Adenylyl cyclase 3 mediates prostaglandin E(2)-induced growth inhibition in arterial smooth muscle cells.

Arterial smooth muscle cell (SMC) proliferation contributes to a number of vascular pathologies. Prostaglandin E(2) (PGE(2)), produced by the endothelium and by SMCs themselves, acts as a potent SMC growth inhibitor. The growth-inhibitory effects of PGE(2) are mediated through activation of G-protein-coupled membrane receptors, activation of adenylyl cyclases (ACs), formation of cAMP, and subsequent inhibition of mitogenic signal transduction pathways in SMCs. Of the 10 different mammalian AC isoforms known today, seven isoforms (AC2-7 and AC9) are expressed in SMCs from various species. We show that, despite the presence of several different AC isoforms, the principal AC isoform activated by PGE(2) in human arterial SMCs is a calmodulin kinase II-inhibited AC with characteristics similar to those of AC3. AC3 is expressed in isolated human arterial SMCs and in intact aorta. We further show that arterial SMCs isolated from AC3-deficient mice are resistant to PGE(2)-induced growth inhibition. In summary, AC3 is the principal AC isoform activated by PGE(2) in arterial SMCs, and AC3 mediates the growth-inhibitory effects of PGE(2). Because AC3 activity is inhibited by intracellular calcium through calmodulin kinase II, AC3 may serve as an important integrator of growth-inhibitory signals that stimulate cAMP formation and growth factors that increase intracellular calcium.

Expression of type I adenylyl cyclase in intrinsic pathways of the hippocampal formation of the macaque (Macaca nemestrina).

The mossy fiber pathway of the hippocampal formation and type 1 adenylyl cyclase (AC1) have been implicated in long-term potentiation and memory function. Using immunohistochemical labeling and light microscopy we demonstrated intense labeling of AC1 in the mossy fibers and less intense labeling in the molecular layers of both the dentate gyrus and fields CA1, CA2 and CA3 of the hippocampus, i.e. in terminal fields of the perforant pathway. These findings indicate that, in the non-human primate, AC1 is found in the mossy fibers and in terminal fields of the perforant pathway where it may play a role in long term potentiation similar to that demonstrated in the rodent.

Evaluation of the ability of lysozyme and nisin to control meat spoilage bacteria.

The antimicrobials lysozyme, nisin, and mixtures of the two were studied to ascertain their abilities to control the growth of the meat-borne spoilage bacteria, Brochothrix thermosphacta B2 and Carnobacterium sp. 845. The goal was to optimize an antimicrobial for potential use in preservation of fresh meats. Their efficacies were evaluated in APT broth, in a meat juice extract and on cores of lean and fat pork tissue. Both lysozyme and nisin alone as well as mixtures of the two effectively inhibited B. thermosphacta B2 at 250 microg/ml in APT broth, the lowest concentration evaluated, for 10 days at 2 degrees C. In the presence of 500 microg/ml lysozyme, B. thermosphacta B2 grew after 12 days incubation. Only 125 microg of antimicrobial/ml was required to inhibit B. thermosphacta B2 for 27 days at 2 degrees C in pork juice. An estimated surface concentration of 130 microg/cm2 of each of the antimicrobials effectively inhibited B. thermosphacta B2 on inoculated cores of fat and lean pork tissue when the cores were incubated in vacuum packages for 6 weeks at 2 degrees C. In APT broth and in pork juice, lysozyme showed no antimicrobial activity against Carnobacterium sp. 845 at concentrations of 500 and 1000 microg/ml, respectively. Nisin and mixtures of the two antimicrobials inhibited Carnobacterium sp. 845 so that its numbers were at least 3 log units lower than untreated samples after 26 and 27 days incubation for APT and pork juice, respectively. The antimicrobial effect was concentration dependent. On lean pork tissue, numbers of Carnobacterium sp. 845 were significantly lower than untreated samples or samples treated with 195 microg/cm2 lysozyme when 260 microg/cm2 of a 1:3 (w/w) ratio of nisin to lysozyme was introduced to the cores. The inhibitory effect lasted for 14 of 42 days incubation in vacuum at 2 degrees C. On fat tissue, both lysozyme alone and the 1:3 nisin/lysozyme mixture inhibited Carnobacterium sp. 845 for 21 days storage in vacuum at 2 degrees C. On fat and lean tissue, mixtures of nisin and lysozyme would be more effective antimicrobials than either nisin or lysozyme alone.

