The role nitric oxide and other neurotransmitter in canine penile erection

The role on nitric oxide and its relative factors (cGMP, cAMP, methylene blue) was studied in canine erection induced by stimulating pelvic nerves, and the effect of cholinergic neuroeffectors and the sinusoidal endothelium was also observed in this experiment. The results indicate that intracavernous injection of nitric oxide can evoke a penile tumescence, which is similar to that of the neurostimulation. The results also suggest that the cholinergic nerves and the sinusoidal endothelium are involved in erection, and the effect of the former must depend on mediation of the latter. The study supports that cholinergic and nonadrenergic noncholinergic (NANC) neuroeffectors take part in penile erection, and nitric oxide may be one of chief NANC neurotransmitters.     

Effect of Cu2+ on relaxations to the nitrergic neurotransmitter, NO and S-nitrosothiols in the rat gastric fundus

1. The effects of superoxide anion generators before and after treatment with inhibitors of Cu/Zn superoxide dismutase (Cu/Zn SOD) and the effects of thiol-modulating agents were investigated on nitrergic relaxations to electrical stimulation of non-adrenergic non-cholinergic (NANC) nerves of the rat gastric fundus and on relaxations to authentic nitric oxide (NO) and nitroglycerin. 2. The superoxide anion generators, pyrogallol (30 microM) and duroquinone (30-60 microM), significantly inhibited the relaxations to NO (0.03-3 microM) but not nitrergic relaxations to NANC nerve stimulation (0.5-8 Hz) or those to ATP (10 microM). Treatment of the rat gastric fundus with the inhibitors of Cu/Zn SOD, diethyldithiocarbamate (DETC, 1 mM for 2 h) or triethylenetetramine (TETA, 100 microM for 2 h) had no effect on the relaxations to NANC nerve stimulation (1-8 Hz), NO (0.03-3 microM) or on those to ATP (10 microM). 3. After treatment of the rat gastric fundus with DETC (1 mM) but not after treatment with TETA (100 microM), pyrogallol (30 microM) and duroquinone (30-60 microM) significantly inhibited the nitrergic relaxations to electrical stimulation (0.5-8 Hz) and those to NO (0.03-3 microM). This inhibitory effect of pyrogallol and duroquinone was prevented by addition of exogenous SOD (250 units ml-1). Pyrogallol but not duroquinone also inhibited the NO-independent relaxations to ATP (10 microM). 4. The thiol modulators, buthionine sulphoximine (1 mM for 2 h) and ethacrynic acid (30 microM for 2 h), significantly inhibited the relaxations to nitroglycerin (0.03-3 microM) but had no effect on the nitrergic relaxations to electrical stimulation (0.5-8 Hz) or on those to NO (0.03-10 microM) and ATP (10 microM). The thiol modulators, sulphobromophthalein (100 microM for 2 h) and diamide (30-100 microM for 2 h) did not affect the relaxations to nitroglycerin, or those to NANC nerve stimulation and NO. 5. In summary, thiol modulators significantly inhibited the thiol-dependent relaxations to nitroglycerin but not those to NANC nerve stimulation or NO. Relaxations to nitrergic stimulation were decreased by superoxide anion generators only after inhibition of Cu/Zn SOD. These results suggest that the nitrergic NANC neurotransmitter in the rat gastric fundus is not a nitrosothiol but more likely free NO, which is protected from breakdown by tissue SOD.     

Effects of nitric oxide synthase gene knockout on neurotransmitter release in vivo

Previous studies suggest that low bone mass is a complication of alcoholic liver disease. Nevertheless, little is known about bone mass and bone metabolism in viral cirrhosis. To evaluate the prevalence and magnitude of hepatic osteopenia in these patients, bone remodeling status, and its relationship with the severity of liver disease and serum levels of insulin-like growth factor I (IGF-I), we studied 32 consecutive patients with viral cirrhosis and no history of alcohol intake. Bone mineral density (BMD) was measured by dual x-ray absorptiometry in the lumbar spine (LS) and femoral neck (FN), and the values were expressed as the z score. Bone metabolism markers and hormone profiles were measured. Patients with viral cirrhosis showed reduced BMD in all sites (LS: -1.27 +/- 1.06, P < .001; FN: -0.48 +/- 0.96; P < .01). Of the 32 patients, 53% met the diagnostic criteria for osteoporosis. In patients, urine deoxypyridinoline (D-Pyr) as a marker of bone resorption and serum bone alkaline phosphatase (b-AP) as a marker of bone formation were significantly higher than in control subjects (P < .001 and P < .01, respectively). Serum IGF-I was lower than in control subjects (P < .001), and significant differences were also found between patients with and without osteoporosis (P < .05). BMD in LS correlated with severity of the disease, with serum levels of IGF-I, and with urine D-Pyr. Our findings show that viral cirrhosis is a major cause of osteoporosis in men, and that low serum IGF-I levels seem to play a role in the bone mass loss in these patients. The biochemical markers of bone remodeling suggest high-turnover osteoporosis in patients with viral cirrhosis.     

