Distributional characterizations and testing for differences of relatedness and inbreeding of a subpopulation of American Hereford bulls.

Beta distributions are characterized by two determining parameters and a parameter space from 0 to 1, and may be useful for examining population genetic parameters such as the relationship or inbreeding coefficients. Often subpopulations exist within breeds that are congregated around particular lineages of cattle or ancestors that breeders value. These subpopulations are more related to each other than to the majority of other animals; they may have higher inbreeding as well. Value may be added to these subpopulations because of their relatedness with important or renowned ancestors. The objectives of this work were to compare the relatedness and inbreeding of a group of 26 modern bulls from a subpopulation of the American Hereford breed relative to 1) 30 males with the most descendants present in the pedigree, 2) 15 renowned American Hereford bulls considered important individuals in the breed's history, and 3) 19 prominent subpopulation male ancestors. Conformance of the mean relationship coefficients of the bulls with the three groups and the mean inbreeding coefficient with all pedigree animals to beta distributions was assessed by 1) visually determining the parameters of the beta distributions based on the entire pedigree, 2) testing the mean relationship coefficient or inbreeding coefficient of the group of subpopulation bulls for its positional inclusion in those distributions, and 3) bootstrap sampling methodology. The mean relationship coefficients of the 26 Trask bulls with the 30 bulls with the most descendants, the 15 renowned ancestors, and the 19 Trask male ancestors were 0.15, 0.132, and 0.208, respectively. Testing of these means in beta distributions indicated that the group of 26 Trask bulls were no more related to the three groups of bulls than all of the animals in the pedigree (0.06 < P < 0.25). Bootstrap sampling indicated that the 26 bulls were more related to the three groups of male ancestors than the remainder of the animals in the pedigree (P < 0.0001). The mean inbreeding coefficient of the 26 bulls (0.13) did not differ from the overall inbreeding coefficient (0.056) when tested using a beta distribution; however, bootstrap sampling indicated otherwise (P < 0.0001). Results may indicate the inadequacy of visually parameterizing a beta distribution. Quantification of pedigree relatedness of a group of animals to key ancestors, especially with no DNA available, may add value to that group and individuals.

Oxygen delivery to the brain before and after birth.

We studied the relationship between cerebral oxygen consumption and cerebral oxygen delivery (cerebral blood flow x arterial oxygen content) in fetal, newborn, and adult sheep, Relative to the amount of oxygen consumed, cerebral oxygen delivery in the fetus exceeds that in the lamb and adult by 70 percent. This may represent a protective advantage for the fetus or simply a necessary adaptation to the low arterial oxygen pressure in the intrauterine environment.

Desensitization of the inhibition of the M-current in sympathetic neurons: effects of ATP analogs, polyanions, and multiple agonist applications.

Desensitization occurs when the response to a neurotransmitter receptor agonist wanes in the continued presence of agonist. In amphibian sympathetic neurons, both muscarinic and peptidergic receptor agonists inhibit a K+ current, the M-current (IM), and this inhibition desensitizes. We have studied the desensitization to substance P (SP) by whole-cell recordings from dissociated sympathetic neurons from bullfrogs. When ATP in the recording pipette was replaced with AMP-PNP, SP still inhibited IM, but no desensitization was observed, indicating that ATP hydrolysis is required for desensitization. Desensitization inhibitors of beta-adrenergic receptors did not block desensitization to SP. When a low dose of muscarine sufficient to inhibit IM, but not to elicit desensitization, was applied simultaneously with a desensitizing dose of SP, IM remained depressed and did not desensitize. Thus, there may be separate systems controlling desensitization for different agonists, or the enzyme(s) involved is "compartmentalized."

Muscarinic suppression of the M-current is mediated by a rise in internal Ca2+ concentration.

The role of intracellular Ca2+ in the muscarinic suppression of M-current was examined. Intracellular injection of Ca2+ buffer into cells in the intact ganglion reduced the response to muscarinic agonist. In similar experiments on isolated cells, Ca2+ buffer was introduced into the cytoplasm using a perfused recording pipette. Ca2+ buffer (20 mM) with the free Ca2+ concentration set to normal resting levels produced a reversible reduction of the muscarinic response. In a second line of investigation, it was found that pharmacological procedures designed to deplete internal stores of Ca2+ produced a decrease in the muscarinic response. These results, taken together with previous work, support the hypothesis that the muscarinic suppression of M-current is mediated by the release of Ca2+ from intracellular stores.

