Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons.

Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.

Identification and localization of ryanodine binding proteins in the avian central nervous system.

Ryanodine binding proteins of the CNS have been identified using monoclonal antibodies against avian skeletal muscle ryanodine binding proteins. These proteins were localized to intracellular membranes of the dendrites, perikarya, and axons of cerebellar Purkinje neurons using laser confocal microscopy and immunoelectron microscopy. Ryanodine binding proteins were not found in dendritic spines. Immunoprecipitation and [3H]epiryanodine binding experiments revealed that the cerebellar ryanodine binding proteins have a native molecular weight of approximately 2000 kd and are composed of two high molecular weight (approximately 500 kd) polypeptide subunits. A comparable protein having a single high molecular weight polypeptide subunit was observed in the remainder of the brain. If the ryanodine binding proteins in muscle and nerve are similar in function, then the neuronal proteins may participate in the release of calcium from intracellular stores that are mechanistically and spatially distinct from those gated by inositol trisphosphate receptors.

Distribution of ryanodine receptors in the chicken central nervous system.

The ryanodine receptor (RR), an intracellular calcium release channel, has been identified in the nervous system but its contributions to neuronal function are unknown. We have utilized immunohistochemical techniques to establish the distribution of RRs in the central nervous system (CNS) of the chick as a step toward elucidating the function of RRs in this system. RR immunoreactivity is observed throughout the brain, most prominently in large neurons. The strongest immunoreactivity is found in cerebellar Purkinje neurons, but nuclei in the motor, visual and vestibular systems are also intensely labeled, and immunoreactive neurons are observed the olfactory bulb and the hippocampus. In these neurons, labeling is prominent in cell bodies, dendrites and axons, but is not observed in the dendritic spines or in plasma membranes. The neuronal RRs bind [3H]ryanodine with high affinity and this activity is regulated by calcium, caffeine, MgCl2/ATP and ionic strength. Multiple forms of the RRs are found in the chicken CNS. Immunoprecipitation and localization studies using RR isoform specific monoclonal antibodies reveal major differences in their distribution. The predominant species in the cerebellum is similar to the skeletal muscle isoform while there is a lower level of expression of either the cardiac or beta skeletal isoforms. In the remainder of the brain, the predominant isoform is similar to the cardiac or beta skeletal muscle isoforms. The broad distribution of RRs in the CNS suggests that calcium release events mediated by these proteins may have a functional role in a diverse array of neurons. Moreover within the populations of neurons expressing RR's, the presence of specific RR isoforms may correlate with specialization in the calcium release events mediated by these proteins.

Metacognitive and frontal lobe processes: at the interface of cognitive psychology and neuropsychology.

During the past several decades, research in both cognitive psychology and neuropsychology has become increasingly concerned with the processes that monitor and control human cognition. In the area of cognitive psychology, this research has focused on metacognition or metacognitive processes, and in the area of neuropsychology, it has focused on frontal lobe processes. It is evident that both areas have been referring to very similar concepts, with some variations, but with notably little acknowledgement of each other. The principal purpose of this study was to conduct an analysis of the main research within each of these areas and to examine the continuities and discontinuities in the theoretical premises and empirical results between the two areas. Results indicated that the two areas are remarkably similar in both theoretical premises and empirical results, but that cognitive psychology emphasizes monitoring and control, whereas neuropsychology tends to emphasize control only. A synthesis that unites the work in cognitive psychology and neuropsychology would supply many new directions for research.

A comparison of somatic chromosomal instability in tissue culture regenerants from Medicago media Pers.

