COVID-19-Associated Bifacial Weakness with Paresthesia Subtype of Guillain-Barré Syndrome.

We report a case of bifacial weakness with paresthesia, a recognized Guillain-Barré syndrome subtype characterized by rapidly progressive facial weakness and paresthesia without ataxia or other cranial neuropathies, which was temporally associated with antecedent coronavirus 2019 (COVID-19). This case highlights a potentially novel but critically important neurologic association of the COVID-19 disease process. Herein, we detail the clinicoradiologic work-up and diagnosis, clinical course, and multidisciplinary medical management of this patient with COVID-19. This case is illustrative of the increasingly recognized but potentially underreported neurologic manifestations of COVID-19, which must be considered and further investigated in this pandemic disease.

Admission delays' magnitude of traumatized patients in the emergency department of a hospital in Egypt: a cross-sectional study.

BACKGROUND: Injury is an escalating public health problem, representing about 9% of global mortality, which disproportionately impacts lower- and middle-income countries. There are approximately 12,000 annual fatalities from road traffic injuries in Egypt, but a little information about delays in seeking emergent care is available. OBJECTIVES: To measure the time interval between sustaining an injury and presentation to the emergency department of Ain Shams University Surgery Hospital and to identify possible causes of these delays. METHODS: We conducted a cross-sectional, facilitated survey of a convenience sample of trauma patients presenting to the emergency department of Ain Shams University Surgery Hospital from 1 February to 31 May 2014. Data obtained included: demographic information, trauma incident details, and injury assessment. RESULTS: The average reported transport time for patients from injury to hospital arrival was 3.8 h, while the mean ambulance response time was 45 min. Referral from other hospitals was revealed to be a significant cause of delay (P = 0.004), while ignorance of the local ambulance phone number could not be confirmed as a cause (P = 0.2). CONCLUSION: This study demonstrated that trauma patients at our hospital experience more than 3 h of delay until they reach the ED. It also identified the possible causes accounting for that delay. However, additional nationwide research is needed to establish the clear causation or association of these causes with the delay intervals.

Hospital discharge instructions: comprehension and compliance among older adults.

BACKGROUND: Little is known regarding the prevalence or risk factors for non-comprehension and non-compliance with discharge instructions among older adults. OBJECTIVE: To quantify the prevalence of non-comprehension and non-compliance with discharge instructions and to identify associated patient characteristics. RESEARCH DESIGN: Prospective cohort study. SUBJECTS: Four hundred and fifty adults aged ≥ 65 admitted to medical and surgical units of a tertiary care facility and meeting inclusion criteria. MEASURES: We collected information on demographics, psycho-social factors, discharge diagnoses, and medications using surveys and patient medical records. Domains within discharge instructions included medications, follow-up appointments, diet, and exercise. At 5 days post-discharge, we assessed comprehension by asking patients about their discharge instructions, and compared responses to written instructions from medical charts. We assessed compliance among patients who understood their instructions. RESULTS: Prevalence of non-comprehension was 5 % for follow-up appointments, 27 % for medications, 48 % for exercise and 50 % for diet recommendations. Age was associated with non-comprehension of medication [odds ratio (OR) 1.07; 95 % confidence interval (CI) 1.04, 1.12] and follow-up appointment (OR 1.08; 95 % CI 1.00, 1.17) instructions. Male sex was associated with non-comprehension of diet instructions (OR 1.91; 95 % CI 1.10, 3.31). Social isolation was associated with non-comprehension of exercise instructions (OR 9.42; 95 % CI 1.50, 59.11) Depression was associated with non-compliance with medication (OR 2.29; 95 % CI 1.02, 5.10) and diet instructions (OR 3.30; 95 % CI 1.24, 8.83). CONCLUSIONS: Non-comprehension of discharge instructions among older adults is prevalent, multi-factorial, and varies by domain.

HGF/SF induces mesothelial cell migration and proliferation by autocrine and paracrine pathways.