Anterograde signaling by nitric oxide: characterization and in vitro reconstitution of an identified nitrergic synapse

Nitric oxide (NO) is recognized as a signaling molecule in the CNS where it is a candidate retrograde neurotransmitter. Here we provide direct evidence that NO mediates slow excitatory anterograde transmission between the NO synthase (NOS)-expressing B2 neuron and an NO-responsive follower neuron named B7nor. Both are motoneurons located in the buccal ganglia of the snail Lymnaea stagnalis where they participate in feeding behavior. Transmission between B2 and B7nor is blocked by inhibiting NOS and is suppressed by extracellular scavenging of NO. Furthermore, focal application of NO to the cell body of the B7nor neuron causes a depolarization that mimics the effect of B2 activity. The slow interaction between the B2 and B7nor neurons can be re-established when the two neurons are cocultured, and it shows the same susceptibility to NOS inhibition and NO scavenging. In cell culture we have also examined spatial aspects of NO signaling. We show that before the formation of an anatomical connection, the presynaptic neuron can cause depolarizing potentials in the follower neuron at distances up to 50 micro(m). The strength of the interaction increases when the distance between the cells is reduced. Our results suggest that NO can function as both a synaptic and a nonsynaptic signaling molecule.     

Neuronal nitric oxide synthase in the canine prostate: aging, sex steroid, and pathology correlations

OBJECTIVE: To clarify some anatomical controversies of the fascial structures and lymph node development of the inguinal region through an embryological study in relation to the surgical techniques of groin lymphadenectomy. METHODS: Sections of the femoral triangle belonging to four fetuses whose crown-rump (CR) length ranged from 70 to 310 mm, corresponding to a developmental age of 11 and 35 weeks, were studied. RESULTS: The femoral fascia is formed of one layer and is not divided into superficial and deep layers. The cribriform fascia has a morphogenetic origin different from that of the femoral fascia and it is defined by the thickening of the connective tissue filling the fossa ovalis and therefore would be more correctly named lamina cribrosa. The deep inguinal lymph nodes originate directly from the superficial lymphatic tissue located in the fossa ovalis. This last observation supports the fact that no lymph nodes are present beneath the femoral fascia distal to the lower margin of the fossa ovalis. CONCLUSIONS: The results of this study, from a surgical point of view, support the technique of total or radical inguinal-femoral lymphadenectomy with preservation of the femoral fascia and, from an anatomical point of view, resolve some of the contradictory statements reported in the anatomical literature regarding morphogenesis and terminology of the structures of the Scarpa's triangle. In addition, the present study provides useful anatomic and terminological landmarks to those surgical oncologists (gynecologist, urologist, dermatologist, etc.) dealing with malignant diseases requiring groin dissection practices. In addition, it could represent a useful background for a future more precise surgical terminology which represents a vital issue for institutional studies with multiple surgeons as well as for large multi-institutional studies.     

Neurogenic nitric oxide mediates relaxation of canine corpus cavernosum

PURPOSE: The aim of the present study is to analyze mechanisms underlying neurogenic relaxation of the corpus cavernosum which are believed to participate in penile erection. MATERIALS AND METHODS: Mechanical responses to nerve stimulation by electrical pulses and nicotine were measured in strips of canine corpus cavernosum precontracted with phenylephrine. Cyclic guanosine monophosphate (GMP) contents in the strips were also measured by radioimmunoassay. Immunohistochemistry for nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) was performed. RESULTS: Transmural electrical stimulation and nicotine produced relaxations in the isolated canine corpus. The neurogenic relaxation was abolished by N omega-nitro-L-arginine, a NOS inhibitor, and the inhibition was reversed by L-arginine. Relaxations induced by nerve stimulation and exogenous nitric oxide (NO) were depressed by oxyhemoglobin and methylene blue. Vasoactive intestinal polypeptide (VIP)-induced relaxations were not influenced by these inhibitors. In the controls strips and those made unresponsive to VIP by its repeated application, the responses to nerve stimulation did not differ. The content of cyclic GMP in the tissue increased in response to nicotine, the effect being abolished by the NO synthase inhibitor. Immunohistochemical study demonstrated neurons containing NOS and VIP. CONCLUSIONS: It appears that the relaxation induced by nerve stimulation is mediated solely by NO liberated from the nerve that activates soluble guanylate cyclase and increases the production of cyclic GMP in smooth muscle, whereas VIP does not play a role in the regulation of muscle tone under the experimental conditions used.     

