Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters.

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus in oysters using the quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) are well established, and various studies using RT-qPCR have detected norovirus in a considerable proportion of oyster samples, both in the UK and elsewhere. However, RT-qPCR detects viral genome, and by its nature is unable to discriminate between positive results caused by infectious viruses and those caused by non-infectious remnants including damaged virus particles and naked RNA. As a result, a number of alternative or complementary approaches to RT-qPCR testing have been proposed, including the use of infectious viral indicator organisms, most frequently F-specific RNA bacteriophage (F-RNA phage). In this study, we investigated the relationships between F-RNA phage and norovirus in digestive tissues from two sets of oyster samples, one randomly collected at retail (630 samples), and one linked to suspected norovirus illness outbreaks (nine samples). A positive association and correlation between PCR-detectable levels of genogroup II F-RNA bacteriophage (associated with human faecal contamination) and norovirus was found in both sets of samples, with more samples positive for genogroup II phage, at generally higher levels than norovirus. Levels of both viruses were higher in outbreak-related than retail samples. Infectious F-RNA phage was detected in 47.8% of all retail samples, and for a subset of 224 samples where characterisation of phage was carried out, infectious GII phage was detected in 30.4%. Infectious GII phage was detected in all outbreak-related samples. Determination of infectivity ratios by comparing levels of PCR-detectable (copies/g) and infectious GII phage (pfu/g) revealed that in the majority of cases less than 10% of virus detected by RT-qPCR was infectious. Application of these ratios to estimate infectious norovirus levels indicated that while 77.8% of outbreak-related samples contained > 5 estimated infectious norovirus/g, only 13.7% of retail samples did. Use of a combination of levels of PCR-detectable norovirus and infectious F-RNA phage showed that while only 7.0% of retail samples contained both > 100 copies/g norovirus and > 10 pfu/g F-RNA phage, these combined levels were present in 77.8% of outbreak-related samples, and 75.9% of retail samples with > 5 estimated infectious norovirus/g. We therefore suggest that combining RT-qPCR testing with a test for infectious F-RNA phage has the potential to better estimate health risks, and to better predict the presence of infectious norovirus than RT-qPCR testing alone.

Expression of diverse Na+ channel messenger RNAs in rat myocardium. Evidence for a cardiac-specific Na+ channel.

This study examined the diversity of Na+ channel gene expression in intact cardiac tissue and purified myocardial cells. The screening of neonatal rat myocardial cell cDNA libraries with a conserved rat brain Na+ channel cDNA probe, resulted in the isolation and characterization of a putative rat cardiac Na+ channel cDNA probe (pCSC-1). The deduced amino acid sequence of pCSC-1 displayed a striking degree of homology with the eel, rat brain-1, and rat brain-2 Na+ channel, thereby identifying pCSC-1 as a related member of the family of Na+ channel genes. Northern blot analysis revealed the expression of a 7-kb CSC-1 transcript in rat cardiac tissue and purified myocardial cells, but little or no detectable expression of CSC-1 in rat brain, skeletal muscle, denervated skeletal muscle, or liver. Using RNase protection and Northern blot hybridization with specific rat brain Na+ channel gene probes, expression of the rat brain-1 Na+ channel was observed in rat myocardium, but no detectable expression of the rat brain-2 gene was found. This study provides evidence for the expression of diverse Na+ channel mRNAs in rat myocardium and presents the initial characterization of a new, related member of the family of Na+ channel genes, which appears to be expressed in a cardiac-specific manner.

Characterization of the binding of Bolton-Hunter labeled [125I]C5a to human neutrophil, monocyte and U-937 cell membranes.

The fifth component of the complement cascade, C5a, was iodinated using the Bolton-Hunter reagent. Results from the present study, using the high affinity ligand, [125I]Bolton-Hunter-labeled C5a ([125I]BH-C5a), revealed a single binding site on membranes prepared from human neutrophils, U-937 cells and human monocytes. Saturation studies demonstrated Bmax values in these cells of 11.5, 47.3 and 16.6 fmol/10(6) cells, respectively. The C5a receptor demonstrated a very high affinity for [125I]BH-C5a of approximately 4 pM in each cell type. Competition studies using analogs of C5a generated a similar order of potency in each of the cell types of C5a > or = C5a(1-74), Ser66Ala > C5a(1-73) > C5a(1-69). These studies indicate that [125I]BH-C5a labels a similar receptor in neutrophil, U-937 cell and monocyte membranes. Furthermore, C5a(1-73) produced shallow inhibition curves in competition experiments in each cell type. Computer analysis of the binding data revealed two components of binding. When 10 nM unlabeled C5a was used to initiate dissociation of [125I]BH-C5a binding in neutrophil membranes, two binding components were observed. In addition, dissociation of [125I]BH-C5a binding by 10 nM unlabeled C5a in the presence of 1 mM GppNHp decreased the percentage of binding to the slowly dissociating, high affinity binding component from 84 to 58%. These results suggest that multiple states of the C5a receptor exist.