Identification of inadequately cleaned equipment used in a sheep carcass-breaking process.

Aeromonads deposited on pork during a carcass-breaking process were recovered on hydrophobic grid membrane filters placed on ampicillin-dextrin-ethanol agar. Isolates from 85 honey-yellow colonies from filters on that medium were Aeromonas hydrophila (95%) or Vibrio sp. (5%). Presumptive aeromonads, coliforms, and Escherichia coli in swab samples from product passing through a sheep carcass-breaking process were enumerated by hydrophobic grid membrane filtration techniques with a detection level of 1 CFU/100 cm2. Total aerobic counts were determined by a spread plate procedure with a detection level of 1 CFU/cm2 The numbers of aerobes, coliforms, and E. coli in the product were apparently unaffected by the carcass-breaking process, although coliforms and E. coli appeared to be redistributed from shoulder to loin and leg portions. However, the numbers of aeromonads on product increased by about two orders of magnitude as a result of the process. Few bacteria were recovered from most cleaned, large items of equipment, but aerobes, coliforms, and aeromonads were recovered at log total numbers of 5.25, 3.96, and 3.26, respectively, from most of 25 samples from bars supporting a conveyor belt. Also, aerobes, coliforms, E. coli, and aeromonads were recovered from 25 supposedly cleaned steel mesh gloves at log total numbers of 10.14, 5.54, 4.73, and 8.30, respectively. Those findings indicate that inspection of cleaned equipment and microbiological sampling of only food-contacting surfaces, as is the current practice at meat plants, cannot provide assurance that the cleaning of carcass-breaking equipment is adequate. Instead, enumeration of indicator organisms on product passing through a process seems to be required as well, with subsequent sampling of equipment to identify sources of contaminants if increases in numbers during processing are observed. For surety of adequate cleaning, enumeration of several types of indicator organism may be necessary, as increases during processing in the numbers of organisms that are present in relatively large numbers on product entering a process may be difficult to detect.

Regulation and immunohistochemical localization of betagamma-stimulated adenylyl cyclases in mouse hippocampus.

Specific forms of synaptic plasticity such as long-term potentiation (LTP) are modulated by or require increases in cAMP. The various adenylyl cyclase isoforms possess unique regulatory properties, and thus cAMP increases in a given cell type or tissue in response to converging signals are subject to the properties of the adenylyl cyclase isoforms expressed. In most tissues, adenylyl cyclase activity is stimulated by neurotransmitters or hormones via stimulatory G-protein (Gs)-coupled receptors and is inhibited via inhibitory G-protein (Gi)-linked receptors. However, in the hippocampus, stimulation of Gi-coupled receptors potentiates Gs-stimulated cAMP levels. This effect may be associated with the regulatory properties of adenylyl cyclase types 2 and 4 (AC2 and AC4), isoforms that are potentiated by the betagamma subunit of Gi in vitro. Although AC2 has been shown to be stimulated by betagamma in whole cells, reports describing the sensitivity of AC4 to betagamma in vivo have yet to emerge. Our results demonstrate that Gs-mediated stimulation of AC4 is potentiated by betagamma released from activated Gi-coupled receptors in intact human embryonic kidney (HEK) 293 cells. Furthermore, we show that the AC2 and AC4 proteins are expressed in the mouse hippocampal formation and that they colocalize with MAP2, a dendritic and/or postsynaptic marker. The presence of AC2 and AC4 in the hippocampus and the ability of each of these enzymes to detect coincident activation of Gs- and Gi-coupled receptors suggest that they may play a crucial role in certain forms of synaptic plasticity by coordinating such overlapping synaptic inputs.

Phosphorylation and inhibition of olfactory adenylyl cyclase by CaM kinase II in Neurons: a mechanism for attenuation of olfactory signals.

