Neutrophil function in whole blood and after purification: changes in receptor expression, oxidase activity and responsiveness to cytokines. Neutrophil function and plasma membrane receptor expression was measured in cell suspensions isolated by two separate procedures and in unfractionated whole blood. When cells were prepared by a combined dextran/ficoll procedure, their ability to generate reactive oxidants in response to fMet-Leu-Phe was greater than in corresponding cells isolated by a one-step procedure on Mono-Poly Resolving Medium (M-PRM). Cells prepared by both methods could be primed in vitro by rGM-CSF, but the priming ratio was greater in cells prepared by the latter method. The ability of neutrophils in whole blood to generate reactive oxidants in response to fMet-Leu-Phe was extremely low, but this was increased by more than 10 fold if the blood was pre-incubated with rGM-CSF. Similarly, expression of CD 11b and CD 16 was very low (or undetectable) in neutrophils in whole blood, but this was rapidly increased upon priming. Activation by PMA resulted in a down regulation of CD 16 expression as the receptor was shed from the cell surface. Neutrophils isolated by either the dextran/ficoll or the M-PRM method showed increased expression of receptors compared with those in whole blood, although this expression was lower in cells isolated by the latter method. These data indicate that the isolation procedures used to obtain purified neutrophils prime both receptor expression and oxidase function, although these effects are minimalised in isolation procedures using M-PRM. Furthermore, as CD 16 expression on neutrophils in whole blood is rapidly up-regulated during priming, it seems likely that, as for complement receptors, rapidly-mobilisable intracellular stores of this receptor exist.


Amino acid uptake regulation by cell growth in cultured hepatocytes isolated from fetal and adult rats. Amino acid uptake mediated by system A was studied in cultured fetal and adult hepatocytes, subjected to growth stimulation by EGF and insulin, or to growth inhibition by high cell density. The mitogenic stimulation induced a strong transport increase only in fetal cells, while the cell density-dependent growth inhibition, probably mediated by molecules present on adult hepatocyte membranes, provoked the decrease of amino acid uptake only in the adult cells. The results indicate that the different modulation of amino acid transport by cell growth is dependent on the age and the differentiation stage of hepatocytes.


Evaluation of in vivo insulin action and glucose metabolism in milk-fed rats. Milk diet has long been recommended in the management of gastrointestinal pathologies. Since milk feeding represents a high fat-low carbohydrate diet and it is acknowledged that insulin resistance is one of the consequences of high fat feeding, it is important to know whether or not chronic milk feeding leads to an impairment of the insulin-mediated glucose metabolism. To examine this question, adult female rats were given raw cow's milk (50% of total calories as lipids) for 18 days. They were compared to rats raised in parallel and fed the standard laboratory diet (15% of total calories as lipids). At the end of the 18 day period, body weight, daily caloric intake, basal plasma glucose and insulin levels in the milk-fed rats were similar to those in the control rats. In vivo insulin action was assessed with the euglycemichyperinsulinemic clamp technique in anesthetized animals. These studies were coupled with the 2-deoxyglucose technique allowing a measurement of glucose utilization by individual tissues. In the milk fed rats: 1) the basal rate of endogenous glucose production was significantly (p < 0.01) reduced (by 20%); 2) their hepatic glucose production was however normally suppressed by hyperinsulinemia; 3) their basal glucose utilization rate was significantly (p < 0.01) reduced (by 20%); 4) their glucose utilization rate by the whole-body mass or by individual tissues was normally increased by hyperinsulinemia. These results indicate that insulin action in adult rats is not grossly altered after chronic milk-feeding, at least under the present experimental conditions.


In vitro secretion of transforming growth factor alpha (TGF-alpha): a comparison of the A431 cell line with three human oesophageal squamous cell carcinoma lines. Transforming growth factor alpha (TGF-alpha) is a single chain polypeptide which exists in a variety of forms differing in molecular weight. These forms are variously present in normal and neoplastic cells. Of particular interest are TGF-alpha's well-known mitogenic properties. The transition from a normal to a neoplastic cellular state results from signalling defects that may depend upon, inter alia, abnormal levels of expression and secretion of TGF-alpha. It is known that the secretion of TGF-alpha may be enhanced appreciably by agents such as phorbol 12-myristate 13-acetate (PMA), serum factors and epidermal growth factor (EGF). Here, we compare the efficacy of these three agents in the elevation of TGF-alpha secretion in the well studied A431 cell line with their previously undocumented efficacy in certain interesting, but little known, human oesophageal squamous cell carcinoma (SCC) lines.


A domain of the alpha-subunit of rabbit phosphorylase kinase shows homologies with regions of rabbit alpha-tropomyosin, human EGF receptor, and the alpha chain of bovine S-100 protein. Sequence comparison of the alpha-subunit of phosphorylase kinase with alpha-tropomyosin revealed 32% identity, and 49% similarity, between the region of alpha-tropomyosin coded by exon 5 and a 39 amino acid segment of the kinase subunit. A subsequence of the alpha-subunit segment and a sequence overlapping the same alpha-subunit region are homologous with: (a) a region of the cytoplasmic domain of EGF receptor (50% identity) and (b) a Ca(2+)-binding domain of the alpha chain of S-100 protein (50% identity) respectively. Statistical analysis shows that these homologies are significant. The biological implication of the above similarities is discussed.


Is calcium or ephedrine superior to placebo for emergence from cardiopulmonary bypass? To determine whether ephedrine or CaCl2 improves hemodynamics in cardiac surgery patients emerging from cardiopulmonary bypass, three sequential doses of either CaCl2 (200 mg/dose; n = 12), ephedrine (5 mg/dose; n = 12), or placebo (n = 12) were administered in a prospective, randomized, double-blind fashion. Thermodilution volumetric catheters were used to calculate right ventricular (RV) volumes and ejection fraction. The first dose of ephedrine improved RV stroke volume from 57 +/- 3 to 63 +/- 4 mL/beat (P < 0.05) and ejection fraction from 44 +/- 2% to 49 +/- 2% (P < 0.05). Subsequent doses maintained this improvement but without further change. In contrast, placebo and CaCl2 had minimal effects on RV end-systolic volume, stroke volume, and ejection fraction. After the third injection of ephedrine, mean arterial pressure had significantly increased from 78 +/- 2 to 93 +/- 4 mmHg (P < 0.05) in contrast to insignificant increments with placebo and CaCl2. Serum ionized calcium increased by 6% to 8% after each CaCl2 bolus but remained stable in the ephedrine and placebo groups. CaCl2 failed to improve RV performance in mildly hypocalcemic patients during separation from cardiopulmonary bypass. In patients with normal preoperative ventricular function, ephedrine more effectively improved RV performance and arterial blood pressure than placebo or CaCl2, and is a suitable short-acting drug to assist separation from cardiopulmonary bypass.