Myosin light chain kinase occurs in bullfrog sympathetic neurons and may modulate voltage-dependent potassium currents.

A polyclonal antibody against myosin light chain kinase (MLCK) of chicken gizzard recognized a 130 kd peptide of bullfrog sympathetic ganglia as MLCK. MLCK immunoreactivity was confined to the neuronal cell body. A synthetic peptide corresponding to an inhibitory domain of MLCK (Ala783-Gly804) was applied intracellularly to isolated sympathetic neurons during whole-cell recordings of ionic currents. The peptide inhibitor reversibly decreased M-type potassium current (IM) while not affecting A-type of delayed rectifier-type potassium currents. Intracellular application of an active fragment of MLCK enhanced IM, whereas application of an inactive MLCK fragment did not. The results suggest that IM can be modulated by MLCK-catalyzed phosphorylation.

Localization and function of NK(3) subtype tachykinin receptors of layer V pyramidal neurons of the guinea-pig medial prefrontal cortex.

The NK(3) subtype of tachykinin receptor has been implicated as a modulator of synaptic transmission in several brain regions, including the cerebral cortex. The localization and expression of NK(3) receptors within the brain vary from species to species. In addition, the pharmacology of NK(3) receptor-specific antagonists shows significant species variability. Among commonly used animal models, the pharmacology of the guinea-pig NK(3) receptor most closely resembles that of the human NK(3) receptor. Here, we provide anatomical localization studies, receptor binding studies, and studies of the electrophysiological effects of NK(3) receptor ligands of guinea-pig cortex using two commercially available ligands, the NK(3) receptor peptide analog agonist senktide, and the quinolinecarboxamide NK(3) receptor antagonist SB-222,200. Saturation binding studies with membranes isolated from guinea-pig cerebral cortex showed saturable binding consistent with a single high affinity site. Autoradiographic studies revealed dense specific binding in layers II/III and layer V of the cerebral cortex. For electrophysiological studies, brain slices were prepared from prefrontal cortex of 3- to 14-day-old guinea pigs. Whole cell recordings were made from layer V pyramidal neurons. In current clamp mode with a K(+)-containing pipette solution, senktide depolarized the pyramidal neurons and led to repetitive firing of action potentials. In voltage clamp mode with a Cs(+)-containing pipette solution, senktide application produced an inward current and a concentration-dependent enhancement of the amplitude and the frequency of spontaneous excitatory postsynaptic potentials. The glutamatergic nature of these events was demonstrated by block by glutamate receptor antagonists. The effects of senktide were blocked by SB-222,200, an NK(3) receptor antagonist. Taken together, these results are consistent with a functional role for NK(3) receptors located on neurons in the cerebral cortex. In layer V pyramidal neurons of the medial prefrontal cortex, activation of the NK(3) receptor system plays an excitatory role in modulating synaptic transmission.

A time-dependent and voltage-sensitive K+ current in single cells from frog atrium.

A quantitative description of the time-dependent and voltage-sensitive outward currents in heart has been hampered by the complications inherent to the multicellular preparations previously used. We have used the whole-cell patch-clamp technique to record the delayed outward K+ current, IK, in single cells dissociated from frog atrium. Na+ currents were blocked with tetrodotoxin and Ca2+ currents with Mn2+ or Cd2+. After depolarizations from -50 mV to potentials positive to -30 mV, a time-dependent outward current was observed. This current has been characterized according to its steady state activation, kinetics, and ion transfer function. The current is well described as a single Hodgkin-Huxley conductance. The deactivation of the current is a single exponential. Activation of the current is sigmoid and is fitted well by raising the activation variable to the second power. The reversal potential of IK is near EK and shifts by 57 mV/10-fold change in [K+]o. This suggests that the current is carried selectively by K ions. The threshold for activation is near -30 mV. IK is maximally activated positive to +20 mV and shows no inactivation. The fully activated current-voltage relationship is linear between -110 and +50 mV. Neither Ba2+ (250 microM) nor Cd2+ (100 microM) affects IK.

Otorhinolaryngological manifestations of the mucopolysaccharidoses.

