Mining the human genome using microarrays of open reading frames.

To test the hypothesis that the human genome project will uncover many genes not previously discovered by sequencing of expressed sequence tags (ESTs), we designed and produced a set of microarrays using probes based on open reading frames (ORFs) in 350 Mb of finished and draft human sequence. Our approach aims to identify all genes directly from genomic sequence by querying gene expression. We analysed genomic sequence with a suite of ORF prediction programs, selected approximately one ORF per gene, amplified the ORFs from genomic DNA and arrayed the amplicons onto treated glass slides. Of the first 10,000 arrayed ORFs, 31% are completely novel and 29% are similar, but not identical, to sequences in public databases. Approximately one-half of these are expressed in the tissues we queried by microarray. Subsequent verification by other techniques confirmed expression of several of the novel genes. Expressed sequence tags (ESTs) have yielded vast amounts of data, but our results indicate that many genes in the human genome will only be found by genomic sequencing.

Synthesis of dopamine and octopamine in the crustacean stomatogastric nervous system.

The spiny lobster stomatogastric nervous system synthesizes dopamine and octopamine in vitro from exogenous [3H]tyrosine. Each amine accumulates with a specific distribution among 9 separately analyzed regions within the system. Synthesis of other catecholamines was not observed. [3H]Dopamine is found in nerves, ganglia, and identified commissural ganglion cell bodies in which catecholamine histofluorescence has been demonstrated. The biosynthetic and histochemical data together indicate that dopaminergic cells send axons from the commissural ganglia to the stomatogastric ganglion neuropil along the same pathway followed by fibers that activate the pylroic motor network. The results support the hypothesis that dopamine mediates activation of the pyloric system in vivo, as observed in vitro. [3H]Octopamine accumulates primarily in the commissural and stomatogastric ganglia, where it may modulate neuronal activity, but octopaminergic cells and release sites within the stomatogastric system have not been identified.

Nerve growth-promoting factor produced in culture media conditioned by specific CNS tissues of the snail Helisoma.

Medium conditioned by tissue from the CNS of the snail, Helisoma, is capable of promoting neurite outgrowth in isolated neurons from adult central ganglia. The conditioning factor(s) (CF), contained in conditioned medium (CM), is produced only by central ganglionic rings and buccal ganglia and not by other tissues, including hemolymph. CF requires a minimum of 24 h to be produced or released into the medium. At 12 h growth-promoting activity was not detectable. CF binds tightly to the polylysine substratum and its activity is not mimicked by addition of various sera, NGF or fibronectin. CF activity is abolished by chymotrypsin, trypsin or heating to 100 degrees C, but is stable to DNase and RNase treatment. The percentage of cells exhibiting neurite outgrowth is approximately linear with the amount of neural tissue used to condition the medium up to 2 ganglionic rings/ml. Addition of more ganglia fails to stimulate a greater response. This apparent plateau of CM activity appears to be a function of production and/or release of CF, rather than a saturation effect on plated cells, since dose-response curves for dilutions of CM are approximately linear regardless of the number of ganglia used for conditioning. In addition, anisomycin inhibits 35% of CF appearance under conditions of over 90% protein synthesis inhibition in the ganglia used to produce the CM. Under these conditions anisomycin has no apparent effect on the maintenance of electrical excitability. The inhibitor data suggest that 65% of CF is derived from a pre-existing storage pool and that the remainder is synthesized during the 72 h conditioning period.

Muscarinic antagonist enhances one-trial food-aversion learning in the mollusc Pleurobranchaea.