Dobutamine increases heart rate more than epinephrine in patients recovering from aortocoronary bypass surgery. To determine whether epinephrine might prove to be a cost-effective substitute for dobutamine, two 8-minute infusions of either epinephrine (10 and 30 ng/kg/min, n = 28) or dobutamine (2.5 and 5 micrograms/kg/min, n = 24) were administered to 52 patients recovering in the intensive care unit (ICU) after aortocoronary bypass (CABG) surgery. At the higher dose, both drugs significantly (P < .05) increased cardiac index (CI), epinephrine from 2.8 +/- 0.1 at baseline to 3.3 +/- 0.1 L/min/m2, and dobutamine from 3.2 +/- 0.1 at baseline to 4.1 +/- 0.2 L/min/m2. Epinephrine increased CI significantly less than dobutamine. Both drugs significantly increased stroke volume index (SVI), epinephrine from 32 +/- 1 at baseline to 36 +/- 1 mL/beat/m2, and dobutamine from 36 +/- 1 at baseline to 40 +/- 2 mL/beat/m2. At the higher dose, the effects of the two drugs on SVI were indistinguishable. On the other hand, while the higher dose of both drugs significantly increased heart rate (HR), epinephrine from 88 +/- 2 at baseline to 90 +/- 2 beats/min and dobutamine from 89 +/- 2 at baseline to 105 +/- 3 beats/min, the increase following the higher dose of dobutamine was significantly greater than that seen after epinephrine. Effects of the two drugs on mean arterial pressure, central venous pressure, pulmonary artery occlusion pressure, systemic vascular resistance, pulmonary vascular resistance, and left-ventricular stroke work did not significantly differ. Similar results were obtained in the subset of patients with baseline CI less than 3 L/min/m2 who more closely resembled patients who might acutely require inotropic drug administration.(ABSTRACT TRUNCATED AT 250 WORDS)


Amrinone and dobutamine as primary treatment of low cardiac output syndrome following coronary artery surgery: a comparison of their effects on hemodynamics and outcome. This study was undertaken in order to compare the effectiveness of amrinone and dobutamine as primary treatment of a low cardiac output (CO) after coronary artery bypass graft (CABG) surgery. Thirty patients with preoperative left ventricular dysfunction participated in this open-label randomized study. Patients were included if they failed to separate from cardiopulmonary bypass (CPB) without inotropic support or if they had a cardiac index (CI) less than 2.4 L/min/m2 after CPB regardless of the blood pressure, in the presence of adequate filling pressures. The treatment objectives were to separate from CPB and achieve a CI > or = 2.4 L/min/m2 and a mean arterial pressure > or = 70 mmHg. Patients treated with amrinone received 0.75 mg/kg followed by 10 micrograms/kg/min; when the objectives were not achieved within five minutes, another 0.75 mg/kg was given. Patients treated with dobutamine received an initial infusion of 5 micrograms/kg/min increased stepwise to 15 micrograms/kg/min if necessary. Eleven of 15 amrinone versus 6 of 15 dobutamine patients achieved the predefined treatment objectives with the test drug alone (P = NS). Comparisons of hemodynamics in patients treated solely with amrinone (n = 7) or dobutamine (n = 6) after CPB showed no significant differences between the treatment groups. The incidence of myocardial ischemia as detected by Holter monitor was 36% with amrinone and 33% with dobutamine. Two patients suffered ventricular fibrillation and two had significant supraventricular tachyarrhythmias (heart rate > 130/min) during treatment with dobutamine alone, whereas no significant arrhythmias occurred in the amrinone group (P = NS). Six dobutamine patients (40%) had postoperative myocardial infarction (MI) as opposed to none among the amrinone patients (P = 0.017). These results indicate that amrinone compares favorably with dobutamine as a primary treatment of low CO after CABG. Further study in a larger number of patients will be required in order to determine if the lower incidence of MI in the amrinone group was due to the treatment drug.


Intravenous milrinone following cardiac surgery: I. Effects of bolus infusion followed by variable dose maintenance infusion. The European Milrinone Multicentre Trial Group. The hemodynamic and adverse effects of intravenous milrinone were studied in 99 adult patients (66 men) following elective myocardial revascularization, mitral and/or aortic valve surgery. All patients had a low cardiac output (cardiac index [CI] mean 1.93, range, 1.11 to 2.5 L/min/m2) despite adequate cardiac filling pressure (mean pulmonary capillary wedge pressure [PCWP] 11.5 mmHg, range, 8 to 20 mmHg). Following a period of baseline stability (mean 17.8 minutes, range, 10 to 50 minutes), patients received a bolus infusion of 50 micrograms/kg over 10 minutes. A continuous maintenance infusion of 0.375 (low), 0.5 (mid) or 0.75 (high) micrograms/kg/min was administered for a minimum of 12 hours. Patients were allocated to each dosage group sequentially, not randomly. Hemodynamic measurements were made before the start of milrinone and 15 minutes after the bolus infusion. Further measurements were made at 30, 45, and 60 minutes, and at 3, 6, and 12 hours after the start of treatment. Measurements were also made at 2 and 4 hours after treatment was stopped. The bolus infusion caused significant increases in CI, heart rate (HR), and stroke index (SI), (P < 0.001), and significant falls in PCWP, right atrial pressure (RAP), mean pulmonary artery pressure (mPAP), pulmonary vascular resistance (PVR), mean arterial pressure (MAP), and systemic vascular resistance (SVR) (P < 0.001). These effects were maintained to a significant degree by each of the three maintenance infusion regimens, although the pulmonary vasodilator effects appeared less predictable and more dose dependent. Eighteen patients (19%) had arrhythmias; 16 of these were judged not to be serious events. Two were deemed serious; these were both episodes of fast atrial fibrillation (AF).(ABSTRACT TRUNCATED AT 250 WORDS)


Intravenous milrinone following cardiac surgery: II. Influence of baseline hemodynamics and patient factors on therapeutic response. The European Milrinone Multicentre Trial Group. Further analysis of the data from 99 adult patients who received an intravenous infusion of milrinone following elective cardiac surgery was done. All patients received a bolus infusion of 50 micrograms/kg over 10 minutes, followed by a maintenance infusion of either 0.375, 0.5 or 0.75 microgram/kg/min for a period of 12 hours. Hemodynamic measurements were made after the bolus infusion (15 minutes), and then after 30, 45, and 60 minutes at 3, 6, and 12 hours, and 4 hours after treatment was stopped. The following was found: (1) pretreatment cardiac index (CI) affected the response to treatment. Patients with a low CI (1.59 L/min/m2) had a 54% increase after the bolus infusion compared to a 27% increase in patients with a higher pretreatment value (2.2 L/min/m2) (P < 0.05); (2) patients with a high resting level of pulmonary vascular resistance (PVR > 200 dynes.sec.cm-5) had a greater response to treatment (26% fall in PVR) than the remainder (9% fall in PVR) after 60 minutes; (3) patients with a low pretreatment mean arterial pressure (MAP) (n = 17, MAP 64 mmMg, range, 52 to 70) showed no fall in MAP following treatment, but showed a significant increase in CI (+55%). A good therapeutic response was found that was similar in patients undergoing valve replacement surgery or coronary artery bypass graft surgery, and in patients in sinus rhythm or atrial fibrillation before treatment. It is concluded that the therapeutic response to intravenous milrinone following cardiac surgery is partially determined by pretreatment hemodynamics.


Left ventricular filling as assessed by pulsed Doppler echocardiography after coronary artery bypass grafting. Diastolic filling of the left ventricle, as assessed by transesophageal pulsed Doppler echocardiography during and in the early phase following coronary artery bypass grafting, was investigated in nine patients without valvular disease or left ventricular hypertrophy. The ratio between the maximal heights of the early diastolic flow-velocity peak and the late (atrial) diastolic flow-velocity peak, the E:A ratio, and also the deceleration time of the early peak were calculated as indices of left ventricular filling. The E:A ratio decreased from 1.01 +/- 0.06 after induction of anesthesia to 0.46 +/- 0.06 on arrival in the intensive care unit (ICU). The E:A ratio then increased and reached 0.87 +/- 0.08 after 6 hours in the ICU. This increase in E:A ratio in the ICU was due to an increasing peak velocity of the E wave. The deceleration time decreased after surgery and increased from 111 +/- 25 to 145 +/- 12 milliseconds in the ICU. Pulsed Doppler indices of diastolic filling are known to be altered by changes in hemodynamic parameters, to be load-dependent, and to vary with heart rate. In the ICU, pulmonary capillary wedge pressure remained unchanged, heart rate decreased by approximately 12%, and systemic vascular resistance decreased by approximately 40%. The changes in hemodynamic parameters could have affected the E:A ratio, but it is unlikely that they could explain the marked increase in the E:A ratio that occurred in the ICU. The results, therefore, imply the presence of impaired diastolic filling immediately after cardiopulmonary bypass with gradual, but not complete, recovery during the first 6 hours in the ICU.