Dinitrosyl iron complexes and S-nitrosothiols are two possible forms for stabilization and transport of nitric oxide in biological systems

The physicochemical properties, mechanisms of synthesis and decomposition of dinitrosyl iron complexes (DNICs) with thiol-containing ligands and of S-nitrosothiols (RS-NO), and the potential role of these compounds in storage and transport of NO in biological systems are reviewed. Special attention is given to the phenomenon of mutual transformation of DNIC and RS-NO catalyzed by Fe2+. Each Fe2+ binds two neutral NO molecules in the DNICs, catalyzes their mutual oxidation--reduction with formation of nitrous oxide and nitrosonium ions appearing in the DNICs. These ions S-nitrosate thiol-compounds with RS-NO formation. Fe2+ binds two RS-NO molecules and catalyzes their mutual oxidation--reduction followed by decomposition of the resulting molecules. Mutual conversion of DNICs and RS-NO regulated by iron, thiol, and NO levels is suggested to provide NO transport in cells and tissues.     

Inhibition of angiotensin-converting enzyme increases the nitric oxide levels in canine ischemic myocardium

Since angiotensin-converting enzyme (ACE) produces angiotensin II in the heart, ACE inhibitors may prevent coronary vasoconstriction and increase coronary blood flow. On the other hand, since ACE inhibitors also inhibit kininase II which results in reduced degradation of bradykinin, ACE inhibitors may increase cardiac nitric oxide (NO) levels via stimulation of bradykinin receptors. This study was undertaken to test whether ACE inhibitors increase the cardiac NO levels and coronary blood flow in the ischemic myocardium. In 34 open-chest dogs, the left anterior descending coronary artery was perfused through an extracorporeal bypass tube from the left carotid artery. When either imidaprilat or cilazaprilat of 3 microg/kg/min was infused into the bypass tube for 10 min after reduction of coronary blood flow due to partial occlusion of the bypass tube, coronary blood flow increased from 31 +/- 1 to either 45 +/- 5 or 43 +/- 4 ml/100 g/min despite no changes in coronary perfusion pressure (43 +/- 2 mmHg). During an infusion of either imidaprilat or cilazaprilat, bradykinin and the end-products of NO (nitrate + nitrite) concentrations of coronary venous blood were markedly increased, which were attenuated by either HOE-140 (an inhibitor of bradykinin receptors) or by N(omega)-nitro-L-arginine methyl ester (an inhibitor of NO synthase). We also observed increases in cardiac bradykinin and NO levels due to either imidaprilat or cilazaprilat in the low constant coronary blood flow condition. It is concluded that ACE inhibitors can increase cardiac NO levels via the accumulation of bradykinin in the ischemic myocardium.     

Redox modulation of L-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols

The effects of NO-related activity and cellular thiol redox state on basal L-type calcium current, ICa,L, in ferret right ventricular myocytes were studied using the patch clamp technique. SIN-1, which generates both NO. and O2-, either inhibited or stimulated ICa,L. In the presence of superoxide dismutase only inhibition was seen. 8-Br-cGMP also inhibited ICa,L, suggesting that the NO inhibition is cGMP-dependent. On the other hand, S-nitrosothiols (RSNOs), which donate NO+, stimulated ICa,L. RSNO effects were not dependent upon cell permeability, modulation of SR Ca2+ release, activation of kinases, inhibition of phosphatases, or alterations in cGMP levels. Similar activation of ICa,L by thiol oxidants, and reversal by thiol reductants, identifies an allosteric thiol-containing "redox switch" on the L-type calcium channel subunit complex by which NO/O2- and NO+ transfer can exert effects opposite to those produced by NO. In sum, our results suggest that: (a) both indirect (cGMP-dependent) and direct (S-nitrosylation/oxidation) regulation of ventricular ICa,L, and (b) sarcolemma thiol redox state may be an important determinant of ICa,L activity.     