The mucopolysaccharidoses (MPS) are a family of related inherited metabolic disorders where, due to specific lysosomal enzyme deficiencies, partially degraded glycosaminoglycans (GAGs) accumulate in the body's cells. Due to the ubiquitous nature of GAGs in the body this deposition can occur in many tissue types and may interfere with cellular function. Although these conditions are rare, there is a propensity for the disease process to cause problems with the function of the ears, noses and throats of affected patients. In this review, we present an overview of the clinical manifestations of MPS in general and highlight the problems specifically presenting in the field of otorhinolaryngology.

Ontogeny of erythrocyte insulin binding in the sheep.

The ontogeny of insulin binding in the sheep was studied using the erythrocytes (RBCs) of 31 fetuses, 10 lambs, and 5 adult animals. Six fetuses were studied on three occasions over a 2-week period from 120--135 days of gestation to provide longitudinal data on changes in insulin binding. Maximal percent binding of [125I]iodoinsulin and receptor concentration decreased significantly as the age of the animal increased (r = 0.76, P less than 0.001 and r = --0.49, P less than 0.001, respectively). Total loss of insulin binding to RBCs was estimated to occur in the second postnatal month, and the RBCs from the adult sheep showed no specific insulin binding. The osmotic fragility of RBCs in each developmental group of animals was also studied to assess possible differences in RBC membrane properties. RBC osmotic fragility was significantly lower in fetuses than in adult sheep (osmotic fragility 50 = 0.55% phosphate-buffered saline vs. 0.76% phosphate-buffered saline, respectively; P less than 0.001). The data suggest that fetal RBCs of lower osmotic fragility and high insulin binding capacity are progressively replaced during late prenatal and early postnatal life by adult-type RBCs of increased osmotic fragility and lacking binding capacity for insulin. The timing of the disappearance of insulin binding to RBCs coincides with the final transition in the animals from a monogastric to a ruminant metabolic state, and may reflect a change in the need for insulin with age.

An endogenous 'hypertensive factor' enhances the voltage-dependent calcium current.

The effects of an immunoaffinity-purified putative endogenous hypertensive factor (HF) on voltage-dependent calcium current in frog cardiac myocytes were assessed. In 9 out of 10 cells, HF reversibly increased the peak amplitude of the calcium current. HF increased peak calcium current density at -5 mV from a control level of 1.8 +/- 1.3 pA/pF (mean +/- SD) to 4.4 +/- 2.0 pA/pF. HF shifted the peak of the calcium current-voltage relationship in the hyperpolarizing direction. HF shifted the voltage dependence of the inactivation of the calcium current to more negative potentials with prepulses from -80 to 0 mV, but the inactivation was not affected with prepulses more positive than 0 mV. Modulation of the voltage-dependent calcium current by HF may be the mechanism underlying its pressor effects.

Solution structures in SDS micelles and functional activity at the bullfrog substance P receptor of ranatachykinin peptides.

A set of novel tachykinin-like peptides has been isolated from bullfrog brain and gut. These compounds, ranatachykinin A (RTKA), ranatachykinin B (RTKB), and ranatachykinin C (RTKC), were named for their source, Rana catesbeiana, and their homology to the tachykinin peptide family. We present the first report of the micelle-bound structures and pharmacological actions of the RTKs. Generation of three-dimensional structures of the RTKs in a membrane-model environment using (1)H NMR chemical shift assignments, two-dimensional NMR techniques, and molecular dynamics and simulated annealing procedures allowed for the determination of possible prebinding ligand conformations. RTKA, RTKB, and RTKC were determined to be helical from the midregion to the C-terminus (residues 4-10), with a large degree of flexibility in the N-terminus and minor dynamic fraying at the end of the C-terminus. The pharmacological effects of the RTKs were studied by measuring the elevation of intracellular Ca(2+) in Chinese hamster ovarian cells stably transfected with the bullfrog substance P receptor (bfSPR). All of the RTKs tested elicited Ca(2+) elevations with a rank order of maximal effect of RTKA >/= SP > RTKC >/= RTKB. A high concentration (1 microM) of the neuropeptides produced varying degrees of desensitization to a subsequent challenge with the same or different peptide, while a low concentration (1 pM) produced sensitization at the bfSPR. Our data suggest differences in amino acid side chains and their charged states at the C-terminal sequence or differences in secondary structure at the N-terminus, which do not overlap according to the findings in this paper, may explain the differing degree and type of receptor activation seen at the bfSPR.