Acute desensitization of olfactory signaling is a critical property of the olfactory system that allows animals to detect and respond to odorants. Correspondingly, an important feature of odorant-stimulated cAMP increases is their transient nature, a phenomenon that may be attributable to the unique regulatory properties of the olfactory adenylyl cyclase (AC3). AC3 is stimulated by receptor activation and inhibited by Ca2+ through Ca2+/calmodulin kinase II (CaMKII) phosphorylation at Ser-1076. Since odorant-stimulated cAMP increases are accompanied by elevated intracellular Ca2+, CaMKII inhibition of AC3 may contribute to termination of olfactory signaling. To test this hypothesis, we generated a polyclonal antibody specific for AC3 phosphorylated at Ser-1076. A brief exposure of mouse olfactory cilia or primary olfactory neurons to odorants stimulated phosphorylation of AC3 at Ser-1076. This phosphorylation was blocked by inhibitors of CaMKII, which also ablated cAMP decreases associated with odorant-stimulated cAMP transients. These data define a novel mechanism for termination of olfactory signaling that may be important in olfactory responses.

Assessment of the hygienic performance of a sheep carcass dressing process.

Swab samples were obtained from the surfaces of randomly selected carcasses passing through a sheep carcass-dressing process. A single sample was obtained from a randomly selected site on the surface of each carcass. Twenty-five such samples were collected at each of four stages in the process. The aerobic bacteria, coliforms, and Escherichia coli recovered from each sample were enumerated. Values for the mean log and standard deviation of each set of 25 log10 values were calculated on the assumption that the log values were normally distributed. The log of the arithmetic mean was estimated from the mean log and standard deviation values for each set. The results showed that bacteria, including coliforms that were largely E. coli, were deposited in high numbers during skinning operations, mainly on the butts and shoulders of carcasses. The mean numbers of coliforms and E. coli on carcasses were little affected by eviscerating and trimming operations, although they were redistributed from the sites they occupied after skinning. Total counts were redistributed and augmented by eviscerating and trimming operations. Washing reduced the log numbers of all of the bacteria by approximately 0.5. The general hygienic characteristics of the sheep carcass dressing process were similar to those of a previously examined beef carcass-dressing process.

Stimulation of type 1 and type 8 Ca2+/calmodulin-sensitive adenylyl cyclases by the Gs-coupled 5-hydroxytryptamine subtype 5-HT7A receptor.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important regulatory role in developing and adult nervous systems. With the exception of the 5-HT3 receptor, all of the cloned serotonin receptors belong to the G protein-coupled receptor superfamily. Subtypes 5-HT6 and 5-HT7 couple to stimulation of adenylyl cyclases through Gs and display high affinities for antipsychotic and antidepressant drugs. In the brain, mRNA for 5-HT6 is found at high levels in the hippocampus, striatum, and nucleus accumbens. 5-HT7 mRNA is most abundant in the hippocampus, neocortex, and hypothalamus. To better understand how serotonin might control cAMP levels in the brain, we coexpressed 5-HT6 or 5-HT7A receptors with specific isoforms of adenylyl cyclase in HEK 293 cells. The 5-HT6 receptor functioned as a typical Gs-coupled receptor in that it stimulated AC5, a Gs-sensitive adenylyl cyclase, but not AC1 or AC8, calmodulin (CaM)-stimulated adenylyl cyclases that are not activated by Gs-coupled receptors in vivo. Surprisingly, serotonin activation of 5-HT7A stimulated AC1 and AC8 by increasing intracellular Ca2+. 5-HT also increased intracellular Ca2+ in primary neuron cultures. These data define a novel mechanism for the regulation of intracellular cAMP by serotonin.

Communicate with patients using common office equipment.

Computer telephony is the technology associated with the electronic transmission of voice, fax, or other information using a computer via the telephone. Medical practices are increasingly under pressure to raise the level of customer service they offer while controlling or cutting expenses. Properly used, interactive voice technology will help a practice expand customer contact, reduce operating expenses, and increase staff efficiency. This article provides an overview of one of the most effective uses of telephony for a medical practice-patient communication systems. It looks at the benefits of such a system, lists the reasons for and advantages of using appointment reminders, and provides tips and criteria for buying a system, judging sound quality, justifying the cost, and keeping patients satisfied.