Two cultivars (Heinrichs, Reaver) and two breeding lines (Br1, Le1) from Medicago media were cultured in a media protocol consisting of a high concentration 2,4-D induction step. Regenerants were produced from all four stocks. Representative samples from each regenerant population along with the corresponding control population were cytologically analyzed for chromosomal and pollen abnormalities. While numerical changes in chromosome numbers were found in all regenerant populations, there was considerable variation between the four stock groups. Heteroploidy was observed for both hypo and hyper aneuploid regenerants, but there were no differences in pollen stainability between hypo and hyper aneuploid regenerants and 'euploid' regenerants. Tissue culture regenerants generally produced a lower pollen stainability percent as compared to control populations grown from seeds. Gross and cryptic changes in chromosomes, or hormonal carry over effects or both were considered causes for poor pollen stainability in tissue culture regenerants. Cytological analyses indicate that the cultivar might play an important role in the cytological stability or instability of regenerant populations. Exploitation of this naturally existing situation to produce 'euploid' regenerants for field experiments and to obtain gross cytological stability in somaclones is discussed.

Stimulus-dependent pacemaker activity in the distal canine lower esophageal sphincter.

Electrical and mechanical properties of the distal canine lower esophageal sphincter were studied in vitro to investigate possible means of inducing pacemaker activity. Both direct excitation and block of potassium conductance were investigated. The acetylcholine analog, carbachol, induced tissue depolarization and increase in tone but no electrical slow waves. Tetraethylammonium (TEA) chloride induced depolarization and evoked continuous spiking activity and increase in tone. BaCl did not depolarize the tissue but low amplitude spiking activity developed and increased tone. The putative potassium channel blocker, aminacrine at 2 X 10(-4) M, induced electrical slow wave activity in the distal lower esophageal sphincter, with or without superimposed spikes, accompanied by phasic contractile activity. This activity closely resembled the spontaneous pacemaker activity observed previously in the proximal lower esophageal sphincter. The aminacrine-induced activity was abolished by calcium influx blockers. Aminacrine, but not TEA or BaCl, abolished the nonadrenergic nerve-mediated inhibitory junction potentials. In conclusion, block of inhibitory innervation, and induction of electrical slow waves as a control mechanism for phasic contractile activity, seems to require blockade of an aminacrine- but not TEA-sensitive potassium conductance.

Embryo-callus-regenerated hybrids and their colchicine-induced amphiploids between Elymus canadensis and Secale cereale.

Intergeneric hybrids recovered through plant regeneration from embryo callus culture and their colchicine-induced amphiploids were obtained from a cross of Elymus canadensis with Secale cereale (cv 'Gazeller'). The embryo-callus-regenerated F1 plants grew vigorously to maturity and regrew well after clipping, while the embryo-rescued F1 died of hybrid necrosis before maturity. Most of the morphological characters of the sterile F1 hybrids were intermediate between the parents, but tiller number and dry matter yield were higher than the parents. Amphiploids from these F1 plants had improved fertility but were less vigorous than the F1 plants. The predominance of univalents in the F1 and bivalents in the amphiploids indicated that the genomes S, H, and R were distinct. However, the occasional occurrence of multivalents reflected a random, intergenomic or intragenomic pairing. The mean chromosome associations of the F1 (2n=21, SHR), the C0 amphiploids (2n=42, SSHHRR), and the C1 amphiploid (2n=40) at metaphase I (MI) were 16.51I+2.05II+0.06III+0.02IV+0.02V, 2.20I+19.87II +0.02IV, and 7.10I+16.37II+0.04III+0.02IV, respectively. These amphiploids could be exploited as new germplasm for forage crop improvement by controlled introgression and backcrosses to the parents.

Embryogenesis and plant regeneration from tissue culture of Canada wildrye, Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.

Pacemaker activity in the proximal lower oesophageal sphincter of the dog.