Left ventricular regional function and relaxation: effects of inotropic stimulation. The effects of increasing doses of two inotropes, isoproterenol and calcium chloride (CaCl2), on left ventricular regional myocardial function and isovolumic relaxation were studied in six anesthetized sheep. After baseline data, CaCl2 was given as intravenous boluses to yield doses of 10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg, and 160 mg/kg. After a second series of baseline data were obtained, constant infusions of isoproterenol were begun with doses of 0.025 micrograms/kg/min, 0.05 micrograms/kg/min, 0.1 micrograms/kg/min, 0.2 micrograms/kg/min, and 0.4 micrograms/kg/min. During each stage of the protocol with both inotropes, data were recorded during acute constriction of the descending thoracic aorta. Left ventricular relaxation was assessed by analysis of peak negative left ventricular (LV) dP/dt and the time constant of isovolumic left ventricular relaxation (Trelax). Regional myocardial function showed little change in either apical or basal segments until high doses of the inotropes. Peak negative LV dP/dt significantly changed from baseline (775 +/- 60 mmHg/s) with 0.2 micrograms/kg/min (1780 +/- 400 mmHg/s, P < 0.05 v baseline) and 0.4 micrograms/kg/min (2,220 +/- 380 mmHg/s, P < 0.05 v baseline) of isoproterenol, and was unchanged by CaCl2. Trelax was significantly decreased by all doses of isoproterenol, whereas only one dose of CaCl2 decreased Trelax. Trelax was increased with afterloading and this effect was altered by isoproterenol. It is concluded that isoproterenol hastens, whereas CaCl2 does not alter, left ventricular relaxation. This may reflect beta-adrenergic modulation of calcium fluxes during isovolumic relaxation.


Effect of prostaglandin E1 on myocardial contractility in dogs anesthetized with halothane: load-independent and noninvasive assessment using transesophageal echocardiography. The presence of an inotropic action of prostaglandin E1 (PGE1) in vivo is controversial, and there are conflicting results obtained by various indices of myocardial contractility. In this study, a direct effect of PGE1 on contractility was investigated in dogs by use of a load-independent contractile index: left ventricular end-systolic wall stress (LVESWS) versus the velocity of circumferential fiber shortening with rate-corrected (Vcfc) relationship using transesophageal echocardiography (TEE). Hemodynamics, arterial blood gas, and TEE data were obtained before PGE1 infusion (control), and with a 10%, 20%, and 30% decrease in mean arterial pressure (MAP) following intravenous PGE1 administration. PGE1 infusion rates were 0.19 +/- 0.03 at 10%, 0.82 +/- 0.17 at 20%, and 2.32 +/- 0.36 micrograms/kg/min at a 30% decrease in MAP. Pulmonary capillary wedge pressure, systemic vascular resistance index, and left ventricular stroke work index significantly decreased, and heart rate, cardiac index, and stroke volume index were not significantly altered. Analysis of the TEE data showed LVESWS (index of afterload) significantly decreased from 92.0 +/- 11.2 g/cm2 to 72.7 +/- 7.8 at 10%, 59.3 +/- 7.8 at 20%, and 44.6 +/- 6.2 at a 30% decrease in MAP, and Vcfc significantly increased from 0.595 +/- 0.065 circ/sec of control value to 0.670 +/- 0.056 at 10%, 0.824 +/- 0.049 at 20%, and 0.939 +/- 0.070 at a 30% decrease in MAP. In the LVESWS versus Vcfc relationship, no significant difference could be detected between the control state and the 10%, 20%, and 30% decrease in MAP, and no inotropic effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)


Canine end-systolic pressure-length relationships: depressed by diltiazem, invalidated by ischemia. This study was designed to determine whether the end-systolic pressure-length relationship (ESPLR) reflects changes in regional contractility during the imposition of graded ischemia, and whether it is modified by diltiazem during propofol anesthesia. Seven beagles were anesthetized and instrumented to measure left ventricular pressure and subendocardial segment lengths (sonomicrometry) in the region of the left anterior descending (LAD) and circumflex (LC) arteries. Afterload was increased by the tightening of a snare around the descending thoracic aorta. Pressure-length loops were constructed and the slope of the ESPLR and the x-axis intercept, Lo, were calculated. Graded ischemia of the apical myocardium only was accomplished by the tightening of a micrometer-controlled snare around the LAD to produce Critical Constriction (CC), Ischemia 1 and 2 (I1, I2), and Total Occlusion (TO). In the basal LC region, LAD ischemia had no effect on either the ESPLR slope or Lo. In contrast, the ESPLR slope in the LAD area was decreased by ischemia at I1 (-40%), increased at TO (+69%), and unchanged at CC and I2, and was reduced by diltiazem at CC and I2 (-31% and -36%, respectively). The LAD ESPLR Lo was increased by ischemia by 64% and 61% at I2, and 91% and 122% at TO, before and after diltiazem, respectively. In the LC region, diltiazem decreased systolic shortening and the ESPLR slope. These results indicate that diltiazem has negative inotropic properties in both ischemic and nonischemic areas. Also, Lo is not a constant and must always be redetermined for every intervention. In the absence of ischemia, the ESPLR may be a reliable measure of myocardial contractility.(ABSTRACT TRUNCATED AT 250 WORDS)


Brainstem Fos expression following acute unilateral labyrinthectomy in the rat. Detection of Fos protein expression with a polyclonal antibody was used to identify brainstem neurons responding to acute (24 h) effects of a unilateral sodium arsanilate chemical labyrinthectomy in Long-Evans rats. Asymmetrical expression was apparent in the medial and inferior vestibular nuclei, the prepositus hypoglossi, the dorsolateral central gray, and the inferior olivary beta subnucleus. These data suggest different distributions of neural activation compared with previous electrophysiological and 2-deoxyglucose results. In addition, there was some Fos expression bilaterally in the olivary dorsomedial cell column, interstitial nucleus of Cajal and the Darkschewitsch nucleus. These results support the concept of multiple systems participating in vestibular compensation and further define some specific nuclei involved in the acute stage.


Apomorphine disrupts odour-induced patterns of glomerular activation in the olfactory bulb. The olfactory bulb of adult male rats stimulated with propionic acid vapours displays a characteristic focus of high metabolic activity in the dorso-medial glomeruli, as revealed by the 2-deoxyglucose (2DG) method. Injection of the dopaminergic agonist apomorphine (1.5 mg kg-1) prior to odour stimulation completely abolishes this selective pattern of glomerular activation, while the metabolic activity of other bulbar areas is not significantly altered. This effect is abolished by a previous injection of the dopamine antagonist haloperidol (0.1 mg kg-1). These observations emphasize the probable involvement of dopamine and dopamine receptors in the bulbar processing of olfactory information.


Quantitative autoradiographic mapping of a novel serotonin receptor-5-HT1S. The spinal cord distribution of a recently identified, high affinity 3H-5-HT binding site, the 5-HT1S receptor was studied employing quantitative autoradiography with microdensitometry. The laminar distribution and density of other high affinity 3H-5-HT binding sites present in rat spinal cord, 5-HT1A, 5-HT1B and 5-HT1C, was also determined. The greatest density of 5-HT1S receptors was found in the superficial dorsal horn laminae, the autonomic nuclei around the central canal and the ventral motor nuclei. Smaller, variable densities of 5-HT1S receptors were observed in other spinal cord regions. A similar spatial pattern of receptor density was observed for 5-HT1A, 5-HT1B and 5-HT1C receptors. The distribution of 5-HT1S receptors suggests functional modulation of somatosensory input to spinal cord as well as control of visceral and somatomotor activity.


Nitric oxide mediates Fos expression in the spinal cord induced by mechanical noxious stimulation. Immunocytochemical localization of Fos protein was used to analyze the involvement of nitric oxide (NO) in the expression of Fos in the spinal cord, induced by mechanical noxious stimulation (NS). Mechanical NS was applied to the left hindpaw 30 minutes after intrathecal administration of the NO synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME) and the resulting Fos expression in the spinal cord dorsal horn was compared with that obtained in rats exposed only to the mechanical NS. Pretreatment with L-NAME but not its stereoisomer N omega-nitro-D-arginine methyl ester (D-NAME), produced a dose-dependent suppression of Fos expression induced by mechanical noxious stimulation. These results indicate that NO modulates the expression of Fos in the dorsal horn induced by mechanical noxious stimulation and further support the hypothesis that NO is involved in nociceptive events occurring in the spinal cord in response to a peripheral noxious stimulus.