Localization and characterization of nitric oxide synthase in the rat suprachiasmatic nucleus: evidence for a nitrergic plexus in the biological clock

Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH-diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of L-[3H]arginine to L-[3H]-citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca2+/calmodulin-dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.     

Comparison among the effects of arginine, a nitric oxide precursor, isosorbide dinitrate and molsidomine, two nitric oxide donors, on hormonal secretions and blood pressure in man

A bulky tumor of the first lumbar vertebra is described. The case is the 21st to be reported. The tumor resembled an aneurysmal bone cyst radiologically. It was resected without incident. The previously reported cases are reviewed and the literature discussed.     

Inhibition of nitric oxide synthesis reveals non-cholinergic excitatory neurotransmission in the canine proximal colon

1. Neuromuscular transmission in the circular muscle of the canine proximal colon was examined, in the presence and absence of nitric oxide synthase inhibitors, by use of mechanical and intracellular microelectrode recording techniques. 2. Electrical field stimulation (EFS; 0.1-20 HZ) produced frequency-dependent contractions of circular muscle strips which reached a maximum at 15 Hz. These responses were enhanced by NG-monomethyl-L-arginine (L-NMMA; 300 microM) and reduced by atropine (1 microM). The effects of L-NMMA were reversed by L-arginine (3 mM). All responses to EFS were abolished by tetrodotoxin (1 microM). 3. In the presence of atropine, phentolamine and propranolol (all at 1 microM; 'non-adrenergic, non-cholingergic (NANC) conditions'), EFS evoked frequency-dependent inhibition of phasic contractions which reached a maximum at 5 Hz. At higher frequencies of EFS, inhibition diminished, and these responses were followed by post-stimulus excitation. 4. Under NANC conditions and in the presence of L-NG-nitroarginine methyl ester (L-NAME; 200 microM), EFS evoked contractions at frequencies of 5 Hz or greater. These contractions were reduced by co-incubation with L-arginine (2 mM) and abolished by tetrodotoxin (1 microM). 5. In the presence of atropine (1 microM), EFS (5-20 Hz) caused frequency-dependent inhibition of electrical slow waves. In the presence of L-NAME (100 microM) and atropine, the inhibitory response to EFS was abolished and an increase in slow wave duration was seen at stimulation frequencies greater than 5 Hz. The effects of EFS on slow wave duration were abolished by tetrodotoxin (1 microM). 6. Atropine-resistant contractions to EFS were enhanced by indomethacin (10 microM) and reduced or abolished by the non-selective NK1/NK2 tachykinin receptor antagonist D-Pro2, D-Trp7,9 SP, and by the selective NK2 receptor antagonist MEN 10,376 (10 microM).7. Exogenous tachykinins mimicked non-cholinergic excitatory electrical and mechanical responses. The rank order of potency for contraction was neurokinin A>neurokinin B>substance P, suggesting a predominance of the NK2 sub-type of tachykinin receptors on colonic smooth muscle cells. Low concentrations of neurokinin A also increased the amplitude and duration of electrical slow waves.8. These results suggest that: (i) in previous studies, non-cholinergic excitatory responses were masked by the simultaneous release of NO; (ii) non-cholinergic excitatory responses occur throughout the period of stimulation and are not manifest only as 'rebound' excitation; (iii) one or more tachykinins, possibly,acting via NK2 receptors, may mediate non-cholinergic excitatory responses.     

The role of nitric oxide in heart failure. Potential for pharmacological intervention

There is now considerable evidence that nitric oxide (NO) production and action are abnormal in patients with heart failure. Spontaneous NO release from the vascular endothelium is preserved or enhanced in patients with heart failure and this may help to maintain tissue perfusion by blunting the vasoconstriction induced by various neurohumoral factors. On the other hand, endothelial NO release in response to various stimuli including exercise appears to be diminished and this may contribute to the impaired exercise capacity of patients with heart failure. It is now apparent that NO produced within the heart plays an important role in the modulation of cardiac contractility under physiological conditions. In patients with heart failure, however, increased myocardial NO production in response to cytokines such as tumour necrosis factor-alpha may contribute to reduced contractility and myocyte injury. Our understanding of the role of NO in the control of vascular tone has provided an explanation for the efficacy of nitrovasodilators in heart failure and has stimulated novel approaches to augmenting endogenous vascular NO production. There is also evidence that ACE inhibitors act to restore normal endothelial function in patients with heart failure. Increased NO production within the heart, particularly that produced via the pro-inflammatory inducible NO synthase, may be detrimental. It remains to be determined whether selective inhibition of inducible NO synthase can favourably modify the course of this lethal condition.     