The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.

Effect of chronic treatment with clorgyline on the relative concentration of specific proteins in the hippocampus and parietal cortex of the rat.

The effect of the chronic administration of clorgyline, a type A inhibitor of monoamine oxidase, on the relative concentration of proteins from the brain of the rat was examined by analysis of two-dimensional electrophoretic gels. The results from this study showed that the administration of clorgyline for 3 weeks produced a significant elevation in the relative concentration of two proteins in the parietal cortex (mol. wt 23,000 and 30,000) and one protein in the hippocampus (mol. wt 25,000). In contrast, the relative concentration of three proteins (mol. wt 31,000, 42,000 and 45,000) was significantly reduced in the parietal cortex by chronic treatment with clorgyline. No protein in the hippocampus was found to be significantly reduced by treatment with clorgyline. Since a previous study has indicated that the relative concentration of three different proteins were significantly altered by the repeated administration of desipramine, the results from the present experiment indicate that different changes in proteins are produced by repeated treatment with the type A monoamine oxidase inhibitor, clorgyline, as compared to those produced by the tricyclic antidepressant, desipramine. These results support previous suggestions that different classes of antidepressant compounds may exert their effects through different mechanisms of action.

2H-[1]benzopyrano[3,4-b]pyridines: synthesis and activity at central monoamine receptors.

Two general synthetic approaches to a novel series of 2H-[1]benzopyrano[3,4-b]pyridines are described together with their receptor binding profile at a variety of monoamine receptors in mammalian brain tissue. The biologically active members of this series fall into into one of two broad classes: 3,4,4a,5-tetrahydro-2H-[1]benzopyrano[3,4-b]pyridines or trans-1,3,4,4a,5,10b-hexahydro-2H-[1]benzopyrano[3,4-b]pyridines. By appropriate pharmacophoric modification potent selective ligands for D2, alpha-2, 5HT1A, and 5HT2 receptors may be obtained. The previously published in vivo data on certain key representatives of these series are also summarized.

Synthesis, opioid receptor binding profile, and antinociceptive activity of 1-azaspiro[4.5]decan-10-yl amides.

A series of azaspiro[4.5]decanyl amides were prepared by a novel cyclization route and examined for opiate receptor binding and antinociceptive activity. Selected tertiary amides in this series showed potent selective mu-receptor binding and antinociceptive activity, in contrast to the less conformationally restricted secondary amides, which showed relatively weak activity. Although structurally similar to the kappa-agonist U-50488H (1), these compounds showed virtually no tendency to bind to the kappa-receptor. An X-ray crystal structure of compound (21) confirms that the spirocyclic amine does not cause distortion away from the chair conformation of the cyclohexane ring. Either this receptor has very specific requirements for the orientation of the two nitrogens of these compounds or this ring system fills a portion of space more readily tolerated by the mu- and delta-receptors.

Benzofuro[2,3-c]pyridin-6-ols: synthesis, affinity for opioid-receptor subtypes, and antinociceptive activity.

A general synthetic approach to a novel series of cis-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ols is described together with their receptor-binding profile on opioid-receptor subtypes (mu, kappa, delta). In addition, their in vivo antinociceptive activity was assessed. A number of the analogues synthesized showed potent affinity for opioid receptors and have potent antinociceptive activity in a mouse phenylquinone abdominal stretching model. In addition, the SAR for nitrogen substitution in the above series is explored with respect to the overall opioid receptor subtype binding profile. In general it was found that substituents which enhanced mu and kappa binding affinity in the benzomorphan series had a similar effect in the benzofuropyridine series described in this manuscript. An overlap hypothesis topologically connecting the benzomorphan nucleus to the cis-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridine nucleus is also presented.

Identification of a receptor-binding region in the core segment of the human anaphylatoxin C5a.

