Transforming electrocortical mapping data into standardized common space.

Subdural grid electrodes are implanted routinely for the pre-surgical work up of epilepsy. While different approaches are available, many centers, including ours, visualize electrode locations by co-registering pre-operative 3-D MR images with post-implantation 3-D CT images. This method allows the determination of the electrode positions in relation to the individual patient's anatomy, but does not easily allow comparison across patients. The goal of this study was to develop and validate a method for transforming electrode positions derived from 3-D CT images into standardized space. We analyzed data from twelve patients with subdurally implanted electrodes. Volumetric CT and MRI images were co-registered and then normalized into common stereotactic space. Electrode locations were verified statistically by comparing distances between the anterior commissure and a representative sampling of 8 electrode sites per patient. Results confirm the accuracy of our co-registration method for comparing electrode locations across patients.

Structure and receptor binding of PYY analogs.

Differences in the structure of PYY and two important analogs, PYY [3-36] and [Pro34]PYY, are evaluated. Y-receptor subtype ligand binding data are used in conjunction with structural data to develop a model for receptor subtype selective agonists. For PYY it is proposed that potent binding to Y1, Y4 and Y5 receptors requires the juxtaposition of the two termini while Y2 binding only requires the C-terminal helix. Further experiments that delineate between primary and tertiary structure contributions for receptor binding and activation are required to support the hypothesis that tertiary structure is stable enough to influence the expression of PYY's bioactivity.

Domain 1.1 of the sigma(70) subunit of Escherichia coli RNA polymerase modulates the formation of stable polymerase/promoter complexes.

The sigma 70 (sigma(70)) subunit of Escherichia coli RNA polymerase specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma(70) is present within polymerase holoenzyme, two of its domains, 2.4 and 4.2, interact with sequences within the -10 and -35 regions, respectively, of promoter DNA. However, in free sigma(70), DNA binding is prevented by domain 1.1, the N-terminal domain of the protein. Previous work has demonstrated that the presence of domain 1.1 is required for efficient transcription initiation at the lambda promoter P(R). To investigate whether this is a general property of domain 1.1, we have used five promoters to compare polymerases with and without domain 1.1 in in vitro transcription assays, and in assays assessing the formation and decay of stable, pretranscription complexes. We find that the absence of domain 1.1 does not render the polymerase defective at all of these promoters. Depending on the promoter, the absence of domain 1.1 can promote or inhibit transcription initiation by affecting the formation of stable pretranscription complexes. However, domain 1.1 does not affect the stability of these complexes once they are formed. For polymerases containing domain 1.1, the efficiency of stable complex formation correlates with how well the -10 and -35 regions of a promoter match the ideal sigma(70) recognition sequences. However, when domain 1.1 is absent, having this match becomes less important in determining how efficiently stable complexes are made. We suggest that domain 1.1 influences initiation by constraining polymerase to assess a promoter primarily by the fitness of its -10 and -35 regions to the canonical sequences.

Simultaneous measures of contraction and intracellular calcium in single, cultured smooth muscle cells.

Simple methods are presented for quantitating contraction and intracellular calcium simultaneously in single, cultured smooth muscle cells. These methods are the first to demonstrate that reliable velocities of cell shortening can be measured in cultured smooth muscle cells and that cells in vitro exhibit shortening velocities comparable to those measured in the fastest phasic muscles in situ. Temporal relationships between changes in intracellular calcium and shortening within single cells were determined with a resolution of 100 ms and were consistent with measures in more "classical" preparations. Intracellular calcium rose quickly and transiently 10-fold above the basal level of 80-90 nM in response to the muscarinic agonist, carbachol. Shortening of the cells occurred 200 ms after intracellular calcium began to rise. The sensitivity and reliability of these methods allowed the effects of different stimuli to be easily resolved. The present report demonstrates that genuine contractility need not be ignored in cultured smooth muscle cells and that the temporal relations between shortening and intracellular calcium mobilization can be quantitatively assessed in controlled in vitro environments.

Effects of amino acid substitutions at conserved and acidic residues within region 1.1 of Escherichia coli sigma(70).

Amino acid substitutions in Escherichia coli sigma(70) were generated and characterized in an analysis of the role of region 1.1 in transcription initiation. Several acidic and conserved residues are tolerant of substitution. However, replacement of aspartic acid 61 with alanine results in inactivity caused by structural and functional thermolability.

A mutation in region 1.1 of sigma70 affects promoter DNA binding by Escherichia coli RNA polymerase holoenzyme.

