Interactions between the benzodiazepine receptor antagonist Ro 15-1788 (flumazepil) and the inverse agonist beta-CCE: behavioral studies with squirrel monkeys

The effects of Ro 15-1788 and ethyl-beta-carboline-3-carboxylate (beta-CCE) were studied alone and in combination on the behavioral performances of squirrel monkeys. Under one procedure, performances maintained by food were suppressed by electric shock presentation (punishment or "conflict" procedure). Under a second procedure, responding was maintained either by food or electric shock delivery under a 5-min fixed-interval schedule. Doses of beta-CCE between 0.1 and 3.0 mg/kg, i.m., produced graded decreases in punished responding which were reversed by pretreatment with Ro 15-1788 (1.0 - 10.0 mg/kg, i.m.). Low doses of beta-CCE (0.03 - 0.3 mg/kg, i.m.) increased responding of monkeys maintained by shock presentation, but did not affect food-maintained responding; higher doses of beta-CCE decreased responding under both schedules. These effects of beta-CCE are opposite those produced by the benzodiazepines under this procedure. Ro 15-1788 (1.0 mg/kg i.m.) antagonized the effects of beta-CCE, producing a shift to the right in the dose-response curves. These findings provide further support for the view that beta-CCE and Ro 15-1788 produce effects mediated by the same benzodiazepine receptor recognition site.     

Detection of a high frequency RsaI polymorphism in the human pro alpha 2(I) collagen gene which is linked to an autosomal dominant form of osteogenesis imperfecta.

Screening of the pro alpha 2(I) collagen genes of Southern African populations for restriction fragment length polymorphisms (RFLPs) has revealed a locus polymorphic for the restriction enzyme RsaI. The frequency of the RFLP was 0.38 in Afrikaners, but much lower in indigenous Southern African populations, which suggests that it is of European origin. The polymorphism was used to study 19 affected and non-affected individuals in a four generation family with the autosomal dominant disorder, osteogenesis imperfecta (OI) type I. Co-inheritance of the loss of the RsaI site and the OI phenotype was observed with a lod score of 3.91 at a recombination fraction (theta) of zero, indicating strong linkage. This suggests that the defect in this family is caused by a structural mutation within or close to the pro alpha 2(I) collagen gene. The use of this high frequency RFLP together with other recently described polymorphisms at this locus will facilitate the analysis of the role of this gene in OI and other inherited disorders of connective tissue.     

Laryngeal influences on breathing pattern and posterior cricoarytenoid muscle activity

Receptors responding to transmural pressure, airflow, and contraction of laryngeal muscles have been previously identified in the larynx. To assess the relative contribution of these three types of receptors to the reflex changes in breathing pattern and upper airway patency, we studied diaphragmatic (DIA) and posterior cricoarytenoid muscle (PCA) activity in anesthetized dogs during spontaneous breathing and occluded efforts with and without bypassing the larynx. Inspiratory duration (TI) was longer, mean inspiratory slope (peak DIA/TI) was lower, and PCA activity was greater with upper airway occlusion than with tracheal occlusion (larynx bypassed). Bilateral section of the superior laryngeal nerves eliminated these differences. When respiratory airflow was diverted from the tracheostomy to the upper airway the only change attributable to laryngeal afferents was an increase in PCA activity. These results confirm the importance of the superior laryngeal nerves in the regulation of breathing pattern and upper airway patency and suggest a prevalent role for laryngeal negative pressure receptors.     

Assessment of pharyngeal airway stability in normal and micrognathic infants

A current hypothesis for obstructive sleep apnea states that 1) negative airway pressure during inspiration can collapse the pharyngeal airway, and 2) neural control of pharyngeal airway-dilating muscles is important in preventing this collapse. To test this hypothesis we performed nasal mask occlusions to increase negative pharyngeal airway pressures during inspiration in eight normal and five micrognathic infants. Both groups developed midinspiratory pharyngeal obstruction, but obstruction was more frequent in micrognathic infants and varied in some infants with sleep state. The airway usually reopened with the subsequent expiration. The occasional failure to reopen was presumably due to pharyngeal wall adhesion. If airway obstruction occurred in sequential breaths during multiple-breath nasal mask occlusions in normal infants, there was a breath-by-breath change in the airway pressure associated with airway closure (airway closing pressure); the airway closing pressure became progressively more negative. Micrognathic infants showed less ability to improve the airway closing pressure, but this ability increased with age. These findings suggest that nasal mask occlusion can test the competence of the neuromuscular mechanisms that maintain pharyngeal airway patency in infants. Micrognathic infants had spontaneous midinspiratory pharyngeal airway obstructions during snoring. Their episodes of obstructive apnea began with midinspiratory pharyngeal obstruction similar to that seen during snoring and nasal mask occlusions. These findings imply a similar pathophysiology for snoring, spontaneous airway obstruction, and obstruction during snoring.     