In order to identify regions of C5a that contribute to receptor binding and functional activity of the anaphylatoxin, a series of peptides was synthesized in which core segments have been attached to C-terminal segments via native peptidic or disulfide bonds. It has been found that residues Arg40 and Arg46 in the loop-3 region of the core induce a 1000-fold increase in the affinity of the disordered C-terminal segment of C5a. The results obtained from this work lead to the conclusion that the loop-3 region is most likely the core binding site of C5a.

New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis.

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values < 0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

The associations among pediatricians' knowledge, attitudes, and practices regarding emergency contraception.

OBJECTIVES: To quantify practitioner administration of the emergency contraceptive pill (ECP) among adolescent patients, and to determine if such administration is associated with physician knowledge and attitudes regarding efficacy, side effects, and appropriate use. DESIGN: Survey of pediatricians. SETTING: The survey address list was generated from a database of active Fellows of the American Academy of Pediatrics in the District of Columbia metropolitan area. MAIN OUTCOMES MEASURES: Prescription of the ECP in the previous 12 months, or counseling of an adolescent patient about the ECP. RESULTS: Of the 236 questionnaires distributed, 143 (61%) were returned and 121 (51%) were usable. Twenty-four pediatricians (20%) reported prescribing the ECP, and 29 (24%) had counseled adolescent patients about the ECP. Of the practice-related variables surveyed, both the number of adolescents seen per week and the practice setting were significantly associated with these outcomes. Of the knowledge-related variables surveyed, knowledge of the timing and the Food and Drug Administration-labeled status of the ECP were significantly associated with outcomes. None of the attitude-related variables surveyed were associated with outcomes. CONCLUSIONS: This study demonstrates that knowledge deficits, not attitude-related variables, are significantly associated with the low level of ECP administration and counseling among District of Columbia pediatricians. Because knowledge deficits are amenable to educational interventions, our data suggest that informing pediatricians about the ECP may increase its administration among their adolescent patients.emergency contraceptive pill, pediatricians, adolescents.

Pediatric Milliman and Robertson length-of-stay criteria: are they realistic?

OBJECTIVE: Guidelines for inpatient length of stay (LOS) have been developed by several groups; among the most widely applied are those published by Milliman and Robertson (M&R). Few published reports have examined the relationship of actual practice to such guidelines, none in pediatric populations. This study was designed to compare pediatric practice in a large and defined population to M&R LOS criteria. METHODS: Administrative data from New York State in 1995 were used to examine LOS for discharges corresponding to 16 selected pediatric diagnoses for which M&R publishes guidelines. Outliers, defined as the 2% of discharges with the longest LOS, were eliminated. The distribution of LOS for each diagnosis was compared with M&R LOS guidelines. RESULTS: In New York State during 1995, pediatric LOS was markedly divergent from M&R guidelines. In general, the percentage of discharges in excess of the criterion LOS was less for nonmandatory admissions (croup: 23%, gastroenteritis: 44%, and pneumonia: 48%) than for those requiring surgery (uncomplicated appendectomy: 67%, pyloromyotomy: 62%, and major but noncritical burns: 64%) or prolonged treatment with antibiotics (bacterial meningitis: 91% and osteomyelitis: 86%). CONCLUSIONS: In New York State during 1995, LOS for selected pediatric conditions was generally in excess of published M&R guidelines. This raises concern about the potential effects of such guidelines on both patients and the hospitals caring for them. While endorsing the need for cost-effective practice, we call attention to the methods used to develop and validate guidelines.length of stay, pediatrics, managed health care, administrative data, practice guidelines.

Identification of a high-affinity anti-phosphoserine antibody for the development of a homogeneous fluorescence polarization assay of protein kinase C.

In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions. Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available. Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 +/- 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO(4))LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO(4))LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates. Phosphatidylserine, which enhances the kinase activity of PKC in a Ca(2+)-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay. The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.

Regional cardiac adrenergic function using I-123 meta-iodobenzylguanidine tomographic imaging after acute myocardial infarction.