The sigma subunit of eubacterial RNA polymerase is essential for initiation of transcription at promoter sites. It directs recognition of DNA sequences by holoenzyme (alpha2betabeta'sigma) and facilitates subsequent steps in the initiation pathway. The primary sigma factor from Escherichia coli, sigma70, has four regions that are conserved among members of the sigma70 family. Previous work has shown that region 1.1 modulates DNA binding by regions 2 and 4 when sigma is separated from the core subunits, and is required for efficient progression through the later steps of initiation in the context of holoenzyme. In this report, we show that an amino acid substitution at position 53 in region 1.1, which converts isoleucine to alanine (I53A), creates a sigma factor that associates with the core subunits to form holoenzyme, but the holoenzyme is severely deficient for promoter binding. The I53A phenotype can be suppressed by truncation of five amino acids from the C-terminus of sigma70. We propose that the behavior of sigma70-I53A is a consequence of impaired ability to undergo a critical conformational change upon binding to the core subunits, which is needed to expose the DNA-binding domains and confer promoter recognition capability upon holoenzyme.

Development and implementation of evidence-based guidelines: a multisite demonstration project.

The development of alternative patient care delivery systems is being explored by health care providers, managed care corporations, insurance companies, and the government. Pathways, guidelines, care maps, and algorithms are techniques that assist in the development of patient care delivery systems with the potential to both decrease cost and ensure quality. This article reviews our experience with a program designed to transport clinical guidelines and pathways developed from evidence-based scientific knowledge to 7 acute care facilities located throughout the United States.

Vitronectin regulates smooth muscle contractility via alphav and beta1 integrin.

Previous work from this laboratory has established a method for maintaining physiological contractility of dissociated avian smooth muscle in a defined medium at low density. The present report emphasizes the dramatic potency of serum to alter smooth muscle phenotype and induce a loss of contractility. Vitronectin, a molecule purified from plasma, mimicked these effects of serum via an integrin that is RGD-sensitive. Studies utilizing blocking antibodies against vitronectin demonstrated that the presence of this specific adhesion molecule was necessary for the serum-induced loss of contractility. Based on the actions of function-blocking antibodies and RGD-containing peptides, the integrin alphavbeta1 appears to be the primary receptor involved in vitronectin's ability to induce phenotypic transformation in amniotic smooth muscle. The influence of vitronectin on smooth muscle contractility is particularly relevant, because this molecule is abundant in whole blood and plasma (approx. 400 microg/ml). The results suggest that smooth muscle needs to be continually protected from normal blood constituents in vivo. The implications of these results for smooth muscle-related diseases like atherosclerosis, restenosis and Kaposi's sarcoma are discussed.

Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

Substance P responsiveness of smooth muscle cells is regulated by the integrin ligand, thrombospondin.

The extracellular factors that determine a cell's responsiveness to neurotransmitters are of particular relevance for pharmacologically diverse cell types such as neurons and smooth muscle. We previously demonstrated that matrix-associated factors are capable of dramatically and specifically suppressing the responsiveness of smooth muscle to the neuropeptide, substance P. We now demonstrate that this influence of extracellular matrix on the pharmacological phenotype of smooth muscle cells can be blocked specifically by an Arg-Gly-Asp (RGD)-containing antagonist of integrins. Of a battery of integrin ligands tested, only thrombospondin mimicked the effect of the extracellular matrix on substance P responsiveness. This effect of thrombospondin was dose dependent, RGD sensitive, and blocked by an antibody directed against the RGD-containing region of thrombospondin. Because the mRNA for thrombospondin is present in the cells of the chicken amnion, this extracellular factor may normally suppress substance P responsiveness in amniotic smooth muscle. The results suggest a role for matrix-associated integrin ligands in the regulation of cellular responses to specific neurotransmitters and hormones and in the development and maintenance of tissue-specific pharmacological properties.

Superfluous neurotransmitters?

In recent years, studies have suggested that the complexity of eukaryotic gene regulation, with its recurring and interacting motifs of cis and trans-acting regulatory elements, might result in superfluous gene expression. This conclusion is supported by a variety of experimental results that suggest that non-adaptive gene expression might be common. However, with few exceptions, the practical ramifications of unnecessary gene expression for cell biologists have not been addressed directly; this is particularly true for peptidergic neurophysiology, a field that might be plagued more than most with the consequences of this phenomenon. In this article, Chauncey W. Bowers discusses the superfluous expression of neuropeptides in the nervous system in the context of gene regulation extrapolated from studies in Drosophila.

Choroid tissue supports the survival of ciliary ganglion neurons in vitro.

