xylulose phosphorylation. The wild-type gene in a centromeric plasmid complemented defective growth of xks1 S. cerevisiae mutant strains in xylulose. By contrast, overexpression negatively influenced cell growth in this carbon source.
Amino Acid Sequence↗
Selective activation of carotid nerve fibers by acetylcholine applied to the cat petrosal ganglion in vitro.
The petrosal ganglion innervates carotid body chemoreceptors through the carotid (sinus) nerve. These primary sensory neurons are activated by transmitters released from receptor (glomus) cells, acetylcholine (ACh) having been proposed as one of the transmitters involved in this process. Since the perikarya of primary sensory neurons share several properties with peripheral sensory endings, we studied the electrical responses of the carotid nerve and glossopharyngeal branch to ACh locally applied to the cat petrosal ganglion superfused in vitro. Ganglionar applications of AChCl (1 microg-1 mg) generated bursts of action potentials conducted along the carotid nerve, while only a few spikes were exceptionally recorded from the glossopharyngeal branch in response to the largest doses. Carotid nerve responses to ACh were dose-dependent, the higher doses inducing transient desensitization. Application of nicotine to the petrosal ganglion also evoked dose-dependent excitatory responses in the carotid nerve. Responses to ACh were reversibly antagonized by adding hexamethonium to the superfusate, more intense and prolonged block of ACh responses being produced by mecamylamine. Ganglionar applications of gamma-amino butyric acid and serotonin, in doses of up to 5 mg, did not induce firing of action potentials in any of the branches of the glossopharyngeal nerve. Our results indicate that petrosal ganglion neurons projecting through the carotid nerve are selectively activated by ACh acting on nicotinic ACh receptors located in the somata of these neurons. Thus, cholinosensitivity would be shared by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.
Acetylcholine↗
HLA-DPbeta residue 69 plays a crucial role in allorecognition.
To investigate the contribution to allorecognition of the individual polymorphic positions Glu 69 and Val 36 from the DPB1*02012 allele, DPB1*02012 cDNA was subjected to site-directed mutagenesis and alleles expressing Lys at 69 and Ala at 36 were generated. The lymphoblastoid cell line (LCL) 45.EM1, a previously generated mutant B-LCL which expresses normal levels of DPA mRNA but is not able to transcribe DPB, was transfected with wild-type or mutant DPB1*02012 cDNAs. The ability of two HLA-DPw2 alloreactive CD4+ cytotoxic T-lymphocyte (CTL) clones to lyse the panel of DPB1*02012 wild-type and site-directed mutant B-cell lines was tested. Both CTL clones (8.3 and 8.9) lysed the B-LCL 45.1, which is haploid for HLA and expresses wild-type DPB1*02012, and transfectants expressing Ala at 36 instead of Val, indicating that this polymorphic residue is not critical for T-cell recognition. However, the change of Glu to Lys at 69 prevented recognition by clones 8.3 and 8.9. These data demonstrate that the residue at peptide-binding position 69 is crucial for T-cell receptor recognition and suggest the requirement for a negatively charged residue at this position for allostimulation of these T-cell clones. The side chain of DPbeta-69 is predicted to point into the peptide-binding groove, and the existence of positive(Lys) or negative (Glu) residues probably leads to substantial differences in the allo- or auto-DP-bound peptides or to differences in the conformation of the peptide-MHC complex, which would therefore be responsible for specific DPw2 allorecognition. The binding of a panel of monomorphic and polymorphic anti-HLA-DP monoclonal antibodies (mAbs) to these transfectants was also tested by flow cytometry. The changes at Glu 69 and Val 36 did not affect recognition by any of the monomorphic antibodies tested. However, the binding pattern of some of the polymorphic mAbs was clearly modified. Therefore, even though it is not crucial for T-cell allorecognition, polymorphic residue 36 must be involved in epitopes recognized by some polymorphic anti-DP antibodies, while residue 69 of the DPB molecule is crucial both for T-cell allorecognition and recognition by some mAbs.
Alleles↗
[Contraceptive behavior of young Latin-American adults living in Montreal].
Recent research on the contraceptive practices of Hispanics adolescents living in Latin America and the United-States indicates that their practices are inadequate. However, contraceptive practices can vary according to levels of traditionalism and acculturation. In order to verify these hypotheses, the contraceptive behaviors of a group of 114 young adults from both genders of Hispanic origin were evaluated. Results indicate that half of the participants did not use any contraceptive method during the first intercourse and that condoms were the most common method. Contrary to American results, acculturation levels were not significant in contraceptive use. Degree of traditionalism in sexual roles was more significant.
Adult↗