One hour before training, in two replicate studies on the sea slug Pleurobranchaea californica, all animals (N = 114) received body-cavity injections of scopolamine, oxotremorine, or the equivalent volume of the saline/seawater vehicle that was used to inject the drugs. The low drug doses (2 mumol/kg) were near the threshold for generating observable neurophysiological responses, but did not affect feeding thresholds arising to a stimulus derived from beer (Sbr) and to one derived from squid (Ssq). Before training, the animals did not discriminate between Sbr and Ssq, as indicated by similar thresholds to both stimuli. During training, experimental animals in each injection group received Sbr alone for 10 sec and then paired with electric shocks for 50 sec; control animals received shocks 1 hr after Sbr. Postconditioning tests began 12 hr after training and were repeated once daily thereafter. After training, all experimental groups exhibited the expected aversive behavior to Sbr, as indicated by 100- to 1000-fold increases in feeding thresholds, and retained low thresholds to Ssq, but the scopolamine animals were better able to discriminate between Sbr and Ssq than either of the other experimental groups. The aversive responses to Sbr increased over a 3-day period, but there appeared to be no difference between injection groups on such a long-duration "consolidation" phase. Of the control groups, only the scopolamine animals exhibited low feeding thresholds to both Sbr and Ssq. The other control groups exhibited similar behavior as the experimental animals, indicating that associative factors relating to the 1-hr separation between Sbr and shocks may have produced the behavior in these control animals. Thus, by comparison to the other injections, scopolamine 1) increased the ability of the experimental animals to make the discrimination between Sbr and Ssq, and 2) prevented learning to avoid Sbr in the control animals. An accompanying paper provides a detailed characterization of muscarinic receptor pharmacology in Pleurobranchaea.

Security of capsular fixation: small-versus large-hole plate-hepatic lenses.

PURPOSE: To assess the effect of relatively large positioning holes on the security of capsular bag fixation of plate-haptic silicone intraocular lenses (IOLs). SETTING: Center for Research on Ocular Therapeutics and Biodevices, Department of Ophthalmology, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA. METHODS: This study tested the hypothesis that larger holes allow ingrowth of lens material, fibrous tissue, or both through them, which helps fixate the lens more firmly in the capsular bag. Five rabbits had bilateral continuous curvilinear capsulorhexis, phacoemulsification, and implantation of a plate-haptic silicone IOL. An IOL with a small, round positioning hole (Staar AA-4203V) was implanted in the right eye in each rabbit, and a large-hole IOL (Staar AA-4203VF) was implanted in the left eye. After 2 months, all rabbits were killed. The force required to extract one haptic from the capsular bag was measured with a digital force meter. All eyes had histopathological analysis. RESULTS: It was slightly more difficult to extract a large-hole IOL from the capsular bag, although this trend was not statistically significant. However, histopathological analysis consistently showed 360 degree synechia formation through the holes, showing that the IOL could be securely fixed in position. CONCLUSIONS: Proliferation of lens epithelial cells through a large positioning hole in a plate-haptic silicone IOL may improve the long-term security of capsular bag fixation. This will help reduce the incidence of IOL decentration and dislocation.

Mini-haptics to improve capsular fixation of plate-haptic silicone intraocular lenses.

PURPOSE: To evaluate the effects of a new mini-haptic design on the strength and stability of capsular bag fixation of plate-haptic silicone intraocular lenses (IOLs) and determine whether this design encourages the growth of regenerating lens material or fibrous tissue around the haptic biomaterial and thus improves lens fixation in the capsular bag. SETTING: Center for Research on Ocular Therapeutics and Biodevices, Storm Eye Institute, Medical University of South Carolina, Department of Ophthalmology, Charleston, South Carolina, USA. METHODS: Six rabbits had bilateral continuous curvilinear capsulorhexis, phacoemulsification, and plate-haptic silicone IOL implantation. Each rabbit had a small-hole plate IOL (Chiron C10UB) implanted in the right eye and a mini-haptic plate IOL (Chiron C40UB) in the left eye. All rabbits were killed at 2 months. The force required to extract one haptic from the capsular bag was measured with a digital force gauge. Histopathologic analysis was performed on all specimens. RESULTS: The mini-haptic style IOLs required significantly more extraction force than the small-hole design (P = .011). Histopathologically, proliferating lens epithelial cells were observed growing circumferentially around the mini-haptics, causing a 360 degree synechia formation. This formation did not occur with the conventional small-hole plate IOLs used as the control. CONCLUSIONS: Lens epithelial cell proliferation around the mini-haptics significantly improved capsular bag fixation of the plate-haptic silicone IOL. This should decrease the incidence of clinical decentration and dislocation.