It is well established that during in vivo development the neurons of the avian ciliary ganglion are dependent for their survival on structures in the eye. Separate neuron populations innervate intraocular smooth and striated muscle targets. All ciliary neurons survive when cocultured with striated muscle. We demonstrate that when ciliary ganglion neurons are plated on explants of the choroid coat (a smooth muscle-containing target tissue) using a defined medium (N2), the neurons survive and grow vigorously into the tissue, forming contacts between axons and target cells identified as smooth muscle. Conditioned medium from choroid explants also rescues all the neurons, as does coculturing ciliary ganglion neurons with dissociated choroid cells. However, the presence of horse serum and chick embryo extract in the medium inhibits the choroid's ability to support ciliary neurons. The effects of these additives on the phenotypic expression of the smooth muscle may explain the inability of previous investigators to demonstrate target-derived support from smooth muscle preparations. Because the choroid contains cell types other than smooth muscle (e.g., fibroblasts and endothelial cells), we could not identify smooth muscle as the only cell type responsible for the release of the soluble trophic factor present in the target tissue. However, indirect evidence using avian primary fibroblast cultures, a fibroblast cell line, and an anatomically simple smooth muscle preparation, the avian amnion, suggests that smooth muscle cells are sufficient to account for the observed trophic activity, and that similar target-derived molecules support the survival of both types of ciliary ganglion cells.

Maintenance of contractility in dissociated smooth muscle: low-density cultures in a defined medium.

The loss of contractility in long-term cultures of dissociated smooth muscle is such an established observation that the lack of contractility of cultured smooth muscle cells is often not even noted. This report describes methods of dissociating and culturing smooth muscle cells from the avian amnion that maintain contractility for > 1 mo in a defined medium. Because contractility was assessed by monitoring the contractions of individual cells to neurotransmitter-related substances, it is clear that these cells maintained both contractility and pharmacological responsiveness. However, when amniotic smooth muscle cells were dissociated with enzymes containing impurities or cultured in the presence of serum, they flattened and lost contractility, as reported for many other types of smooth muscle.

Mechanisms of synaptic fatigue in a developing autonomic ganglion.

Synaptic transmission in developing systems has often been noted to exhibit depression or failure at moderate frequencies of stimulation. While this is often presumed to be a transient, nonspecific inability of developing systems to meet the demands of synaptic transmission, this report demonstrates that such failure in the choroidal neurons of the embryonic ciliary ganglion is due to muscarinically mediated inhibition. Although the ganglion is composed of both choroid and ciliary neurons, only the choroid neurons exhibit the muscarinic depression, and only during embryonic development. The pharmacological properties of the relevant receptor are different from those of the muscarinic receptor involved in presynaptic inhibition in adult autonomic systems. Receptor-mediated, synaptic failure during development may serve to protect immature postsynaptic neurons from potentially toxic overstimulation.

Extracellular matrix regulates smooth muscle responses to substance P.

Little is known about the extracellular factors that determine a cell's responsiveness to neurotransmitters. This is a particularly important issue for pharmacologically diverse cell types such as neurons and smooth muscle. This report demonstrates that the contractile responses of amniotic smooth muscle to a specific neuropeptide, substance P, is controlled by a molecule(s) intimately associated with the extracellular basement membrane. This molecule(s) normally represses the expression of substance P responsiveness in this tissue. When the amniotic smooth muscle is separated from the basement membrane by dissociation, normally unresponsive cells exhibit a progressive increase in responsiveness to substance P, beginning within the first 24 hr in culture. The induction of substance P responses was completely inhibited when the cells were plated onto isolated amniotic basement membrane rather than onto polyornithine or collagen I. Similar changes in the responsiveness to another agonist, histamine, did not occur. The data demonstrate that extracellular matrix exerts a major instructive influence in determining the responsiveness of avian amniotic smooth muscle to specific ligands. We suggest that similar regulatory mechanisms may operate in other tissues.

Expression of functional neurotransmitter receptors in an uninnervated tissue: avian amnion.

The smooth muscle of the avian amnion is unusual because it is normally never innervated. However, as assessed by contractile response, this tissue expressed at least 11 different types of receptor for neurotransmitter substances including acetylcholine, norepinephrine, histamine, 5-hydroxytryptamine, vasoactive intestinal peptide, urotensin II, neurotensin, and somatostatin-28. Three neurotransmitters, histamine, 5-hydroxytryptamine, and norepinephrine, each acted via 2 separate and antagonistic types of receptors. The amnion also responded to prostaglandin E2. On the other hand, the tissue did not respond to substance P or bradykinin, 2 peptides that are known to affect smooth muscle contractility in a variety of other systems. Studies with organ-cultured amnion demonstrated that the smooth muscle can be cultured early in development and will differentiate in vitro. Some, but not all, of the amniotic responses developed in a defined medium. The results indicate that this novel smooth muscle preparation will be useful for identifying epigenetic factors that control the expression of functional receptors.