Fos and jun in rat central amygdaloid nucleus and paraventricular nucleus after stress. The present paper describes the effect of capsaicin-induced stressful stimulus on the expression of immediate early genes (IEGs) c-fos, c-jun, junB and junD in the hypothalamic paraventricular nucleus (PVN) and the central amygdaloid nucleus (ACe) using in situ hybridization. Stress caused an intense expression of c-fos, c-jun and junB especially in the PVN and ACe and also a clear induction of junD was observed in the PVN. This suggests that the PVN and the ACe are two major targets of stress in the brain. The intense expression of the IEGs in the ACe and PVN suggests that stress may affect neurotransmitter gene expression through Fos and Jun proteins in both these nuclei.


Decortication and striatal mRNA: increases of mRNA for fibronectin, but not of NCAM or alpha-1 tubulin. Ipsilateral frontal cortex lesions damaged the corticostriatal input. Northern hybridization analysis showed increases in fibronectin (FN) mRNA, but not changes in mRNA for neural cell adhesion molecule (NCAM), neuro-filament-68 (NF-68) or alpha-tubulin (alpha-1T) 72 h post-lesion. In situ hybridization resolved a different spatial-temporal distribution. The superficial cell layer beneath the wound cavity showed transient elevations of FN mRNA that peaked at 72 h post-lesion. However, in the ipsilateral striatum, FN mRNA was maximal at later times than in the wound cavity, at 240 h post-lesion. Changes in NCAM and alpha-tubulin mRNAs in response to decortication occur only around the wound cavity but not in the deafferentated striatum. The different time courses of mRNA revealed by Northern blot analysis and in situ hybridization are most probably due to contamination of the ipsilateral striatum at dissection with superficial tissue adjacent to the wound cavity. These results suggest that cellular responses to ipsilateral decortication consist of two phases: (i) a wound healing process; and (ii) striatal responses to deafferentation.


Different distributions of dystrophin and related proteins at nerve-muscle junctions. The distributions of dystrophin, 'dystrophin-related protein' (DRP) and beta-spectrin were compared with that of acetylcholine receptors (AChRs) at rat nerve-muscle junctions (NMJs) using immunofluorescence techniques. In sections, monoclonal antibodies (MAbs) to dystrophin and beta-spectrin labelled the entire sarcolemma but were concentrated at the NMJs while those to DRP labelled only NMJs. In permeabilized muscle fibres, DRP was precisely co-localized with the AChRs, whereas the zone of high density labelling of dystrophin and beta-spectrin extended 0.3-0.4 microns beyond the AChRs. Within the NMJ, the labelling of DRP appeared as a series of interconnecting lines similar to that of AChRs. However, labelling of dystrophin and beta-spectrin was consistently more punctate. These data suggest DRP is more closely associated with AChRs than are dystrophin or beta-spectrin.


Oncostatin M regulates VIP expression in a human neuroblastoma cell line. Oncostatin-M (OM), a recently described glycoprotein cytokine, is structurally and functionally related to cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) and ciliary neurotrophic factor (CNTF). To determine whether OM, like CDF/LIF and CNTF, possesses trophic or differentiative functions for neurons we examined the effects of recombinant human OM on ciliary neuron survival and neurotransmitter expression in sympathetic neurons. Like CDF/LIF, but in contrast to CNTF, OM had no effect on ciliary neuronal survival at any concentration tested. OM produced small but reproducible increases in choline acetyl transferase (ChAT) activity and vasoactive intestinal peptide (VIP) levels in rat sympathetic neuron cultures, but this effect was significantly less than that of CNTF or CDF/LIF. To determine if human OM would elicit a more robust response from human cells, we utilized a human neuroblastoma cell line, NBFL, that responds to CNTF and CDF/LIF by altering vasoactive intestinal peptide (VIP) levels. OM specifically elevated VIP and c-fos mRNA levels in NBFL cells and was as potent as CDF/LIF in this assay. Our data provides evidence that OM acts on neurons and identifies a neural cell line responsive to OM, CNTF, CDF/LIF.


Wisteria floribunda agglutinin-labelled nets surround parvalbumin-containing neurons. Net-like structures surrounding several types of neurones contain glycoconjugates which are detectable by lectins specific for N-acetylgalactosamine. Wisteria floribunda agglutinin (WFA) was introduced as a further marker for the visualization of such perineuronal nets, which were also revealed in regions of the rat brain where these structures could not be clearly demonstrated using other lectins. The WFA-labelled perineuronal nets resembled in detail those which could be visualized using Vicia villosa agglutinin, colloidal iron or hyaluronectin as markers. Furthermore, WFA-stained perineuronal net components appeared to be similar to proteoglycan-immunoreactive structures. Dual-peroxidase experiments and fluorescence double labelling demonstrated that WFA-binding structures frequently ensheath GABAergic neurons containing the calcium-binding protein parvalbumin in the areas investigated.


Bicommissural neurones in the cerebral cortex of developing hamsters. The trajectory, developmental time course, and origin of callosal fibres that recross through the anterior commissure were studied in developing hamsters, using carbocyanines in fixed brains on different ages. The bicommissural fibres were found in hamsters from E15 through P7, but disappeared after P7. By double labelling it was found that the neurones of origin of these bicommissural fibres were located in the lateral cortex within the region where the callosal zone of origin overlaps that of the anterior commissure. From these experiments, it was concluded that the axons of a group of cells in the lateral cortex of developing rodents are branched and grow transiently through both the callosum and the anterior commissure.


Developmental changes of EAA metabotropic receptor activity in rat cerebellum. The potency but not the efficacy of t-ACPD stimulation of phosphatydil inositide hydrolysis changes in developing rat cerebellum. This suggests that the excitatory amino-acid-stimulated metabotropic receptors and/or their coupling are ontogenically regulated. In this, cerebellum differs from other CNS regions where only efficacy changes were described. Differently from hippocampus, the t-ACPD effect, at all ages, is independent of the activation of the NMDA receptor.


A transient immunoglobulin-like reactivity in the developing cerebral cortex of rodents. Species-specific antisera against rat or mouse immunoglobulins, such as the commercial ones currently used as secondary antibodies in immunohistochemistry, revealed an intense, developmentally regulated immunostaining of the cerebral cortex in the homospecific rodent species. The immunostaining showed evident anatomical restrictions, favouring the subplate in prenatal animals. In Western blots, the antisera recognized the protein moiety of a 93 kDa glycoprotein. Both tissue and Western blot immunostaining were completely prevented by preadsorption of the antisera with the corresponding homospecific immunoglobulin. The present results show that a novel molecule, related to immunoglobulins, is transiently expressed in the developing cerebral cortex of rodents, and stress the need for exquisite controls in immunohistochemical studies.


Neurotrophin-induced trk receptor phosphorylation and cholinergic neuron response in primary cultures of embryonic rat brain neurons. Tyrosine phosphorylation of trk type neurotrophin receptors in primary cultures of embryonic rat brain cells was studied by immunoprecipitation and immunoblotting. In cultures containing basal forebrain cholinergic neurons, but not in cultures of cerebral cortex, nerve growth factor (NGF) treatment for 4 min induced tyrosine phosphorylation of trk family proteins. Stimulation with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), resulted in a very robust phosphorylation signal in basal forebrain and cortical cultures, suggesting actions of these neurotrophins not only on cholinergic cells but probably on most embryonic brain neurons. Trk tyrosine phosphorylation was completely abolished by 5 microM K-252b. Inhibition was rapid, being evident by 30 s following addition of the drug. Corresponding stimulatory and inhibitory effects were seen for phospholipase-C gamma 1 (PLC gamma 1) and extracellular signal-regulated kinase 1 (Erk1), two enzymes involved in second messenger mechanisms. Our findings indicate involvement of trk receptor activation in the NGF response of basal forebrain cholinergic cells and provide evidence for widespread presence of BDNF and NT-3 responsive neurons in the embryonic brain.