The effect of acute myocardial infarction (AMI) on regional cardiac adrenergic function was studied in 27 patients mean +/- standard deviation 10 +/- 4 days after AMI. Regional adrenergic function was evaluated noninvasively with I-123 meta-iodobenzylguanidine (MIBG) using a dedicated 3-detector tomograph. Four hours after its administration, there was reduced MIBG uptake in the region of infarction, 0.38 +/- 0.31 counts/pixel/mCi x 103 compared with 0.60 +/- 0.30 counts/pixel/mCi x 103 and 0.92 +/- 0.35 counts/pixel/mCi x 103 in the zones bordering and distant from the infarct area, respectively, p less than 0.001. In all patients, the area of reduced MIBG uptake after 4 hours was more extensive that the associated thallium-201 perfusion defect with defect scores of 52 +/- 22 and 23 +/- 18%, respectively, p less than 0.001. After anterior wall AMI, the 4-hour MIBG defect score was 70 +/- 13% and the degree of mismatch between myocardial perfusion and MIBG uptake was 30 +/- 9% compared with 39 +/- 17 and 21 +/- 17% after inferior AMI, p less than 0.001 and p = 0.016, respectively. The 4-hour MIBG defect score correlated inversely with the predischarge left ventricular ejection fraction, r = -0.73, p less than 0.001. Patients with ventricular arrhythmia of greater than or equal to 1 ventricular premature complexes per hour, paired ventricular premature complexes or ventricular tachycardia detected during the late hospital phase had higher 4-hour MIBG defect scores, 62.5 +/- 15.0%, than patients with no detectable complex ventricular ectopic activity and a ventricular premature complex frequency of less than 1 per hour, 44.6 +/- 23.4%, p = 0.036.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the binding of [3H]-CGS 19755: a novel N-methyl-D-aspartate antagonist with nanomolar affinity in rat brain.

1. CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid), a rigid analogue of 2-amino-5-phosphonopentanoic acid (AP5), is one of the most potent competitive N-methyl-D-aspartate (NMDA) antagonists described. Using Triton-treated crude synaptic membranes from rat brain, binding studies indicated that [3H]-CGS 19755 bound with high affinity and selectivity to the NMDA-type excitatory amino acid receptor. 2. [3H]-CGS 19755 binding was saturable, reversible, heat-labile, pH-dependent and linear with protein concentration. Specific binding represented 80-85% of the total amount bound. 3. Using a centrifugation assay, saturation experiments revealed two distinct binding components with Kd values of 9 and 200 nM, and corresponding Bmax values of 0.55 and 1.00 pmol mg-1 protein. In contrast, a single binding component with a Kd value of 24 nM and an apparent Bmax value of 0.74 pmol mg-1 protein was observed with a filtration assay. 4. Competition experiments in which both assay techniques were used, showed that [3H]-CGS 19755 selectively labels the NMDA receptor. The most active inhibitors of [3H]-CGS 19755 binding were L-glutamate and CGS 19755 (IC50 values = 100 nM). 5. In the centrifugation assay, a number of excitatory amino acids were found to generate shallow inhibition curves, and computer analysis indicated the presence of two binding components. The quisqualate receptor ligand AMPA (D,L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate), kainic acid and the non-competitive NMDA antagonists, such as phencyclidine, tiletamine and MK-801, were without activity. 6. The high affinity binding obtained with [3H]-CGS 19755 by use of filtration techniques thus permits the more rapid evaluation of compounds as potential NMDA antagonists and agonists. Therefore, this rigid analogue of AP5 is a more suitable radioligand for NMDA receptors than [3H]-CPP (34-+/-)2-carboxypiperazin-4-yl)propyl-1-phosphonic acid), the corresponding analogue of 2-amino-7-phosphonoheptanoic acid (AP7).

Measurement of cdk4 kinase activity using an affinity peptide-tagging technology.

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.

Methylene blue-induced Heinz body hemolytic anemia.

OBJECTIVE: To describe the manifestations of methylene blue toxicity, with a review of the literature. DESIGN: A descriptive analysis of physical findings and significant laboratory tests in patients with methylene blue toxicity. SETTING: A pediatric referral center. PATIENTS: Two infants, one a neonate with trisomy 21 exposed to methylene blue as an intraoperative diagnostic marker and the other a neonate treated with methylene blue for type II glutaric acidemia. INTERVENTIONS: Laboratory tests to define the occurrence of methylene blue toxicity, phototherapy for hyperbilirubinemia, and transfusions for anemia. MEASUREMENTS AND RESULTS: Within hours after exposure to methylene blue, the infants voided green-blue urine, followed by hyperbilirubinemia, recurrent anemia requiring transfusions, and red blood cell dysmorphology, including the appearance of blister cells and Heinz bodies visible in both Wright's- and supravital-stained peripheral blood smears. After the initiation of phototherapy, both infants exhibited cutaneous bullae followed by desquamation. CONCLUSION: Significant neonatal morbidity may occur following postpartum administration of methylene blue. Toxic manifestations include hyperbilirubinemia, Heinz body hemolytic anemia, and possibly desquamation of the skin. In our infants toxicity was secondary to an overdose of methylene blue, as is true for most of the previously reported cases. Methods for defining the mechanism of dye-related hemolysis and simple screening tests for elucidating the unique sensitivity of certain individuals to dye toxicity are suggested.