Mesothelial repair differs from that of other epithelial-like surfaces as healing does not occur solely by centripetal in-growth of cells as a sheet from the wound margins. Mesothelial cells lose their cell-cell junctions, divide, and adopt a fibroblast-like morphology while scattering across and covering the wound surface. These features are consistent with a cellular response to hepatocyte growth factor/scatter factor (HGF/SF). In this study, we examined the ability of mesothelial cells to secrete HGF/SF and investigated its possible role as an autocrine regulator of mesothelial cell motility and proliferation. We found that human primary mesothelial cells expressed HGF/SF mRNA and secreted active HGF/SF into conditioned medium as determined by ELISA and in a scattering bioassay. These cells also expressed the HGF/SF receptor, Met, as shown by RT-PCR and by Western blot analysis and immunofluorescence. Incubation of mesothelial cells with neutralizing antibodies to HGF/SF decreased cell migration to 25% of controls, whereas addition of HGF/SF disrupted cell-cell junctions and induced scattering and enhanced mesothelial cell migration. Furthermore, HGF/SF showed a small but significant mitogenic effect on all mesothelial cell lines examined. In conclusion, HGF/SF is produced by mesothelial cells and induces both motility and proliferation of these cells. These data are consistent with HGF/SF playing an autocrine role in mesothelial healing.

Caffeine citrate: a review of its use in apnoea of prematurity.

UNLABELLED: Apnoea of prematurity is a common condition in neonates born at less than 37 weeks' gestational age; it affects approximately 90% of premature neonates weighing under 1000 g at birth, and 25% of infants with a birthweight of less than 2500 g. Caffeine, a methylxanthine which occurs naturally in many plants, has been used for over 20 years to treat apnoea of prematurity. In a recent double-blind, placebo-controlled trial, apnoea was eliminated or reduced by at least 50% in significantly more neonates receiving caffeine citrate as first-line treatment than those receiving placebo. In a nonblind trial, caffeine citrate was more effective at reducing apnoeic episodes when compared with neonates receiving no treatment. Caffeine as first-line treatment demonstrated similar efficacy to theophylline or aminophylline (theophylline ethylenediamine) in 4 small randomised studies. Caffeine citrate was generally well tolerated in short term clinical trials, with very few adverse events reported. Caffeine was associated with fewer adverse events than theophylline in randomised trials. No differences in the incidence of individual adverse events were reported between caffeine citrate and placebo in a double-blind, randomised trial. Long term tolerability data are not yet available. CONCLUSIONS: Caffeine citrate was generally well tolerated by neonates in clinical trials and it decreased the incidence of apnoea of prematurity compared with placebo. It has demonstrated similar efficacy to theophylline, but is generally better tolerated and has a wider therapeutic index. Caffeine citrate should, therefore, be considered the drug of choice when pharmacological treatment of apnoea of prematurity is required.

Docetaxel: a review of its use in non-small cell lung cancer.

Docetaxel, a semisynthetic member of the taxoid class of antineoplastic agents, is effective in the treatment of patients with locally advanced and metastatic non-small cell lung cancer (NSCLC). In noncomparative trials in patients with NSCLC, docetaxel 75 or 100 mg/m2 produced objective response rates of 20 to 38% and 14 to 25% as a first-line or second-line monotherapy, respectively. In Japan, docetaxel 60 mg/m2 produced objective response rates of 19 to 25% in previously untreated patients. Docetaxel 100 or 75 mg/m2 produced significantly higher response rates than either vinorelbine or ifosfamide in previously treated patients; patients treated with docetaxel 75 mg/m2 had an improved 1-year survival rate compared with those who received vinorelbine or ifosfamide. Docetaxel monotherapy in chemotherapy-naive patients produced survival rates that are similar to those reported for most platinum-containing standard combinations such as cisplatin plus vinorelbine. Combination of docetaxel with one other antineoplastic resulted in objective response rates of 20 to 54% in chemotherapy-naive patients; triple chemotherapy combinations produced responses in 51 and 60% of patients. Promising results from a few small studies and one large phase II study have also indicated a potential role for docetaxel as neoadjuvant therapy. The main dose-limiting adverse event associated with docetaxel is neutropenia, and fluid retention is common in many patients. The tolerability profile is generally acceptable in the majority of patients, although extra care has to be taken in patients with impaired liver function to minimise the risk of severe or febrile neutropenia. Conclusions. Docetaxel is generally well tolerated by patients receiving treatment for locally advanced and metastatic NSCLC, and produces response and survival rates equivalent to many current standard treatment options. Comparative studies have shown that docetaxel monotherapy provides significant survival benefits over best supportive care or treatment with vinorelbine or ifosfamide. Response and 1-year survival rates with docetaxel monotherapy are particularly encouraging in patients refractory or resistant to cisplatin or carboplatin, for whom treatment options are few. Neoadjuvant docetaxel has produced improved survival compared with local treatment alone. Combinations of docetaxel with other antineoplastic agents have produced relatively high response and 1-year survival rates; however, further comparative studies are required to confirm these benefits. In the meantime, docetaxel is a welcome addition to the options available for patients with advanced NSCLC.