Molecular characterization and functional expression of a substance P receptor from the sympathetic ganglion of Rana catesbeiana.

Substance P is an important neuropeptide neurotransmitter in the central, autonomic and enteric nervous systems. In sympathetic ganglia, substance P is thought to play a role in modulating synaptic transmission. Release of substance P by neuronal stimulation or direct application of substance P to ganglionic neurons increases neuronal excitability. An amphibian substance P receptor complementary DNA has been cloned and characterized from bullfrog, Rana catesbeiana, sympathetic ganglion complementary DNA libraries. The deduced primary structure contains features indicative of a seven transmembrane domain G-protein-coupled receptor. The deduced protein sequence shows 69% identity to previously cloned mammalian substance P receptors. In situ hybridization analysis performed on bullfrog sympathetic ganglia using digoxigenin-labelled complementary RNA probe demonstrated that approximately 75% of the principal neurons displayed reaction product above background levels. Radioligand binding studies were performed on stably transfected cells with [(125)I]Tyr-1-substance P as the ligand. Substance P had an IC50 of 16 nM and the agonist potency profile was substance P>neurokinin A >> neurokinin B. The order of potency for three tachykinins to increase intracellular calcium when applied to a stably transfected clonal cell line was substance P>neurokinin A >> neurokinin B. This order of agonist potency also held for inhibition of the M-type potassium current in intact bullfrog sympathetic neurons. The non-peptide substance P antagonists CP-96345 and RP-67580 at concentrations that block mammalian substance P receptors had little or no effect on the responses to substance P at the bullfrog receptor. Overall, these results demonstrate that the cloned sequence has the features consistent with and characteristic of a substance P receptor. The results are discussed with reference to the established pharmacology of the bullfrog substance P receptor and known structure activity relationships of mammalian tachykinin receptors.

Otitis media in the neonatal intensive care unit.

Thirty-eight of 125 premature infants who were hospitalized in a neonatal intensive care unit (NICU) had abnormal tympanic membrane mobility compatible with otitis media. Twenty-five of these 38 had received antibiotics within one week of otoscopic examination and were considered to have either serous otitis or partially treated bacterial otitis media; tympanocentesis was not performed in them. Tympanocentesis was performed in the remaining 13 infants who had not received antibiotics. Bacterial otitis media was confirmed in ten of the 13. Either staphylococcal (six cases) or Gram-negative enteric organisms (four cases) were isolated in cultures obtained by tympanocentesis in these cases. The four cases of Gram-negative infections occurred in infants within six weeks of birth. Nasotracheal intubation for more than seven days was significantly correlated with impaired tympanic membrane mobility compatible with otitis media. Otitis media occurs frequently among premature infants who are hospitalized in an NICU, and it should be looked for in any infant in whom sepsis is clinically suspected.

Inhibition by wortmannin of M-current in bullfrog sympathetic neurones.

1. The actions of wortmannin, an inhibitor of myosin light chain kinase (MLCK), on M-type potassium current of dissociated bullfrog sympathetic neurones have been examined. 2. The amplitude of M-current was measured by whole cell recordings from cells pretreated with wortmannin (0.01-10 microM) or the wortmannin vehicle, dimethylsulphoxide (0.0001-0.1 vol%), for 30 min. Internal (recording pipette) solutions having three different pCa values (6, 7 and 8) were used for the measurements. 3. Irrespective of the pCa, M-current was not detectable when the cells were pretreated with 10 microM wortmannin. Wortmannin, 3 microM, produced 85-95% inhibition of the M-current. Pretreatment with 10-30 nM wortmannin was without effect on M-current. 4. The M-current inhibition by wortmannin at concentrations of 0.1-1 microM depended on the pCa of the internal solution. Inhibition occurred only when the calcium-rich (pCa = 6) internal solution was used. 5. Pre-treatment of the cells with wortmannin (10 microM) did not affect rapidly-inactivating A-type or delayed rectifier-type potassium currents not did it alter inwardly rectifying sodium-potassium current (IH). 6. These observations show that M-current inhibition by wortmannin has two pharmacological profiles. One is calcium-dependent and occurs at lower concentrations (0.1-1 microM), and is attributed to inhibition of MLCK by wortmannin. At higher concentrations (3-10 microM), wortmannin has an additional, calcium-independent action, inhibiting the M-current by an unknown mechanism.