Muscarinic M3 receptors inhibit a leak conductance in rat corticocallosal neurons. Acetylcholine, acting on muscarinic receptors, has a powerful modulatory effect on neurons in the cerebral cortex. Recent evidence that cortical tissue contains at least four different muscarinic receptor subtypes having different pharmacological properties indicates the value of new investigations of the receptor subtypes that mediate electrophysiological responses. Here we studied the ionic nature and pharmacology of the depolarizing response to acetylcholine in identified corticocallosal neurons in primary culture. This response is the result of a non-rectifying 'leak' K+ conductance which is reduced in the presence of acetylcholine. The relative sensitivity of this conductance change to the antagonists pirenzepine and 4-DAMP suggests that it is mediated by the M3 subtype of muscarinic receptor.


Mannitol does not affect energy metabolism in forebrain ischemia. Mannitol has a beneficial effect on ischemic injury following a short-duration forebrain ischemic insult in rats. Using the same animal model, we attempted to show that this effect of mannitol could be traced (via an improvement in cerebral blood flow) to a tempering of the collapse in the high-energy phosphates that occurs during the insult. A 10 min ischemic insult was induced by bilateral carotid artery occlusion followed by a reduction of the blood pressure to 50 mmHg through removal of blood via a tail catheter. 31P NMR spectroscopy was used to monitor the energy state and the pH prior to, during, and after the insult. The studies show that mannitol administered in doses of 0.25 g kg-1 or 1.0 g kg-1 prior to the insult has no significant effect on the high-energy phosphate levels or on the cerebral pH during ischemia, or on their post-ischemia recovery.


Increased monoamine turnover in the subfornical organ area following body fluid depletion. To clarify whether monoaminergic inputs to the subfornical organ (SFO) area participate in fluid regulatory systems, we examined the effects of body fluid depletion on monoamine turnover in the region of the SFO using microdialysis techniques in rats. An iso-osmotic reduction of fluid volume following subcutaneous treatment with polyethylene glycol (PEG) significantly increased dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in the SFO area. 5-hydroxytryptamine (5-HT) in the SFO area could be detected after the PEG treatment, while 5-HT was undetectable before the treatment. The data imply that both dopaminergic and serotonergic systems in the SFO area may be involved in controlling body fluid balance.


Requirement for macrophages in neuronal injury induced by HIV envelope protein gp120. HIV-1-related neuronal injury may involve a complex web of viral proteins and cytokines, but neurons themselves are not infected. The HIV envelope protein gp120 has been shown to engender an early increase in neuronal free calcium followed by delayed excitotoxic-like damage, which is prevented by N-methyl-D-aspartate (NMDA) antagonists. In the present study, we found that the injurious effects of gp120 on retinal ganglion cell neurons require the presence of macrophages in mixed neuronal glial cultures of postnatal retina. Within 24 hours of incubation, 20 pM gp120 injured nearly 40% of retinal ganglion cells in cultures containing macrophages and other glial cells, whereas no deleterious effects of gp120 were noted on retinal ganglion cells in cultures depleted of macrophages. Thus, the toxic effect of gp120 on neurons appears to be an indirect one, mediated by activation of macrophages and perhaps other glial cells.


Ganglioside derivative LIGA20 reduces NMDA neurotoxicity in neonatal rat brain. The semisynthetic ganglioside derivative LIGA20 (II3Neu5-AcGgOse4-2-d-erythro-1,3-dihydroxy-2-dichloro-ac eta mide-4-trans-octadacene) was found to be about ten times more potent than the natural ganglioside GM1 in protecting neurones in culture against glutamate toxicity. Here we show that, in vivo, LIGA20 attenuated toxicity of the glutamate receptor agonist N-methyl-D-aspartate (NMDA). In seven-day-old rats NMDA was injected intracerebroventricularly, while LIGA20 or GM1 were administered subcutaneously. The loss in brain weight, five days following treatment, was used to estimate NMDA toxicity. Significant protection was observed with 2.5 mg kg-1 of LIGA20, while at least ten times this dose was needed for GM1, thus suggesting the superior in vivo pharmacological action of LIGA20.


Dose-response of rt-PA and its combination with aspirin in a rat embolic stroke model. The efficacy and safety of thrombolysis with different dose regimens of recombinant tissue plasminogen activator (rt-PA) and its combination with aspirin was tested in an embolic stroke model. In rats the carotid territory was embolized with a single clot formed in a polyethylene tube and washed with saline. Fifteen minutes after embolization 10 animals were treated with rt-PA 10 mg kg-1; 11 with 15 mg kg-1; 12 with 20 mg kg-1 and 9 with 10 mg kg-1 + 20 mg kg-1 aspirin and 34 animals with saline. Rt-PA 10 mg kg-1 reduced median infarct volume (in percent of the ipsilateral hemisphere volume) from 19.5 to 4.8; rt-PA 15 mg kg-1 to 2.0; rt-PA 20 mg kg-1 to 0.0, while rt-PA 10 mg kg-1 + aspirin resulted in a median infarct volume of 9.5%. Thrombolytic therapy significantly and dose dependently (p = 0.001) reduced the infarct volume, improved the presacrifice clinical score and increased (p = 0.02) angiographically verified reperfusion. There was no additional benefit of coadministration of aspirin.


Evidence for an endogenous cholecystokininergic balance in social memory. The cholecystokinin (CCK) peptide family is involved in a variety of physiological processes, including neurotransmission in the brain. Pharmacological responses to CCK are mediated through at least two receptor subtypes termed CCK-A and CCK-B. Studies with CCK agonists suggest a possible role for CCK in cognition. Using selective antagonists and a behavioural recognition test based on the olfactory discriminative capacities of rats, we found that endogenous CCK acting at CCK-A and CCK-B receptors modulates olfactory recognition positively and negatively, respectively. CCK-B receptor antagonists therefore have facilitatory potentialities on memory processes.


Possible inspiratory off-switch neurones in the ventrolateral medulla of the cat. The location and axonal projection of a type of respiratory neurones (termed bIE neurones), which show burst firing at the time of phase transition from inspiration to expiration, were studied in Nembutal-anaesthetized, paralysed and artificially ventilated cats. The bIE neurones showed maximum firing at the sharp decline of inspiratory activity. All of the bIE neurones tested projected to medullary respiration-related areas of the ventral respiratory group (VRG), the dorsal respiratory group (DRG), and the Bötzinger complex (BOT). The bIE neurones were distributed in the dorso-medial border of the main assembly of respiratory neurones of the VRG and BOT. The possibility that these bIE neurones participate in inspiratory termination is discussed.


Sulpiride anxiogenic-like effect inhibition by a D1 dopamine receptor antagonist. The increased latency to explore a white compartment from a black one into which a mouse has been introduced, appears to be correlated with its anxiety level. In this test, the D2 specific dopamine receptor antagonist (+/-) sulpiride dose dependently (5-40) mg kg-1 i.p.) increased this latency. This effect was suppressed by the D1 specific dopamine receptor antagonist SCH 23390 (25 micrograms kg-1 s.c.) which intrinsically displayed an apparent anxiolytic effect.


Apamin blocks the slow AHP in lamprey and delays termination of locomotor bursts. The effects of apamin on the slow afterhyperpolarization (sAHP) in spinal neurones and on the frequency of rhythmic bursting during fictive locomotion were investigated in the lamprey spinal cord in vitro. Apamin, which is a selective blocker of a small conductance KCa channel responsible for the sAHP in many types of neurones, was also found to reduce the sAHP in lamprey neurones. The summation of the sAHP is considered to be an important burst terminating factor in the spinal locomotor network and thereby to regulate the frequency of fictive locomotion. In support of this view, apamin was found to reduce the frequency of rhythmic bursting during fictive locomotion induced by kainate and NMDA. Serotonin, which has previously been shown to reduce the sAHP and slow the rate of rhythmic bursting, may therefore act, at least in part, on apamin-sensitive KCa channels.