Stress, adaptation, and nitric oxide

The biological role of nitric oxide (NO) has been studied for more than ten years. Nevertheless, the number of investigations in this field continues to increase. It is now suggested that NO is a previously unrecognized, very important regulator of physiological functions and cell metabolism in the body. Through the application of the methods of molecular biology, more and more data are being accumulated on the regulatory role of NO in the mechanism of gene expression and protein biosynthesis. The data presented in this review show an important role of NO in stress and adaptive responses of organisms and thereby expand existing notions on the biological role of this unique molecule. This review substantiates the idea that the system of NO generation is a newly discovered stress-limiting system. The action of this NO-ergic system is based on the capability of NO to limit key links of the stress reaction and to enhance the potency of endogenous defense systems of the organism. The role of NO is considered at the major stages of adaptation: 1) at the urgent stage related with the stress reaction; 2) at the stage of the transition from urgent to long-term adaptation; and 3) at the stage of long-term adaptation characterized by the formation of stable protective effects. It is demonstrated that pharmacological "imitation" of the activated NO-ergic system by administration of NO donors to the organism provides in many instances an efficient protection against stress damage and enhances the adaptive capacity of the organism.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide

There has been confusion as to what role(s) nitric oxide (NO) has in different physiological and pathophysiological mechanisms. Some studies imply that NO has cytotoxic properties and is the genesis of numerous diseases and degenerative states, whereas other reports suggest that NO prevents injurious conditions from developing and promotes events which return tissue to homeostasis. The primary determinant(s) of how NO affects biological systems centers on its chemistry. The chemistry of NO in biological systems is extensive and complex. To simplify this discussion, we have formulated the "chemical biology of NO" to describe the pertinent chemical reactions under specific biological conditions. The chemical biology of NO is divided into two major categories, direct and indirect. Direct effects are defined as those reactions fast enough to occur between NO and specific biological molecules. Indirect effects do not involve NO, but rather are mediated by reactive nitrogen oxide species (RNOS) formed from the reaction of NO either with oxygen or superoxide. RNOS formed from NO can mediate either nitrosative or oxidative stress. This report discusses various aspects of the chemical biology of NO relating to biological molecules such as guanylate cyclase, cytochrome P450, nitric oxide synthase, catalase, and DNA and explores the potential roles of NO in different biological events. Also, the implications of different chemical reactions of NO with cellular processes such as mitochondrial respiration, metal homeostasis, and lipid metabolism are discussed. Finally, a discussion of the chemical biology of NO in different cytotoxic mechanisms is presented.     

Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain

The structures capable of synthesizing cyclic GMP in response to nitric oxide in the rat brain were compared relative to the anatomical localization of neuronal nitric oxide synthase. In order to do this, we used brain slices incubated in vitro, where cyclic GMP-synthesis was stimulated using sodium nitroprusside as a nitric oxide-donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Nitric oxide-stimulated cyclic GMP synthesis was found in cells and fibers, but was especially prominent in varicose fibers throughout the rat brain. Fibers containing the nitric oxide-stimulated cyclic GMP production were present in virtually every area of the rat brain although there were large regional variations in the density of the fiber networks. When compared with the localization of nitric oxide synthase, it was observed that although nitric oxide-responsive and the nitric oxide-producing structures were found in similar locations in general this distribution was complementary. Only occasionally was nitric oxide-mediated cyclic GMP synthesis observed in structures which also contained nitric oxide synthase. We conclude that the nitric oxide-responsive soluble guanylyl cyclase and nitric oxide synthase are usually juxtaposed at very short distances in the rat brain. These findings very strongly support the proposed role of nitric oxide as an endogenous activator of the soluble guanylyl cyclase in the central nervous system and convincingly demonstrate the presence of the nitric oxide-cyclic GMP signal transduction pathway in virtually every area of the rat brain.     

Sleep and nitric oxide: effects of 7-nitro indazole, inhibitor of brain nitric oxide synthase

The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.