Extended-release oxybutynin.

Extended-release oxybutynin (Ditropan XL) uses an osmotic system (OROS) to deliver a controlled amount of oxybutynin chloride into the gastrointestinal tract over a 24-hour period when taken once daily. Oxybutynin binds to M3 muscarinic receptors on the detrusor muscle of the bladder, preventing acetylcholinergic activation and relaxing the muscle. Mean peak plasma concentrations are lower with extended-release oxybutynin 15mg once daily than with conventional immediate-release oxybutynin 5mg taken 3 times daily. Relative bioavailabilities of parent drug and metabolite N-desethoxybutynin are 153 and 69%, respectively, for extended-release oxybutynin when compared with immediate-release oxybutynin. In short (< or =6 weeks) randomised, double-blind clinical trials of patients with detrusor instability, extended-release oxybutynin 5 to 30mg once daily significantly reduced the mean weekly number of urge incontinence episodes by 84 to 90%. Extended-release oxybutynin had similar efficacy to immediate-release oxybutynin. Adverse events reported by patients taking extended-release oxybutynin were dose-related anticholinergic effects, most frequently dry mouth, somnolence, constipation, blurred vision and dizziness. A large noncomparative study demonstrated that approximately two thirds of the patients prescribed extended-release oxybutynin for detrusor instability were still taking the medication 6 months later.

Lidocaine patch 5%.

Lidocaine patch 5% comprises a soft, stretchy adhesive patch (l0 by 14 cm) containing 5% lidocaine (700 mg) for the topical treatment of pain associated with postherpetic neuralgia (PHN). Lidocaine provides analgesic relief by blocking neuronal sodium channels. In clinical trials (conducted over 12 hours to 24 days) involving patients with allodynia associated with PHN, treatment with lidocaine patch 5% resulted in a significant reduction in pain intensity and increased pain relief compared with vehicle patch. Lidocaine patch 5% was associated with few adverse events, the most frequent being mild skin redness or irritation at the application site which occurred with a similar incidence with lidocaine and vehicle patch.

Detection of mRNA species in bulbospinal neurons isolated from the rostral ventrolateral medulla using single-cell RT-PCR.

The rostral ventrolateral medulla (RVL) contains neurons which are critically involved in the tonic and reflex control of blood pressure. Some of these neurons project to the intermediolateral cell column of the thoracolumbar spinal cord and excite preganglionic sympathetic neurons. In order to gain a better understanding of the properties of the RVL neurons at the cellular and molecular level, a protocol was developed utilizing acute dissociation and the reverse transcription-polymerase chain reaction (RT-PCR) to study the expression of several genes in single RVL neurons. Neurons were dissociated from the RVL region of young rats, and classified as spinally projecting or non-spinal by the presence or absence of retrogradely transported fluorescent beads injected into the upper thoracic segments of the spinal cord. Individual neurons were collected by aspiration into a glass micropipette and analysed by RT-PCR. The presence of either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or neuron-specific enolase (NSE) mRNA was used as the criterion for selecting cells for further analysis. A subpopulation (50%) of spinally projecting, GAPDH- or NSE-positive neurons expressed mRNA for tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT), indicative of catecholaminergic or C1 adrenergic neurons, respectively. Some bulbospinal RVL neurons, including those that were TH- or PNMT-positive, were also found to express mRNA for the mineralocorticoid receptor (MR), the glucocorticoid receptor (GR), noradrenaline transporter (NET), and neuronal glutamate transporter (EAAC1). The glial glutamate transporter (GLT), glycine transporter (GLYT2), glutamic acid decarboxylase (GAD67) and gamma-amino butyric acid (GABA) transporter (GAT-1) were not expressed. The single-cell RT-PCR protocol is a powerful, yet simple and relatively rapid method for analysis of mRNA expression in a defined neuronal population. It can be combined with whole-cell patch-clamp recording prior to RT-PCR analysis, allowing linkage of the molecular analysis of mRNA expression to the electrophysiological and pharmacological properties of single neurons. The method is very sensitive, enabling mRNA transcripts in low abundance to be detected, and its application in our recent studies provided novel information about neurons involved in blood-pressure regulation at the molecular and cellular level.

Neurotrophic requirements of rat embryonic catecholaminergic neurons from the rostral ventrolateral medulla.