1. The electrical and mechanical activities of different regions of the canine lower oesophageal sphincter were measured using the single sucrose gap technique. 2. Spontaneous electrical activity was found in the region 0-6 mm oral to the squamocolumnar border. 3. The electrical activity consisted of bursts of spikes superimposed on slow waves. The slow-wave frequency ranged from 0.6 to 5 min-1 in different muscle strips. 4. The slow wave-spike complex and associated contraction were insensitive to tetrodotoxin and atropine. 5. In the pacemaker region, electrical stimulation of intrinsic nerves evoked excitatory junction potentials (atropine sensitive), inhibitory junction potentials (non-adrenergic) and post-stimulus excitation. 6. Increase in the frequency of the slow waves was obtained by muscarinic receptor stimulation (carbachol 10(-7) M) and 10 mM-KCl. 7. The distal lower oesophageal sphincter exhibited a high basal tension but did not show spontaneous electrical activity and stimulation of intrinsic nerves revealed only non-cholinergic, non-adrenergic inhibition. 8. The electrical slow-wave activity observed in the proximal sphincter may constitute the control mechanism for the phasic nature of the contractile activity seen during both the postprandial period and phase III of the interdigestive migrating myoelectric complex. 9. The neural cholinergic activity present in the proximal lower oesophageal sphincter suggests the possibility of neural modulation of the myogenic control activity.

Embryogenesis and plant regeneration of Medicago spp. in tissue culture.

Ten cultivars and breeding lines from two species of alfalfa (Medicago media and M. sativa) were screened for their ability to produce embryos and plantlets from the root and hypocotyl under three different tissue culture protocols. The three protocols differed in basal salt composition, vitamins, hormones and cytokinin additions. That protocol having a high 2-4,D low cytokinin induction step gave the highest percentage of embryogenic calli in some cultivars and lines. M. media cultivars and breeding lines had a high percentage of embryoid formation. M. sativa cultivars gave no embryoid formation. Two M. media breeding lines (Br1 and Le1), which were intermediate in the percentage of embryogenic calli formed from explants, had the highest number of regenerated plants established in soil. The creeping rooted M. media cultivar Heinrichs produced the highest percentage of embryogenic calli from explants but most of these embryoids were abnormal and failed to grow in soil or vermiculite. Accordingly, successful regeneration is directly related to the quality and quantity of the embryoids produced.

Primary cytomegalovirus infection in pregnancy. Incidence, transmission to fetus, and clinical outcome.

We studied 16 218 pregnant women from two income groups to determine the incidence of primary cytomegalovirus (CMV) infection and its consequences for the offspring. In the high-income group, 64.5% of the women were seronegative for CMV and 1.6% had primary CMV infection. In the low-income group, only 23.4% of the women were seronegative for CMV, but 3.7% experienced a primary infection. The rate of transmission in utero was similar in the two groups (39% and 31%). Congenital infections were more frequent in the low-income group; however, primary CMV accounted for 25% of the congenital infections in this group, in contrast to 63% of the high-income cases. Infections acquired early and late in gestation had similar rates of transmission in utero, but three infants (8%) with symptomatic congenital infection and five infants (13.5%) who have developed significant handicaps were exposed in the first half of pregnancy. Primary CMV infection during pregnancy poses a 30% to 40% risk of intrauterine transmission and adverse outcome is more likely when infection occurs within the first half of gestation.

Congenital cytomegalovirus infection: The relative importance of primary and recurrent maternal infection.

We studied the incidence of primary and recurrent cytomegalovirus infection in 3712 pregnant women--2698 of middle to high income and 1014 of low income--to determine whether there were differences in the effects on the fetus. In the higher-income group, 1203 women (45 per cent) did not have antibodies to cytomegalovirus and were therefore susceptible to primary infection, as compared with 179 women (18 per cent) of low income. Congenital infection occurred more often (1.6 vs. 0.6 per cent) in infants in the low-income group. In this group it was associated with recurrent maternal infection more often (in 82 per cent) than with primary maternal infection, whereas in the upper-income group, it was associated with primary maternal infection in half the cases. Altogether, there were 32 cases of congenital cytomegalovirus infection - 16 in each group. Whereas primary maternal infection resulted in fetal infection in only half the cases, it was more likely to ge associated with clinically apparent disease than was recurrent infection. When these cases were combined with 28 cases of congenital infection retrospectively identified at other prenatal clinics, five of 33 infected infants born after primary maternal infection had clinically apparent disease, as compared with none of 27 born after recurrent maternal infection. We conclude that congenital cytomegalovirus infection resulting from primary maternal infection is more likely to be serious than that resulting from recurrent infection, and is more likely to occur in upper socioeconomic groups.