Dynamic palmitoylation of neuromodulin (GAP-43) in cultured rat cerebellar neurons and mouse N1E-115 cells.

We conducted pulse-chase and metabolic labeling experiments to determine directly whether palmitoylation of neuromodulin in neurons is dynamic, and if acylation is regulated. The rates of turnover of neuromodulin protein and associated palmitoyl groups were quantified using cultured cerebellar granule neurons and the neuronal cell line N1E-115. The half-life of [3H]palmitate bound to neuromodulin was approximately 5 h, whereas the half-life of the [35S]methionine-labeled neuromodulin was greater than 50 h. Metabolic and pulse-chase labeling experiments were carried out in the presence of various activators of cellular signaling pathways. Our data indicate that dynamic acylation and deacylation of neuromodulin in neurons are constitutive and are not regulated by G protein activation or other signals that control growth cone dynamics.

Hair cell differentiation in chick cochlear epithelium after aminoglycoside toxicity: in vivo and in vitro observations.

Inner ear epithelia of mature birds regenerate hair cells after ototoxic or acoustic insult. The lack of markers that selectively label cells in regenerating epithelia and of culture systems composed primarily of progenitor cells has hampered the identification of cellular and molecular interactions that regulate hair cell regeneration. In control basilar papillae, we identified two markers that selectively label hair cells (calmodulin and TUJ1 beta tubulin antibodies) and one marker unique for support cells (cytokeratin antibodies). Examination of regenerating epithelia demonstrated that calmodulin and beta tubulin are also expressed in early differentiating hair cells, and cytokeratins are retained in proliferative support cells. Enzymatic and mechanical methods were used to isolate sensory epithelia from mature chick basilar papillae, and epithelia were cultured in different conditions. In control cultures, hair cells are morphologically stable for up to 6 d, because calmodulin immunoreactivity and phalloidin labeling of filamentous actin are retained. The addition of an ototoxic antibiotic to cultures, however, causes complete hair cell loss by 2 d in vitro and generates cultures composed of calmodulin-negative, cytokeratin-positive support cells. These cells are highly proliferative for the first 2-7 d after plating, but stop dividing by 9 d. Calmodulin- or TUJ1-positive cells reemerge in cultures treated with antibiotic for 5 d and maintained for an additional 5 d without antibiotic. A subset of calmodulin-positive cells was also labeled with BrdU when it was continuously present in cultures, suggesting that some cells generated in culture begin to differentiate into hair cells.

Induction of acetylcholine receptor cluster formation by local application of growth factors in cultured Xenopus muscle cells.

A survey of several growth factors was carried out to determine their effect on the formation of acetylcholine receptor (AChR) clusters in cultured Xenopus muscle cells. Factors were locally applied via polystyrene beads and AChR clustering at bead-muscle contacts was assessed. Among the factors tested, basic fibroblast growth factor, insulin and insulin-like growth factor-I, as well as several polycations, were found to be effective in inducing AChR clusters. The action of these molecules appears to be mediated by tyrosine kinase receptors, since it can be blocked by a tyrosine kinase inhibitor. These results suggest that local ligand-mediated tyrosine phosphorylation is involved in the formation of AChR clusters.

Concentration of pp125 focal adhesion kinase (FAK) at the myotendinous junction.