The factors that regulate the ontogeny and differentiation of C1 adrenergic neurons located in the rostral ventrolateral medulla (RVLM) are completely unknown. In the present study, we have investigated the effects of a number of neurotrophic factors on the survival of E18-19 rat C1 adrenergic neurons in culture. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to study the expression of tyrosine hydroxylase (TH), an enzyme present in all catecholaminergic neurons, and of phenylethanolamine N-methyltransferase (PNMT), the final enzyme in the synthesis of adrenalin, as markers for the C1 RVLM neurons. Our results show that GDNF, CNTF BDNF, NT-3 and NT-4/5 increase the number of TH-immunoreactive neurons surviving in vitro. The effects of NGF, TGFbeta and bFGF were not significant. The E18-19 C1 neurons appeared to loose their ability to express PNMT in culture as examined with immunocytochemistry and RT-PCR, and none of the tested neurotrophic factors was able to sustain or induce this expression. Our results indicate that the adrenergic phenotype of C1 neurons, or the survival of these neurons, is determined by environmental factors other than the neurotrophic factors examined in this study.

Localization of the noradrenaline transporter in rat adrenal medulla and PC12 cells: evidence for its association with secretory granules in PC12 cells.

The noradrenaline transporter (NAT) is present in noradrenergic neurons and a few other specialized cells such as adrenal medullary chromaffin cells and the rat pheochromocytoma (PC12) cell line. We have raised antibodies to a 49-residue segment (NATM2) of the extracellular region (residues 184-232) of bovine NAT. Affinity-purified NATM2 antibodies specifically recognized an 80-kDa band in PC12 cell membranes by western blotting. Bands of a similar size were also detected in membranes from human neuroblastoma (SK-N-SH) cells expressing endogenous NAT and human embryonic kidney (HEK293) cells stably expressing bovine NAT. Immunocytochemistry of rat adrenal tissue showed that NAT staining was colocalized with tyrosine hydroxylase in medullary chromaffin cells. Most NAT immunoreactivity in rat adrenal chromaffin and PC12 cells was present in the cytoplasm and had a punctate appearance. Cell surface biotinylation experiments in PC12 cells confirmed that only a minor fraction of the NAT was present at the cell surface. Subcellular fractionation of PC12 cells showed that relatively little NAT colocalized with plasma membrane, synaptic-like microvesicles, recycling endosomes, or trans-Golgi vesicles. Most of the NAT was associated with [3H]noradrenaline-containing secretory granules. Following nerve growth factor treatment, NAT was localized to the growing tip of neurites. This distribution was similar to the secretory granule marker secretogranin I. We conclude that the majority of NAT is present intracellularly in secretory granules and suggest that NAT may undergo regulated trafficking in PC12 cells.

Interaction of Smad complexes with tripartite DNA-binding sites.

The Smad family of transcription factors function as effectors of transforming growth factor-beta signaling pathways. Smads form heteromultimers capable of contacting DNA through the amino-terminal MH1 domain. The MH1 domains of Smad3 and Smad4 have been shown to bind to the sequence 5'-GTCT-3'. Here we show that Smad3 and Smad4 complexes can contact three abutting GTCT sequences and that arrays of such sites elevate reporter expression relative to arrays of binding sites containing only two GTCTs. Smad3/4 complexes bound synergistically to probes containing two of the four possible arrangements of three GTCT sequences and showed a correlated ability to synergistically activate transcription through these sites. Purified Smad3 and Smad4 were both able to contact three abutting GTCT sequences and reporter experiments indicated that either protein could mediate contact with all three GTCTs. In contrast, the Smad4 MH1 domain was essential for reporter activation in combination with Smad1. Together, these results show that Smad complexes are flexible in their ability to interact with abutting GTCT triplets. In contrast, Smads have high affinity for only one orientation of abutting GTCT pairs. Functional Smad-binding sites within several native response elements contain degenerate GTCT triplets, suggesting that trimeric Smad-DNA interaction may be relevant in vivo.

Effects of cyanide and hypoxia on membrane currents in neurones acutely dissociated from the rostral ventrolateral medulla of the rat.