Somatostatin-like immunoreactivity in a subpopulation of nerve terminals in frog sympathetic ganglia.

A somatostatin-like substance was observed specifically in a subset of B-type nerve terminals in frog paravertebral ganglia. The middle ganglia (4th and 5th) of the sympathetic chain contained the highest proportions of somatostatin-positive terminals, with decreasing proportions in more caudal or rostral ganglia. Intracellular recordings from the 6th ganglion demonstrated that it contained both B and C neurons. Although prolonged depolarizing potentials were observed in B and C neurons after bursts of stimuli in C-type preganglionic axons, no slow potentials were observed after stimulating somatostatin-positive B-type axons. In addition, no effects of exogenously applied somatostatin were observed on the membrane potentials of B neurons. The present results are compared to a growing list of autonomic systems where peptidergic transmitters appear to have only subtle or no acute electrical consequences on postsynaptic neurons.

The efferent role of sensory axons in nerve-evoked contractions of bullfrog bladder.

The neural input to the frog bladder was characterized in vitro. The nerve-evoked bladder contraction consists primarily of an early parasympathetic cholinergic component and a later, longer-lasting non-adrenergic non-cholinergic component. This slow non-adrenergic non-cholinergic contraction is not only resistant to cholinergic and adrenergic antagonists, but also to H1 and H2 histaminergic antagonists and to the serotoninergic antagonist, methysergide. It is concluded that the non-adrenergic non-cholinergic contraction is mediated by an efferent action of the sensory system because it is resistant to ganglionic nicotinic antagonists and because it is elicited specifically by stimulation of the peripheral cut end of the dorsal root. 5-Hydroxytryptamine is a potent and specific inhibitor of the sensory non-adrenergic non-cholinergic contraction. Although the bladder smooth muscle is innervated by terminals containing a somatostatin-like substance, somatostatin does not cause a bladder contraction. Luteinizing hormone-releasing hormone, enkephalin, histamine, 5-hydroxytryptamine, adenosine and adenosine 5 monophosphate are also unlikely candidates for the non-adrenergic non-cholinergic transmitter because they do not produce bladder contractions and/or their antagonists are ineffective on the nerve-evoked contraction. A putatively sensory network of fibers containing a substance P-like material is located within the wall of the bladder. Substance P produces bladder contractions at concentrations as low as 10(-9) M and so it, or a related substance, is a viable transmitter candidate in this system. Adenosine 5'-triphosphate (ATP)(10(-5) M) also causes a bladder contraction and remains a possible candidate as well. The data demonstrate that the bladder contraction resulting from electrical stimulation of the bladder nerves is due in large part to the "antidromic" stimulation of sensory axons. The likely presence therefore of potent and releasable substances in the peripheral sensory terminals of the bladder suggests that this sensory system may exert significant local, efferent control of bladder smooth muscle (i.e. independent from the central nervous system).

Identification and purification of an irreversible presynaptic neurotoxin from the venom of the spider Hololena curta.

A search for potent toxins that inhibit neuronal calcium channels in Drosophila melanogaster has resulted in the identification of a presynaptic neurotoxin from the venom of the hunting spider Hololena curta. Using Drosophila neuromuscular junction as an assay, presynaptic inhibitory activity was purified using gel filtration and reverse-phase HPLC. Data from gel electrophoresis indicate that the toxin is composed of two different subunits of Mr 7000 and 9000. At nanomolar concentrations the toxin produced a complete and long-lasting inhibition of synaptic transmission at the Drosophila larval neuromuscular junction without affecting the amplitudes of the spontaneously occurring miniature junction potentials. The block of transmission produced by the toxin was observed even during the direct depolarization of the motor nerve terminal. These physiological results indicate that the terminal is the site of action for the toxin. Indirect evidence using abnormally excitable Drosophila mutants suggests that the toxin is inhibiting transmitter release by altering the electrical properties of the nerve terminal rather than by interfering with nonelectrical events that may occur subsequent to calcium influx. All of the actions of the Hololena toxin can be explained by a specific and direct effect on presynaptic calcium channels in Drosophila motor neurons.