Slow and medium afterhyperpolarizations in maturing rat hippocampal CA1 neurones. The postnatal evolution of the signals underlying the afterhyperpolarization (slow-AHP and medium-AHP) was studied in rat CA1 hippocampal neurones (P10-16, P17-23 and P greater than 26) using in vitro slices. Noradrenaline (NA) and signal subtraction were used to decompose the AHP (m-AHP and s-AHP). The amplitude of the s-AHP was found significant between P10-16 and P17-23 cells; the m-AHP showed no significant change. The time to peak of the s-AHP of the P greater than 26 cells was found to be significantly different from the remaining groups; the m-AHP showed no significant change. The changes in waveform and amplitude of the AHP at this stage were found to be mainly due to the modifications of the slow-AHP.


Elevated gene expression in the red nucleus after spinal cord compression injury. Since the mechanism of injury in the majority of human traumatic spinal cord injuries involves rapid cord compression due to bone displacement or fracture-dislocation, we have used a compression injury model to examine the response of adult rat rubrospinal neurons to traumatic spinal cord injury. We have applied in situ hybridization techniques to examine levels of mRNA for cytoskeletal and growth-associated proteins. We report a population of magnocellular red nucleus neurons with elevated levels of T alpha 1 tubulin and GAP-43 up to four weeks post-lesion. In control animals, both probes had very low hybridization signals indistinguishable from background. These and other findings suggest that kinetic compression injuries of the adult spinal cord can result in regeneration-associated gene expression in intrinsic CNS neurons.


Potentiation of GABA-mediated currents by cAMP-dependent protein kinase. Recent reports described a down-regulation of gamma-aminobutyric acid (GABA)-receptor function in several types of central neurones by cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA). Surprisingly, we found that in cerebellar Purkinje cells (PCs) the membrane permeable-compound 8-bromo-cAMP (500 microM) induced a long-lasting potentiation of both, whole-cell current responses to bath-applied GABA and amplitudes of miniature inhibitory synaptic currents (mIPSCs). When dialyzing the PCs with the specific protein kinase inhibitor peptide (PKIP, 400 micrograms ml-1), the same manipulation failed to induce a potentiation. These results strongly suggest that, in contrast to its action in other types of neurones, activation of PKA up-regulates the GABAA receptor function in cerebellar PCs.


Inhibition of nitric oxide synthesis impairs two different forms of learning. Nitric oxide (NO), an intercellular messenger in the central nervous system of vertebrates, plays an important role in the establishment of synaptic plasticity. In order to investigate the role of NO and synaptic plasticity in learning, we injected rats and rabbits with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) prior to training on two tests of learning. Rats treated with L-NAME were impaired in learning a spatial learning task, while rabbits given the NO synthase inhibitor demonstrated learning deficits in the conditioned eyeblink response. The results support the hypothesis that NO plays a critical role in acquisition of two different forms of learning.


Specific hypothalamic neuropeptide Y variation with diet parameters in rats with food choice. Neuropeptide Y (NPY) preferentially stimulates carbohydrate intake rather than fat intake but there is no information on the effects of food choice on the concentration of NPY in the brain. We measured brain NPY concentrations in male adult rats that had to choose between a high fat and a high carbohydrate diet or were fed a control diet for 2 weeks. In rats with food choice, energy intake increased (+17%). NPY levels increased in the parvocellular part (PVNp) of the PVN and decreased in the lateral hypothalamus and were significantly correlated with the carbohydrate-to-fat energy ratio but not with total energy intake. This suggests that hypothalamic NPY might be involved in food choice and that PVNp is important in the regulation of feeding behaviour by NPY.


Gene expression in the geniculate induced by a nonphotic circadian phase shifting stimulus. We examined the effect of a nonphotic stimulus (running in a novel wheel) on the induction of Fos-related proteins in the two well-established neural components of the circadian clock: the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) of the lateral geniculate complex. There was no induction of Fos immunoreactivity (IR) in the SCN, but the IGL showed distinct Fos IR in animals whose running levels were associated with maximal rhythm shifts. Induction of Fos immunoreactivity was greatest at circadian phases when wheel running induces phase shifts. This temporal and spatial specificity of Fos induction is evidence that the intergeniculate leaflet mediates nonphotic signals to the circadian clock.


Evoked oscillatory cortical responses are dynamically coupled to peripheral stimuli. We report that the response of neurons in rat somatosensory cortex to tactile stimulation consists of two components, a short-latency response and an oscillatory response, observable as up to 8 peaks in the post-stimulus-time-histogram with interpeak intervals in the order of 100 ms (10 Hz). While the first component is always stimulus locked, the second component is strictly stimulus-locked only when elicited from the resting state: once started, the oscillations are only weakly affected by further stimulation. This implies generally that the question of stimulus locking of oscillatory response components is not a yes/no question. Instead, the concept of dynamic coupling is shown to adequately capture the different limit cases. We present a simple dynamic model that exemplifies this point.


Perceptual correlates of massive cortical reorganization. Following long-term deafferentation of one upper limb in adult primates, the cortical areas corresponding to that limb become responsive to stimuli applied to the face. To explore this phenomenon, we studied some patients after upper limb amputation. In patient VQ, stimuli applied to the lower face or 7 cm above the stump evoked precisely localized referred sensations in individual digits which were often modality specific. Similarly, in another patient, WK several complete somatotopic representations of the phantom limb were found, on the face, chest and axilla, indicating the emergence of such maps in regions remote from the stump. These effects may be a direct perceptual correlate of the physiological observations of Merzenick et al (1984), Wall (1977) and Pons et al (1991).


The differential effects of lateral midbrain lesions on the two phases of formalin pain. Lesions were placed in the lateral midbrain (LM) to determine the effects of disruption of paleo- and neospinothalamic systems, at the level of the midbrain, on the biphasic tonic pain response induced by formalin. During the first phase of formalin pain, subjects with LM lesions exhibited significantly less pain responding than did unoperated controls or subjects with lesions at another site, the habenula. During the second phase, there were no significant differences in pain responding among the three groups. The results suggest that the two phases are mediated by different neural substrates and specifically that Phase 2 appears to involve either neural substrates caudal to our LM lesions or sites rostral to the lesions but accessible via a more medial path.


Atropine and naloxone sensitive stimulation produced analgesia from pretectal nucleus in rat. Mild and brief electrical stimulation of sites in the pretectal nucleus (PTN) of rats evoked potent analgesia of long duration, without significant aversions and was unassociated with motor deficit. The present study has analysed effects of opioidergic and cholinergic neurotransmitter antagonists administered intracerebroventricularly (i.c.v.) on this analgesia. Pretreatment either with naloxone or atropine sulphate both in doses of 30 and 50 micrograms each, respectively i.c.v., 10 min prior to subsequent pretectal stimulation, significantly attenuated the increase in tailflick latency. The antagonism of pretectal stimulation produced analgesia (PSPA) by naloxone and atropine, raises the possibility of involvement of both endogenous opioids and cholinergic mechanisms in pretectal analgesia.


Behavioral correlates of action potentials recorded chronically inside the Cone Electrode. Long-term recording of stable action potentials with a new electrode configuration has been achieved for up to 15 months in monkeys. Action potentials are separated from the continuous multi-unit recordings made between two wires inside a glass open ended cone implanted in the motor cortex. Neural tissue is induced to grow into the glass cone by autologous sciatic nerve placed inside prior to implantation. Data presented here show that behavioral correlates of neural activity are related to flexion and extension movements of the contralateral hand and digits. These data suggest that this electrode can be used in both basic and applied studies.