Previous reports suggested that some neurones located in the rostral ventrolateral medulla (RVL) can act as fast oxygen sensors which enhance the sympathetic activity and blood pressure independent of peripheral chemoreceptors. The aim of this study was to compare hypoxic responses of different subpopulations of RVL neurones to ascertain whether the hypoxic sensitivity is restricted to one group of these neurones. Whole-cell patch-clamp recordings were made from acutely dissociated neurones obtained from RVL of P13-P19 rats. Short-lasting hypoxia (1-2 min) was evoked by pressure injection of NaCN or lowering pO2. Cells projecting to the upper thoracic segments were retrogradely labelled with fluorescent beads. Catecholaminergic (CA) or non-catecholaminergic (non-CA) neurones were identified using single-cell reverse-transcription polymerase chain reaction (RT-PCR) or immunocytochemistry. Recordings were made from 38 neurones (26 spinally-projecting, 12 non-spinal) using Cs+/TEA or K+-containing pipettes. In most of the cells tested with slow depolarising ramp commands (78%; including spinally-projecting and non-spinal neurones, as well as CA and non-CA neurones), NaCN or hypoxia evoked a reversible increase of the sustained inward current. Extracellular application of 1 mM Co2+ or 25 nM TTX revealed three components of the hypoxia-sensitive inward current which resembled the persistent sodium (INaP), low threshold calcium (LVA Ca2+) and high threshold calcium (HVA Ca2+) currents. The NaCN or hypoxia induced increase of the current could also be observed during step commands. Recordings with K+-containing pipettes during similar depolarising ramps revealed, in addition, a reversible increase of IK in 78% of tested cells (in all four types of examined neurones). These results are consistent with the concepts that RVL neurones can act as a central oxygen sensor. However, in contrast to the previously published data demonstrating that in pentobarbital anaesthetised rats only the barosensitive and spinally projecting cells were affected by a short-lasting hypoxia, our findings obtained with dissociated RVL neurones indicate that sensitivity to hypoxia is widely distributed within this part of the medulla oblongata.

The tyrosine kinase Abl and its substrate enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance.

Genetic analysis of growth cone guidance choice points in Drosophila identified neuronal receptor protein tyrosine phosphatases (RPTPs) as key determinants of axon pathfinding behavior. We now demonstrate that the Drosophila Abl tyrosine kinase functions in the intersegmental nerve b (ISNb) motor choice point pathway as an antagonist of the RPTP Dlar. The function of Abl in this pathway is dependent on an intact catalytic domain. We also show that the Abl phosphoprotein substrate Enabled (Ena) is required for choice point navigation. Both Abl and Ena proteins associate with the Dlar cytoplasmic domain and serve as substrates for Dlar in vitro, suggesting that they play a direct role in the Dlar pathway. These data suggest that Dlar, Abl, and Ena define a phosphorylation state-dependent switch that controls growth cone behavior by transmitting signals at the cell surface to the actin cytoskeleton.

Noradrenaline transporter expression in the pons and medulla oblongata of the rat: localisation to noradrenergic and some C1 adrenergic neurones.

Catecholaminergic neurotransmission is normally terminated by rapid re-uptake of the neurotransmitter by a high-affinity Na+/Cl--dependent plasma membrane transporter. Specific transporters have been cloned for both dopamine (DAT) and noradrenaline (NAT) in the rat. While DAT has been studied extensively, NAT expression has received less attention, particularly at the protein level. We used an antibody generated against a 49 residue segment of an extracellular loop region of NAT to study expression of the transporter protein throughout the rat pons and medulla oblongata. NAT was expressed in over 95% of noradrenergic neurones in the A1, A2/area postrema, A5, A6/locus subcoeruleus, and A7 noradrenergic groups. Approximately 10% of C1 adrenergic neurones located in the rostral ventrolateral medulla (RVL) also expressed NAT. Expression of NAT mRNA in bulbospinal C1 cells was confirmed using single-cell reverse transcription polymerase chain reaction (RT-PCR) of acutely isolated RVL neurones. Spinally projecting neurones were identified by retrograde labelling with rhodamine beads, and C1 neurones were identified by RT-PCR using primers specific for tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT) mRNAs. Thirteen percent of adrenergic bulbospinal neurones tested expressed NAT mRNA. C1 neurones are potentially important in cardiovascular control and blood pressure regulation, and the identification of NAT expression in a sub-population of these neurones provides further evidence for the heterogeneity of this neuronal population.

Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains.

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

Whole cell patch-clamp study of putative vasomotor neurons isolated from the rostral ventrolateral medulla.