Dietary carnitine did not influence performance and carcass composition of broiler chickens and young turkeys fed low- or high-fat diets.

Two experiments were conducted to determine the influence of supplemental dietary carnitine on performance and carcass composition of young turkeys and broiler chickens. Experiments 1 and 2 were done with poults to 21 d of age and broilers to 45 d of age, respectively. Three dietary concentrations of L-carnitine (0, 50, or 100 mg/kg) were fed in a factorial arrangement with two concentrations of animal-vegetable fat (A-V fat), 2.25 or 8% in Experiment 1 and 1 or 5% in Experiment 2. L-Carnitine did not affect (P > .05) BW gain or feed efficiency in either experiment, irrespective of concentration of dietary fat. Similarly, proximate composition of 21-d-old poults and 45-d-old broilers was not changed by L-carnitine. Increasing levels of fat supplementation improved feed efficiency of poults and improved 45-d BW and feed efficiency of broilers. Carcass fat of poults and broilers was increased (P < or = .05) by supplemental fat at the expense of carcass water and protein.

Research note: vitamin E status of turkey poults as influenced by different dietary vitamin E sources, a bile salt, and an antioxidant.

An experiment was conducted to determine the effects of level and chemical form of dietary vitamin E on alpha-tocopherol status of poults. The effects of a dietary bile salt and an antioxidant on concentrations of alpha-tocopherol in serum and liver were also tested. Six dietary treatments were obtained by supplementing a corn-soybean meal diet with 12 IU of DL-alpha-tocopheryl acetate (TA)/kg (LE), 12 IU of TA plus 800 mg of sodium taurocholate/kg (LB), 12 IU of TA plus 500 mg of ethoxyquin/kg (LS), 12 IU of D-alpha-tocopheryl polyethylene glycol 1,000 succinate (TPGS)/kg (LT), 100 IU of TA/kg (HE), and 100 IU of TPGS/kg (HT). Growth rate and feed efficiency of poults were unaffected (P > .05) by dietary treatments. The HE diet increased alpha-tocopherol in liver (P < .01) at 14 and 21 days of age. Liver and serum alpha-tocopherol concentrations were unaffected by dietary TPGS (LT and HT diets) at any age. Serum alpha-tocopherol concentration was unaffected by dietary treatments at 5 days of age. The HE diet, however, increased (P < .01) serum alpha-tocopherol at 9, 14, and 21 days of age. Age-related changes in alpha-tocopherol concentration were observed. Both liver and serum alpha-tocopherol decreased markedly from 1 to 14 days of age. The HE diet only partly alleviated the reduction of alpha-tocopherol in liver and serum. The water-soluble form of vitamin E, TPGS, dietary sodium taurocholate, or dietary ethoxyquin, did not prevent the marked decline in alpha-tocopherol concentration of liver and serum during the 21-day experiment.

Clinical pharmacy services in a hospital-based home care program.

The Hospital-Based Home Care (HBHC) Program at the Veterans Administration Medical Center in San Francisco, California, is a specialized medical service designed to provide comprehensive continuity of care to the veteran patient in his or her own home through the use of a multidisciplinary team approach. Professional health care services are provided by nurses, dieticians, physical therapists, pharmacists, physicians, and social workers. Professional services provided by the clinical pharmacists include: the evaluation of prescribed medication regimen; product identification; patient counseling and education; drug therapy consultant to the HBHC team; and liaison between the HBHC team and outpatient pharmacy services.

[(3)H]Choline uptake and metabolism in nonsynaptic regions of a crustacean sensory nerve.

The posterior stomach nerve (PSN) is a crustacean sensory nerve containing about 60 cholinergic neurons, which are devoid of synaptic interactions. Kinetic analysis shows that the PSN takes up [(3)H]choline by both low-affinity (K(m) = 163 micron) and high-affinity (Na(¿dependent) (K(m) - 1 micron) processes. The capacity of the high-affinity system is only about 1% that of the low-affinity system. The high-affinity system is not tightly coupled to acetylcholine (ACh) synthesis, and it appears that both ACh and phosphorylcholine are formed from an intracellular pool of choline, which is fed by both uptake systems. There are differences in the rates of [(3)H]choline uptake and (3)H metabolite accumulation between regions of the PSN that contain neuronal cell bodies and those that do not. These differences may arise from differences in the relative proportion of neuronal to nonneuronal tissue in each nerve region.