Focal adhesion kinase is a recently characterized tyrosine kinase that is concentrated at focal contacts in cultured cells. It is thought to play an important role in the regulation of the integrin-based signal transduction mechanism involved in the assembly of this membrane specialization. In this study, we examined the immunocytochemical distribution of focal adhesion kinase in Xenopus skeletal muscle and its role in the formation of two sarcolemmal specializations, the myotendinous junction and the neuromuscular junction, using a monoclonal antibody (2A7) against this protein. Immunoprecipitation of Xenopus embryonic tissues with this antibody demonstrated a single band at a relative molecular mass of 116 kDa. A distinct concentration of immunolabeling for focal adhesion kinase was observed at the myotendinous junction of muscle fibers in vivo. At this site, the labeling for this protein is correlated with an accumulation of phosphotyrosine immunolabeling. Focal adhesion kinase was not concentrated at the neuromuscular junction in muscle cells either in vivo or in vitro. However, it was localized at spontaneously formed acetylcholine receptor clusters in cultured Xenopus myotomal muscle cells, although its distribution was not exactly congruent with that of the receptors. In these cells, the accumulation focal adhesion kinase was induced by polystyrene microbeads. In addition, beads also induce the formation of acetylcholine receptor clusters and myotendinous junction-like specializations. By following the appearance of the focal adhesion kinase relative to the formation of these sarcolemmal specializations at bead-muscle contacts in cultured muscle cells, we conclude that the accumulation of this protein was in pace with the development of the myotendinous junction, but occurred well after the clustering of acetylcholine receptors. These results suggest that focal adhesion kinase may be involved in the development and/or maintenance of the myotendinous junction through an integrin-based signaling system. Although it can accumulate at acetylcholine receptor clusters formed in culture, it does not appear to be involved in the development of the neuromuscular junction.

Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells.

Aggregation of the nicotinic acetylcholine receptor (AChR) at sites of nerve-muscle contact is one of the earliest events to occur during the development of the neuromuscular junction. The stimulus presented to the muscle by nerve and the mechanisms underlying postsynaptic differentiation are not known. The purpose of this study was to examine the distribution of phosphotyrosine (PY)-containing proteins in cultured Xenopus muscle cells in response to AChR clustering stimuli. Results demonstrated a distinct accumulation of PY at AChR clusters induced by several stimuli, including nerve, the culture substratum, and polystyrene microbeads. AChR microclusters formed by external cross-linking did not show PY colocalization, implying that the accumulation of PY in response to clustering stimuli was not due to the aggregation of basally phosphorylated AChRs. A semi-quantitative determination of the time course for development of PY labeling at bead contacts revealed early PY accumulation within 15 min of contact before significant AChR aggregation. At later stages (within 15 h), the AChR signal came to approximate the PY signal. We have reported the inhibition of bead-induced AChR clustering in response to beads by a tyrphostin tyrosine kinase inhibitor (RG50864) (Peng, H. B., L. P. Baker, and Q. Chen. 1991. Neuron. 6:237-246). RG50864 also inhibited PY accumulation at bead contacts, providing evidence for tyrosine kinase activation in response to the bead stimulus. These results suggest that tyrosine phosphorylation may play an important role in the generative stages of cluster formation, and may involve protein(s) other than or in addition to AChRs.

A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells.

During the development of the neuromuscular junction, acetylcholine receptors (AChRs) become clustered in the postsynaptic membrane in response to innervation. In vitro, several non-neuronal stimuli can also induce the formation of AChR clusters. DC electric field (E field) is one of them. When cultured Xenopus muscle cells are exposed to an E field of 5-10 V/cm, AChRs become clustered along the cathode-facing edge of the cells within 2 h. Recent studies have suggested the involvement of tyrosine kinase activation in the action of several AChR clustering stimuli, including nerve, polymer beads, and agrin. We thus examined the role of tyrosine phosphorylation in E field-induced AChR clustering. An antibody against phosphotyrosine (PY) was used to examine the localization of PY-containing proteins in E field-treated muscle cells. We found that anti-PY staining was colocalized with AChR clusters along the cathodal edge of the cells. In fact, cathodal PY staining could be detected before the first appearance of AChR clusters. When cultures were subjected to E fields in the presence of a tyrosine kinase inhibitor, tyrphostin RG-50864, cathodal AChR clustering was abolished with a half maximal inhibitory dosage of 50 microM. An inactive form of tyrphostin (RG-50862) had no effect on the field-induced clustering. These data suggest that the activation of tyrosine kinases is an essential step in E field-induced AChR clustering. Thus, the actions of several disparate stimuli for AChR clustering seem to converge to a common signal transduction mechanism based on tyrosine phosphorylation at the molecular level.

Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an endogenous muscle-derived signalling system.