A distinct subpopulation of neurons in the rostral and ventrolateral part of the medulla oblongata (RVL) plays a key role in controlling sympathetic vasomotor tone. To characterize these neurons under conditions in which all cell-to-cell interactions are eliminated, RVL neurons were acutely dissociated from 13- to 19-day old rats. Cells projecting to the upper thoracic segments were retrogradely labeled with fluorescent beads. Fifty-two percent (17/33) of examined spinally projecting neurons were catecholaminergic, as demonstrated by single-cell reverse transcription-polymerase chain reaction or immunocytochemistry. No spontaneous (capacitive) spikes were revealed in the tight seal cell-attached configuration. Whole cell recordings were made from 54 spinally projecting neurons using Cs(+)- or K(+)-containing pipettes. No spontaneous firing was observed in current-clamp mode with K(+)-based pipettes (membrane potential, -61.5 +/- 2.3 mV). Step depolarizations (300- or 400-ms pulses, up to 100 pA) evoked regular firing or one to four spikes. Several voltage-gated currents, resembling the transient and persistent Na+, delayed rectifier and low- and high-threshold Ca2+, were revealed in voltage-clamp mode. These results show that isolated spinally projecting RVL neurons display no pacemaker-like activity. Because data from the literature indicate that these neurons are capable of generating such activity under different experimental conditions, the factors responsible for different behavior need to be determined. Dissociated RVL neurons provide a useful new model for studying biophysical and other properties of neurons involved in blood pressure control.

Phosphorylation of Enabled by the Drosophila Abelson tyrosine kinase regulates the in vivo function and protein-protein interactions of Enabled.

Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila ena were discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.

Detection of weakly expressed genes in the rostral ventrolateral medulla of the rat using micropunch and reverse transcription-polymerase chain reaction techniques.

1. The present study describes the use of reverse transcription-polymerase chain reaction (RT-PCR) to detect weakly expressed neurotransmitter receptor mRNA in tissue micropunched from the rostral ventrolateral medulla (RVLM) and other discrete areas of the medulla oblongata of the rat. 2. Micropunches were made from 240 microns transverse medullary sections. Punched regions included the RVLM, hypoglossal nucleus (XIIn), ventrolateral subnucleus of the nucleus tractus solitarius (NTS) and spinal trigeminal nucleus (STN). RNA was extracted and reverse transcribed into cDNA, which was probed for the presence of seven genes: glyceraldehyde phosphate dehydrogenase (GAPDH), neuron-specific enolase (NSE), tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), glucocorticoid receptor (GCR), mineralocorticoid receptor (MCR) and the adenosine 5'-triphosphate (ATP) receptor subunit P2X2-1. Each transcript was detected using a semi-nested PCR protocol, which used three primers. 3. Tyrosine hydroxylase was detected in the RVLM and NTS and PNMT was also detected in the RVLM, which agrees with the distribution of catecholamine neurons in the medulla. Expression of GCR mRNA was detected in the RVLM and the XIIn but not in the NTS (it was not probed for in the STN punches). The P2X2-1 receptor message was detected in all areas. Expression of MCR mRNA was detected in the RVLM only. 4. This method offers a simple way to detect the presence of low-abundance receptor mRNA in discrete brain regions.

P2 receptor excitation of rodent hypoglossal motoneuron activity in vitro and in vivo: a molecular physiological analysis.

The role of P2 receptors in controlling hypoglossal motoneuron (XII MN) output was examined (1) electrophysiologically, via application of ATP to the hypoglossal nucleus of rhythmically active mouse medullary slices and anesthetized adult rats; (2) immunohistochemically, using an antiserum against the P2X2 receptor subunit; and (3) using PCR to identify expression of P2X2 receptor subunits in micropunches of tissue taken from the XII motor nucleus. Application of ATP to the hypoglossal nucleus of mouse medullary slices and anesthetized rats produced a suramin-sensitive excitation of hypoglossal nerve activity. Additional in vitro effects included potentiation of inspiratory hypoglossal nerve output via a suramin- and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS)-sensitive mechanism, XII MN depolarization via activation of a suramin-sensitive inward current, decreased neuronal input resistance, and a slow-onset theophylline-sensitive reduction of inspiratory output likely resulting from hydrolysis of extracellular ATP to adenosine and activation of P1 receptors. Immunohistochemically, P2X2 receptors were detected in inspiratory XII MNs that were labeled with Lucifer yellow. These data, combined with identification of mRNA for three P2X2 receptor subunit isoforms within the hypoglossal nucleus (two of which have not been localized previously in brain) and the previous demonstration that P2X receptors are ubiquitously expressed in cranial and spinal motoneuron pools, support not only a role of P2 receptors in modulating inspiratory hypoglossal activity but a general role of P2 receptors in modulating motor outflow from the CNS.