Synthesis of octopamine by insect dorsal median unpaired neurons.

Dorsal unpaired median (DUM) neurons of locusts and grasshoppers inhibit the instrinsic rhythm of contraction of metathoracic extensor tibia muscle fibers. This physiological action is mimicked by very low concentrations of octopamine and by higher concentrations of dopamine and noradrenaline. We have examined the synthesis of biogenic amines from tritiated tyrosine by these neurons. DUM cell bodies and a peripheral nerve containing the DUM axon which terminates in the extensor tibia both produced octopamine, but neither noradrenaline nor dopamine synthesis was detected. The observations support the suggestion that the dorsal unpaired median neurons are octopaminergic.

A neurochemical description of the dopaminergic innervation of the stomatogastric ganglion of the spiny lobster.

The spiny lobster stomatogastric ganglion has been shown to be innervated by catecholaminergic processes which derive from cells of large central ganglia (Kushner and Maynard, 1977). Biochemical evidence had indicated that the stomatogastric system synthesizes dopamine and not norepinephrine from tritiated tyrosine (Barker, Kushner, and Hooper, 1979). Studies reported here document that the stomatogastric ganglion itself contains dopamine, as measured with a sensitive endogenous assay. Moreover, the ganglion can synthesize dopamine from tritiated tyrosine or DOPA. Additionally, when incubated in tritiated dopamine, the ganglion takes up dopamine and protects it from degradation; this process is inhibited by cocaine. When incubated with 3H-tyrosine, small but measurable amounts of tritiated dopamine were detected in the medium surrounding the ganglion.

Synaptic mechanisms that generate network oscillations in the absence of discrete postsynaptic potentials.

Synaptic mechanisms were examined in the pyloric network of the lobster stomatogastric which generate network oscillations in the absence of discrete postsynaptic potentials (PSPs). In normal saline, the unstimulated pyloric network underwent weak bursting in only a few cells. Stimulation of the input nerve, or bath application of the input neurotransmitter dopamine, produced similar vigorous bursting in many pyloric neurons. In saline-containing tetrodotoxin (TTX) plus dopamine, action potentials and corresponding discrete PSPs were blocked, but the underlying slow wave oscillations in network neurons continued. No oscillations occurred in TTX-saline without dopamine. The generation of these nonspiking network oscillations can be explained by the interaction between two synaptic mechanisms which do not produce discrete PSPs: neurotransmitter activation of bursting pacemaker oscillations in a single network neuron, and graded inhibition between network neurons.

Activation by dopamine of patterned motor output from the buccal ganglia of Helisoma trivolvis.

The buccal ganglia of the snail, Helisoma trivolvis, contain an intrinsic system of dopamine-containing neurons (Trimble, Barker, and Bullard, 1983). Dopamine, when bath applied to the isolated buccal ganglia, activates patterned motor output in a dose-dependent fashion. Haloperidol blocks the activating effect of dopamine, but the similar activation evoked by serotonin is not blocked by haloperidol. We suggest that there are two separate mechanisms for activating patterned motor output from the buccal ganglia. One is serotonergic, emanating from identified cerebral ganglion cells (Granzow and Kater, 1977), while the other is dopaminergic, involving neurons intrinsic to the buccal ganglia.

Separate factors produced by the CNS of the snail Helisoma stimulate neurite outgrowth and choline metabolism in cultured neurons.