Evaluations of ventral pallidal dopamine receptor activation in behaving rats. Recent evidence justifies the inclusion of the ventral pallidum/substantia innominata (VP) into the category of dopaminoceptive brain regions. Since the VP is known to mediate both cognitive and motoric processes, the present study employed intracerebral microinjections of dopamine directly into the VP of rats to determine if the catecholamine influences these processes. Dopamine concentrations of up to 10 micrograms were ineffectual in altering performance parameters in a working memory task. However, concentrations as low as 0.01 micrograms increased locomotion in an open field. The magnitude of this response was related to the dopamine dose injected and the effect was attenuated by systemic pretreatment with the dopaminergic antagonist, flupentixol. These studies suggest that dopamine neurotransmission at the level of the VP may be important in the locomotor functions attributed to ascending dopamine systems.


SKPYMRFamide, a novel FMRFamide-related peptide in the snail Lymnaea stagnalis. In Lymnaea stagnalis, three members of the FMRFamide peptide family have been chemically identified in the central nervous system, and other members of the family are predicted by cDNA studies. The present study demonstrates the occurrence of even more FMRFamide-related peptides in this species by identifying a novel member of this family. The peptide was purified from brain extracts by three different HPLC steps. Its amino acid sequence has been determined as Ser-Lys-Pro-Tyr-Met-Arg-Phe-amide (SKPYMRFamide).


Fibronectin staining detects micro-organisms in aged and Alzheimer's disease brain. Filamentous, fibronectin-immunopositive structures, previously described in Alzheimer's disease and control brains were negative for neuronal, glial, and macrophage markers. The present study sought to determine the nature of these entities and to further characterize their morphology, immunoreactivity and distribution between neuropathologies. Ultrastructural analysis shows these formations to be filamentous micro-organisms, which may belong to the actinomycetes. Immunohistochemistry for the cell-stress protein ubiquitin is consistently positive in these organisms. They are also present in Down's syndrome, dementia pugilistica, amyotrophic lateral sclerosis with dementia, and Parkinson's disease. The pattern of tissue distribution implies a pre-mortem invasion of the brain, and, as the micro-organism is present at a four to five-fold higher frequency in Alzheimer's disease, it may act pathogenically in this dementing illness.


Expression of neuronal markers in the so-called ENU-induced schwannomas. The present data show that the so-called ENU-induced malignant schwannomas express neuronal markers. This finding supports the hypothesis that a population of tumor cells in these neoplasms are neuroblastic in nature, and suggests the classification of ENU-induced schwannomas as primitive neuroectodermal tumors.


Drug interactions represented by high resolution computer graphics. The amount of backward walking induced in mice by co-administration of clenbuterol, a beta-adrenoceptor agonist and putative antidepressant, and the benzodiazepine chlordiazepoxide was plotted as a three-dimensional surface, using modified Uniras software. The surface was compared with the theoretical surface to be expected if the interactions between the two drugs were merely additive. The difference between the theoretical and the obtained surfaces was plotted and a functional representation of statistical variability was 'draped' over the difference model. The computer methods can be applied to small amounts of irregularly distributed data, and results are apparent from simple inspection, e.g. the highest peak of backward walking is also the most reliable statistically. The technique is useful for both laboratory and clinical studies.


Effects of a metabotropic glutamate agonist, trans-ACPD, on cortical epileptiform activity. The effect of the metabotropic glutamate receptor agonist trans-1-amino-1,3,cyclopentanedicarboxylic acid (trans-ACPD) on epileptiform activity induced in rat neocortical slices by exposure to Mg(2+)-free medium was examined. Trans-ACPD dose dependently (10-200 microM) decreased the frequency of spontaneous epileptiform events whilst increasing both the duration of afterpotentials and the number of afterbursts associated with single events. This effect on afterpotentials and afterbursting was particularly pronounced in 14-17 day-old rats and was blocked by the sigma ligand ditolyguanidine (DTG) 10 microM. The putative metabotropic glutamate receptor antagonist L-AP3 did not antagonise the actions of trans-ACPD. The results suggest a role for metabotropic glutamate receptors in epilepsy, possibly in the transition from interictal to ictal activity.


5-HT response of rat hippocampal pyramidal cell bodies. Serotonin (5-HT) responses of pyramidal neurones freshly dissociated from rat ventral hippocampal CA1 region were investigated by using nystatin-perforated whole-cell recording. These dissociated neurones lack most of the dendrites and axons. Application of nanomolar concentrations of 5-HT induced outward current with an increase of membrane conductance at a holding potential (VH) of -40 mV. The current was mimicked by alpha-methyl-5-HT (5-HT2 receptor family agonist), but not by 8-OH-DPAT (5-HT1 receptor family agonist). Ketanserin (5-HT2 receptor family antagonist) and spiperone (5-HT1A and 5-HT2 receptor family antagonist) blocked the current in a concentration dependent manner. These results suggests that 5-HT-induced outward current is mediated by the activation of 5-HT2 receptor family in the cell bodies of hippocampal pyramidal neurones.


The effects of orally active enkephalinase inhibitors on morphine withdrawal syndrome. Considerable evidence has accumulated to suggest that intracerebroventricular administration of enkephalinase inhibitors, which do not penetrate the blood-brain barrier, significantly attenuates opioid withdrawal syndrome. Therefore, the aim of this study was to examine the effect of intraperitoneal (i.p.) administration of orally active enkephalinase inhibitors, acetorphan (2.5-20 mg kg-1) and SCH 34826 (15-120 mg kg-1). These drugs significantly decreased the severity of the naloxone precipitated withdrawal syndrome in morphine dependent rats and mice. It therefore appears that these orally active enkephalinase inhibitors are promising tools in studying modulation of opioid dependence phenomena.


Effect of nitecapone and clorgyline, given intracerebro-ventricularly on L-dopa metabolism in the rat brain. A new COMT inhibitor, nitecapone (OR-462) or clorgyline, a MAO-A inhibitor, was infused into the 3rd brain ventricle (i.c.v.) of conscious male rats. None of the enzyme inhibitors given alone alter hypothalamic or striatal levels of L-dopa, dopamine or their metabolites. Most of the rats were pretreated with levodopa/carbidopa (LD/CD, 15/30 mg kg-1 intraperitoneally). Now, the action of nitecapone is localized in the hypothalamus since homovanillic acid (HVA) is decreased there, not in the striatum. The levels of 3-O-methyldopa (3-OMD) are not changed in either brain region, suggesting a lack of the peripheral leakage of nitecapone. Clorgyline (3 and 10 micrograms rat-1) elevates hypothalamic and dopamine levels. Nitecapone and clorgyline decrease prolactin (PRL) levels below those reduced by LD/CD treatment.


Philanthotoxin-343 blocks long-term potentiation in rat hippocampus. The effects of extracellularly-applied synthetic philanthotoxin-343 (PhTX-343) on transmission and long-term potentiation (LTP) at Schaffer-collateral/commissural-CA1 synapses were investigated. PhTX-343 was ineffective in antagonizing CA1 field-EPSPs mediated by AMPA/kainate receptors. However, when a micromolar concentration of the toxin was present during tetanization, the induction of LTP was suppressed. In contrast, when PhTX-343 was applied either immediately after or long after tetanization no effect on LTP could be found. It appears that the synaptic, non-NMDA receptors of the CA1-region are insensitive to PhTX-343. Suppression of LTP induction could result from antagonism of postsynaptic NMDA receptors, but the results do not rule out other possibilities such as presynaptic block.


Reduction of A1 adenosine receptors in cortex, hippocampus and cerebellum in ageing mouse brain. Age related changes in A1 adenosine receptor binding were investigated in mouse brain using the selective agonist, [3H]-cyclohexyladenosine ([3H]CHA). In the cortex, hippocampus and cerebellum of aged mice (28 months old), a significant decrease of about 44%, 50% and 12%, respectively, in [3H]CHA binding compared to young animals (3 months old) was observed. According to the Scatchard analysis of the binding data in the cortex, this decrease was due to a receptor density reduction and not to a Kd change. Since the weight and protein content of each tissue tested did not differ significantly between the old and the young animals, our findings may be partly explained by specific reductions of A1 receptors rather than a general cell degeneration in old age.