Aneural muscle cells in culture often form acetylcholine receptor (AChR) clusters, termed hot spots, which are similar to those found at the postsynaptic membrane both in structure and in molecular composition. Although hot spots form on both dorsal and ventral surfaces of the cell, the ventral ones are better characterized because of their association with sites of cell-substratum contact. To understand the stimuli and mechanisms involved in ventral hot spot formation, native, uncoated polystyrene beads were applied to cultured Xenopus myotomal muscle cells to create local membrane-substratum contacts. These beads were able to induce a postsynaptic-type development as evidenced by the clustering of AChRs and the development of a set of ultrastructural specializations, including membrane infoldings and a basement membrane. Whereas these native beads were effective in inducing clustering, beads coated with bovine serum albumin or treated with serum-containing medium were ineffective. Native beads were also capable of inducing clusters in serum-free medium, indicating that their effect was mediated by endogenous molecules that were locally presented by the beads, rather than by bead adsorption of components in the medium. Heparan sulfate proteoglycan (HSPG) is a major component of the muscle extracellular matrix and our previous study has shown that basic fibroblast growth factor (bFGF), a member of the heparin-binding growth factor (HBGF) family, and its receptor are present in Xenopus myotomal muscle during the period of synaptogenesis. Therefore, we tested the involvement of HBGF in bead induction. The results of this study show the following: (1) preincubation of cultures in heparin, which solubilizes matrix-bound HBGFs, suppressed the bead-induced AChR clustering. (2) Suramin, which interferes with the interaction between several growth factors and their receptors, also inhibited bead-induced clustering. (3) Tyrphostin, which blocks tyrosine kinase activity associated with a number of growth factor receptors, was also inhibitory to the bead effect. (4) The percentage of bead-induced AChR clusters was significantly enhanced by pretreating the cultures with bFGF prior to bead application. This exogenously applied bFGF could be largely removed by treatment of cultures with heparin, suggesting its association with HSPG at the cell surface. (5) An anti-bFGF neutralizing antiserum significantly reduced the efficacy of the bead stimulation. These data suggest that uncoated beads, which adhere to the cell surface and can mimic the cell-substratum interaction, effect a local presentation of HBGFs, such as bFGF, residing with the HSPG to their membrane receptors, thereby locally activating receptor-associated tyrosine kinases.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of synaptic development in cultured muscle cells by basic fibroblast growth factor.

The role of basic fibroblast growth factor (bFGF) in signaling the development of the neuromuscular junction was examined. Beads coated with bFGF induced the formation of acetylcholine receptor (AChR) clusters in cultured Xenopus myotomal muscle cells. Tyrphostin, a tyrosine kinase inhibitor, abolished AChR clustering induced by bFGF beads, suggesting a role of tyrosine kinase activation in AChR clustering. Using specific antibodies, we demonstrated the presence of both bFGF and its receptor in the myotomal muscle in vivo during the period of neuromuscular connection. However, similar tissue from older animals with mature neuromuscular junctions showed an apparently truncated form of the bFGF receptor. These data suggest that bFGF may play a role in signaling synaptogenesis in skeletal muscle.

CT patterns of mesenteric disease.

To evaluate the patterns of mesenteric disease as visualized by computed tomography (CT), we reviewed the scans of 370 patients whose primary diagnoses coincided with diseases known from the pathology literature to have frequent mesenteric involvement. Diagnoses included selected malignancies, inflammatory diseases, and traumatic injuries. Four general patterns of involvement of the mesentery were recognized: (a) rounded masses, (b) "cake-like" masses, (c) ill-defined masses, and (d) stellate mesentery. Of the malignancies reviewed, mesenteric involvement as visualized by CT occurred most commonly with ovarian carcinoma (20/52) and non-Hodgkin's lymphoma (41/134). The incidences of CT evidence of involvement of the mesentery with other common malignancies were carcinoma of the colon (8/68), carcinoma of the pancreas (5/21), and leukemia (5/19). Certain benign and malignant lesions of the mesentery do demonstrate unique CT patterns of involvement. Examples of the individual patterns in common and unusual disease states are illustrated.