Neurons from the snail Helisoma require a brain-derived factor(s) for neurite outgrowth in both organ and isolated cell culture. This factor is released from the CNS of Helisoma when brains are incubated in defined medium, producing a conditioned medium (CM). In addition to its growth-promoting activity, CM also enhances total uptake of 3H-choline and the incorporation of 3H-choline into specific metabolites: acetylcholine, phosphorylcholine and lipid. This choline metabolism-enhancing factor(s) is distinct and separable from neurite growth-promoting factor: 1. Over 95% of neurite growth-promoting activity can be removed from CM by adsorption to a polylysine surface while there is no loss of choline metabolism-enhancing activity. 2. When central ganglia were treated with anisomycin, a potent inhibitor of molluscan protein synthesis, the choline metabolism-enhancing activity was completely absent from the resulting CM, while the growth promoting activity was reduced by only 35%. These results suggest that the Helisoma CNS produces a variety of trophic factors that are involved in regulating the interaction between neuronal growth and metabolism.

Acetylcholine synthesis and accumulation in the CNS of Drosophila larvae: analysis of shibirets, a mutant with a temperature-sensitive block in synaptic transmission.

A radiochemical method is applied to the study of neurotransmitter metabolism in Drosophila. The larval CNS is a favorable system for analyzing acetylcholine (ACh) metabolism, since the pool of [3H]ACh rapidly reaches a steady state with a high ratio of intracellular [3H]ACh to [3H]choline. A temperature-sensitive paralytic mutant, shibirets, shows reduced [3H]ACh accumulation at the restrictive temperature. This reduction is not the result of decreased synthesis of [3H]ACh, but rather an abnormally rapid rate of release, which is not prevented by blocking tetrodotoxin-sensitive nerve activity.

Autoradiography using storage phosphor technology.

We describe the application of photostimulable storage phosphor imaging plates to autoradiography of samples labeled with 32P, 14C and 35S. Imaging plates can detect a 32P-labeled sample at an exposure level of 1 disintegration/mm2, and 14C and 35S at 25 disintegrations/mm2. Compared to X-ray film, imaging plates offer increased sensitivity of 15- to 250-fold for 32P and 20- to 100-fold for 14C and 35S. Resolution is in the 0.3 mm range and is therefore suitable for any gel or blot application.

Migration and differentiation of neural crest and ventral neural tube cells in vitro: implications for in vitro and in vivo studies of the neural crest.

During vertebrate development, neural crest cells migrate from the dorsal neural tube and give rise to pigment cells and most peripheral ganglia. To study these complex processes it is helpful to make use of in vitro techniques, but the transient and morphologically ill-defined nature of neural crest cells makes it difficult to isolate a pure population of undifferentiated cells. We have used several established techniques to obtain neural crest-containing cultures from quail embryos and have compared their subsequent differentiation. We confirm earlier reports of neural crest cell differentiation in vitro into pigment cells and catecholamine-containing neurons. However, our results strongly suggest that the 5-HT-containing cells that develop in outgrowths from thoracic neural tube explants are not neural crest cells. Instead, these cells arise from ventral neural tube precursors that normally give rise to a population of serotonergic neurons in the spinal cord and, in vitro, migrate from the neural tube. Therefore, results based on previously accepted operational definitions of neural crest cells may not be valid and should be reexamined. Furthermore, the demonstration that cells from the ventral (non-neural crest) part of the neural tube migrate in vitro suggests that the same phenomenon may occur in vivo. We propose that the embryonic "neural trough," as well as the neural crest, may contribute to the PNS of vertebrates.

Dopamine in a molluscan nervous system: synthesis and fluorescence histochemistry.

The nervous system of the pond snail, Helisoma trivolvis, was investigated for its ability to synthesize and accumulate 3H-catecholamines from 3H-tyrosine. 3H-Dopamine, but not 3H-norepinephrine, was synthesized by several ganglia. The highest accumulations were found in the cerebral, pedal, and buccal ganglia. The Falck-Hillarp and glyoxylic acid fluorescence histochemical techniques were applied to the buccal ganglia to visualize dopamine-containing cells. Fluorescing cells were found on both dorsal and ventral sides of the ganglion. Peripheral nerves of the buccal ganglia also displayed catecholamine fluorescence and accumulated 3H-dopamine. However, no 3H-dopamine synthesis occurred in the cerebral-buccal connectives, which connect the buccal ganglia with the rest of the central nervous system. Therefore, we conclude that there is a dopaminergic system intrinsic to the buccal ganglia and their peripheral targets.