Acute effect of cigarette smoke on laryngeal receptors

Upper airway exposure to cigarette smoke elicits reflex changes in breathing pattern. To examine whether laryngeal afferents are affected by cigarette smoke, neural activity was recorded from the peripheral cut end of superior laryngeal nerve in anesthetized dogs. A box-balloon system, connected to the breathing circuit, allowed smoke to be inhaled spontaneously through the isolated upper airway while preserving its normal respiratory flow and pressure. Our results showed the following. Inhalation of cigarette smoke (25-50% concentration, 300-400 ml) caused a marked increase in activity of laryngeal irritant receptors which were either silent or randomly discharging during control breathing [their activity increased from a control value of 1.67 +/- 0.50 (mean +/- SE; n = 21) to a peak of 5.03 +/- 0.85 impulses/s in 11-15 s]. The activity of laryngeal cold receptors was reduced to 77.3 and 63.8% of control (n = 9) during the two breaths of smoke inhalation, respectively. After returning toward the base-line activity, a more pronounced inhibition (26.3% of control) occurred at three to nine breaths after the smoke inhalation. A small but significant decrease (88.5% of control) in the inspiratory discharge of laryngeal mechanoreceptors was observed during the first test breath. These effects were independent of the CO2 content of the smoke. Furthermore, there was no difference between the responses of these laryngeal afferents to high- and low-nicotine cigarette smoke.     

Calcimedins: purification and characterization from chicken gizzard and rat and bovine livers

A procedure for the simultaneous extraction and purification of four calcimedins from chicken gizzard, rat liver, and bovine liver is described. These proteins bind to hydrophobic resins in a calcium-dependent manner similar to calmodulin and troponin C. The four calcimedins purified had molecular weights 67,000 (67K), 35,000 (35K), 33,000 (33K), and 30,000 (30K) as determined by SDS polyacrylamide gel electrophoresis. Their ability to bind calcium was demonstrated using the Hummel-Dreyer method. Their tissue concentration ranged between 1-4 mg/100 g wet weight in the three tissues studied. During gel filtration, calcimedins 67K and 35K, had Rf (Ve-Vo/Vt-Vo) values of 0.46 and 0.74, respectively, indicating monomeric structure. However, the 33K and 30K calcimedins had Rf values of 0.26 (molecular weights greater than 90,000) suggesting that they occur as subunit complexes in their native state. Antibodies raised against the 67K and 35K calcimedins showed cross reactivity suggesting possible common origin. However, peptide mapping studies showed that they are independent proteins with considerable peptide homology. Antibodies to 30/33K calcimedins did not cross-react with either 67K or 35K calcimedins. Moreover, their peptide maps were strikingly different from those of 67K and 35K calcimedins indicating that they are unique. At present, the regulatory function of this group of proteins is not clear. Indirect evidences support the possibility that they are involved in membrane associated events, such as endocytosis and secretion.     

Solvent isotope effects on the reaction catalyzed by yeast hexokinase

The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O. In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7. In 2H2O, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5-1.7 for all three parameters. When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5-anhydromannitol which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E X glucose complex.     

Vertebral pathology in the afar australopithecines

Ten vertebral elements from the AL-288 partial hominid skeleton and 11 elements from the AL-333 collection are described. The AL-288 column presents a marked kyphosis at the level of thoracic vertebrae 6 through 10, with pronounced new bone formation on the ventral surfaces of these vertebrae. These features, associated with narrowed disc space and minor osteophytosis, resemble Scheuermann disease in the human. Even though this diagnosis is consistent with a basically human, bipedal locomotor repertoire, the presence of Scheuermann disease suggests that lifting, climbing, or acrobatic activities may have been important in early hominids.