Premature rupture of membranes and preterm labor: neonatal infection and perinatal mortality risks.

The purpose of this study was to analyze neonatal infection rates and perinatal mortality in births complicated by premature rupture of membranes (PROM) and spontaneous labor before term (less than 37 weeks' gestation). Neonatal infection occurred more commonly in preterm compared with term infants. The risk of infection due solely to PROM was insignificant compared with the risk attributable to preterm birth. Perinatal mortality and cause-specific mortality varied inversely with gestational age. These rates were not significantly different between groups with or without PROM or with or without associated development of maternal endometritis. Although the mortality due to infection was higher in preterm compared with term groups, most preterm deaths were attributed to other factors, particularly anoxia and respiratory causes. The diverse fetal risks associated with PROM and the associated maternal infection risks analyzed support expectant management when PROM occurs preterm.

Tachykinin peptide-induced activation and desensitization of neurokinin 1 receptors.

The actions of four tachykinins on inhibition and desensitization of the M-current of bullfrog sympathetic neurons have been characterized. Radioligand binding parameters of the tachykinins were determined at a neurokinin receptor in a heterologous expression system. The correlation between binding, signaling and receptor regulation was investigated. A correlation between receptor binding and signaling was found between the peptides; however, their ability to produce desensitization was not correlated with binding and signaling. These results show that the ability of a tachykinin peptide to induce signal activation is not indicative of its ability to induce receptor regulation.

Actions of 4-aminopyridine on mammalian ganglion cells.

The effects of 4-aminopyridine (4-AP) on membrane electrical properties and synaptic transmission in neurons of the isolated rabbit superior cervical ganglion were investigated. 4-AP (0.03-0.1 mM) increased the amplitude of the fast excitatory postsynaptic potential (f-EPSP) without affecting appreciably either the acetylcholine (ACh) depolarization induced by iontophoresis of ACh or the passive and active membrane properties of the neurons. At concentrations of 1-5 mM, 4-AP reversibly depressed the amplitude of the f-EPSP as well as the ACh depolarization; a slight to moderate prolongation of the action potential duration was observed. In addition to the effects on evoked synaptic potentials, 4-AP induced spontaneous discharges which were abolished reversibly by curare, low Ca solution or Co. The results indicate that 4-AP at low concentrations facilitated evoked as well as spontaneous release of ACh by a presynaptic mechanism, whereas at higher concentrations it exerted a curare-like effect on the postsynaptic membrane.

Enkephalinergic modulation of non-cholinergic transmission in mammalian prevertebral ganglia.

The non-cholinergic excitatory potential elicited in neurons of the isolated inferior mesenteric ganglia was depressed by bath application of Met- or Leu-enkephalin and enhanced by naloxone or naltrexone. The membrane depolarization induced by exogenously applied putative transmitter substance P was not appreciably affected by either enkephalin (Enk) or opiate antagonists. Our results indicate that the non-cholinergic excitatory transmission which may be mediated by substance P in prevertebral ganglia is modulated presynaptically by endogenously released Enk in a manner that may resemble the interaction between these two peptides in the spinal cord.

Amyloid beta peptides act directly on single neurons.

A peptide consisting of residues 25-35 of the amyloid beta protein was applied to single neurons while monitoring membrane current by whole cell voltage clamp recording. Within minutes of direct exposure of a neuron to the amyloid beta peptide, a paroxysmal increase in neuronal membrane conductance was observed. This conductance does not resemble previously described ionic conductances in terms of its time-dependence, voltage-dependence or sensitivity to changes in extracellular or intracellular ionic constituents. The effect of the amyloid beta peptide was not mimicked or blocked by substance P nor was it prevented by low intracellular or extracellular Ca. The increased membrane permeability elicited by the peptides may lead to the neuropathology observed in Alzheimer's disease.

Hyperpolarizing shift of the M-current activation curve after washout of muscarine in bullfrog sympathetic neurons.

The mechanism underlying the over-recovery of an M-type potassium current following the washout of muscarine (20 microM) has been examined. Whole-cell recordings were made from single neurons dissociated from bullfrog sympathetic ganglia. During over-recovery, the maximum M-conductance decreased by about 2.8 nS while the steady-state M-current activation curve was displaced in the hyperpolarizing direction by about 13 mV. These data suggest that a hyperpolarizing shift in the kinetics of M-current causes over-recovery in amphibian autonomic neurons.