Propranolol and methylatropine antagonize the cardiovascular effects produced by microinjection of the TRH analog MK-771 into the preoptic suprachiasmatic nucleus.

Thyrotropin-releasing hormone (TRH) has been shown to increase heart rate as well as blood pressure when administered into rat brain. The present study investigated the mechanism by which the TRH analog MK-771 produces these effects when injected into the preoptic suprachiasmatic nucleus (POSC). MK-771, at a dose of 125 pmol (50 ng), produced significant increases in both heart rate and blood pressure. These effects occurred within 5 minutes of microinjection and lasted approximately 20-30 minutes. Pretreatment with either the beta-adrenergic antagonist propranolol or the muscarinic antagonist methylatropine, administered into the POSC, significantly altered the response produced by MK-771. Propranolol, at a dose of 7 nmol, and methylatropine at a dose of 0.5 nmol, significantly inhibited the tachycardia produced by MK-771. In addition, methylatropine, at a dose of 0.5 nmol, significantly reduced the increase in diastolic pressure produced by the TRH agonist. These results are consistent with the idea that TRH agonists, when administered centrally, produce cardiovascular alterations through the autonomic nervous system.

Autoradiographic distribution of 125I-galanin binding sites in the rat central nervous system.

Galanin (GAL) binding sites in coronal sections of the rat brain were demonstrated using autoradiographic methods. Scatchard analysis of 125I-GAL binding to slide-mounted tissue sections revealed saturable binding to a single class of receptors with a Kd of approximately 0.2 nM. 125I-GAL binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the following areas: prefrontal cortex, the anterior nuclei of the olfactory bulb, several nuclei of the amygdaloid complex, the dorsal septal area, dorsal bed nucleus of the stria terminalis, the ventral pallidum, the internal medullary laminae of the thalamus, medial pretectal nucleus, nucleus of the medial optic tract, borderline area of the caudal spinal trigeminal nucleus adjacent to the spinal trigeminal tract, the substantia gelatinosa and the superficial layers of the dorsal spinal cord. Moderate binding was observed in the piriform, periamygdaloid, entorhinal, insular cortex and the subiculum, the nucleus accumbens, medial forebrain bundle, anterior hypothalamic, ventromedial, dorsal premamillary, lateral and periventricular thalamic nuclei, the subzona incerta, Forel's field H1 and H2, periventricular gray matter, medial and superficial gray strata of the superior colliculus, dorsal parts of the central gray, peripeduncular area, the interpeduncular nucleus, substantia nigra zona compacta, ventral tegmental area, the dorsal and ventral parabrachial and parvocellular reticular nuclei. The preponderance of GAL-binding in somatosensory as well as in limbic areas suggests a possible involvement of GAL in a variety of brain functions.

Increases in heart rate and blood pressure produced by microinjections of atrial natriuretic factor into the AV3V region of rat brain.

Recent studies have provided evidence for the dense localization of atrial natriuretic factor (ANF) in the anteroventral third ventricle (AV3V) region of the rat brain. This area is currently thought to be involved in the regulation of blood pressure and fluid and electrolyte balance. To investigate whether ANF may play a role in central cardiovascular regulation, the effects of microinjection of ANF into the preoptic suprachiasmatic nucleus (POSC), which is located in the AV3V region of the brain, were examined in the present study. Low doses of ANF (2-4 pmol) produced modest elevations in systolic and diastolic pressures, approximately 10-14%, and a small rise in HR of roughly 7%. Higher doses of ANF (20-40 pmol) produced significant increases in systolic (15-19%), mean arterial (12-14%) and pulse (25-36%) pressures. In addition, much larger increases in HR, approximately 20%, were produced by these higher doses of ANF. The onset of effects produced by ANF on BP and HR was seen 15-45 min after injection. Peak effects were usually observed approximately 60-150 min after onset, and the duration of the effect was 2-4 hours, after which time values usually returned to baseline. These studies indicate that ANF produces significant increases in BP and HR when injected at pmol doses into the POSC, and lends support to the idea that this peptide may play an important role in central cardiovascular regulatory mechanisms.