Early functional glutamate receptors in acutely dissociated embryonic raphe cells. We report for the first time, modulation of cytosolic calcium in response to glutamate and specific glutamate receptor agonists in early embryonic rat brain cells (raphe cells taken at gestation days 13 or 14). Metabotropic as well as ionotropic agonists were effective. Cells responding to kainic acid were particularly prominent in caudal raphe. We used very short post-plating delays (2 to 6 h); it may therefore be assumed that functional receptors already exist in the intact embryonic brain by gestation day 13. Since many developmental processes are influenced by cytosolic calcium modulation, glutamate receptors may play a key role in brain development, well before the extensively studied postnatal peak in receptor density.


A novel method to analyse response patterns of taste neurons by artificial neural networks. Despite several notions on the gustatory code proposed over three decades, investigators have not yet reached a consensus. This paper describes a new approach to analyse gustatory neural activities. Three-layer neural networks were trained by the back-propagation learning algorithm, to classify the neural response patterns to four basic taste qualities. The discrimination by the trained networks on taste qualities in the response patterns of rat chorda tympani fibres (CT) and cortical taste neurons (CN) was consistent both with the correlation analysis and with behavioural experiments. By examining the connection weights of each neuron, some input neurons representing CN were 'pruned' without deteriorating the ability of the network to discriminate taste. This characteristic of the network is contrary to a previous hypothesis, that taste neurons are of equal importance in the neural coding.


Immunolocalization of two cytochrome P450 isozymes in rat nasal chemosensory tissue. The NMa and NMb isoforms of cytochrome P450 enzymes are expressed in three nasal chemosensory organs: the olfactory, septal and vomeronasal mucosae. The NMa isoform is widely distributed throughout the nasal mucosa whereas the NMb isoform is present primarily in the chemosensory mucosae. The localization of cytochromes P450 demonstrates that sustentacular cells in the olfactory and septal epithelia, the mucus of the vomeronasal organ and the acinar cells of glands in the lamina propria of all three chemosensory systems engage in xenobiotic metabolism and participate in odorant/pheromone clearance, a perireceptor process associated with chemosensory transduction.


Neutrophil-activating peptide-1/interleukin-8 mRNA is localized in rat hypothalamus and hippocampus. Neutrophil-activating peptide-1/interleukin-8 (NAP-1/IL-8) is a cytokine synthesized by various cell types. In the immune system NAP-1/IL-8 is part of an immune cascade initiated by IL-1 production. NAP-1/IL-8 affects hypothalamic function and its production is suppressed by steroids. Therefore, it might be expected that NAP-1/IL-8 would be produced in brain areas involved in the control of the hypothalamic-pituitary-adrenocortical axis (HPA). NAP-1/IL-8 mRNA was localized by in situ hybridization in the paraventricular nucleus of the hypothalamus and hippocampus. Those areas also express the genes encoding interleukin-1-alpha (IL-1 alpha), IL-1 beta, IL-1 receptors, and IL-1 receptor antagonist (IL-1ra). This suggests that an immune cascade, which is well characterized in the immune system, may exist in brain, in areas of relevance to the regulation of stress-related neuroendocrine function.


Treatment with excitatory amino acids or high K+ and NMDA receptors in cerebellar granule cells. N-methyl-D-aspartate (NMDA) receptor activity was examined, in terms of 45Ca2+ influx, during the development of cerebellar granule cells grown under 'non-trophic' [10 mM potassium (K10)] or 'trophic' conditions [25 mM potassium (K25), NMDA and kainate (KA)]. NMDA receptor activity increased sharply between 2 and 4 days in vitro (DIV) irrespective of growth conditions which upon further cultivation exerted a powerful influence, the NMDA response increasing progressively in K25 and NMDA grown cells, while remaining at a constant level in KA treated cells. In contrast, in K10 grown cells the NMDA response declined by 7 DIV to about 20% of the estimates in K25 at 9 DIV. Trophic conditions are, therefore, essential for the proper functional expression of NMDA receptors.


Preparation for reaching: a PET study of the participating structures in the human brain. The regional cerebral blood flow (rCBF) was measured as an indicator of regional metabolic activity with positron emission tomography (PET) in eight subjects who, after seeing a screen with seven targets prepared themselves with their eyes closed to reach these targets. The preparation phase was associated with increases of rCBF in the prefrontal cortex, several remote visual association areas in the parietal lobe, the supramarginal gyrus, the ventrolateral thalamus and the cerebellar vermis. During the course of learning the activations in the parietal visual areas, the supramarginal gyrus and the prefrontal cortex prevailed as a sign of the visual spatial information; its transformation being kept in working memory. The other activations vanished. No activations were seen in the motor cortices, indicating that reaching is a task which does not require substantial preparatory activity of motor cortices prior to the go signal.


Atypical pyramidal cells in epileptic human cortex: CFLS and 3-D reconstructions. Epileptic temporal cortices, removed from 3 patients with intractable partial epilepsy (IPE) during neurosurgery, were studied. Pyramidal neurons (40-50 per slice) in laminae III, V and white matter, were injected with lucifer yellow. Samples were examined in a confocal laser scanning microscope (Biorad 600) and individual cells scanned at 0.1-1 microns incremental levels. 2-D maximal linear projection was used for overview. Frames (50-60) of scanned neurons were transformed into 3-D volumes, using VoxelView software on a Silicone Graphics workstation and rotated. All samples contained neurons with duplicated apical dendrites, additional basal dendrites or were misplaced in a horizontal position in the white matter. The relation between these preliminary observations and the disease is discussed.


beta-Amyloid induces neuritic dystrophy in vitro: similarities with Alzheimer pathology. beta-Amyloid protein, the major component of neuritic plaques found in Alzheimer's disease, has been implicated as a potential contributor to the disease's progressive neuropathology. We report that within a two day exposure to aggregates of synthetic beta-amyloid peptide, the neurites of cultured rat hippocampal neurons adopt a dystrophic appearance. Observed morphological changes in the neurites include beading, fragmentation, terminal swelling and tortuous growth patterns. The degenerative changes are similar to those observed in neurites associated with neuritic plaques, suggesting that beta-amyloid may induce the neuritic abnormalities of Alzheimer neuropathology.


Differential effects of deprenyl and MPTP on catecholamines and activity in BALB/c mice. The present study examined the effects of deprenyl (1.5 mg kg-1) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg kg-1) on dopamine (DA), norepinephrine (NE) and motor activity in BALB/c mice. Results indicate that pre-treatment with deprenyl injections protected against MPTP's toxicity on DA, NE and motor function. However, if deprenyl was given 3 or 7 days after the MPTP injection (for 3 days in succession), it could not reverse MPTP's toxicity. However, if deprenyl was given for an extended period of 7 days, it reversed MPTP's toxicity on NE, but not on DA and behaviour. If deprenyl was given soon after MPTP each day for 10 days, it protected against MPTP's toxicity on DA and NE, but not on locomotor activity. These results suggest that only prior and repeated deprenyl injection has a satisfactory protective effect against MPTP's neurotoxicity.


Morphine differentially affects domoic acid and kainic acid toxicity in vivo. The effect of systemic injections of morphine on behavioural toxicity and hippocampal (CA3 region) damage produced by both domoic and kainic acids was investigated in mice. Low doses of morphine (2.0 and 4.0 mg kg-1), but not higher doses, significantly antagonized the toxic response to a previously determined TD50 of domoic acid. By contrast, low doses of morphine had either minimal or no effect on the response to an equitoxic dose of kainic acid (TD50), but higher doses (6.0 and 8.0 mg kg-1) resulted in significant potentiation of kainate toxicity. These results provide the first evidence of a pharmacological dissociation between the mechanisms of domoic acid and kainic acid toxicity in vivo, suggesting that these two toxins produce behavioural and hippocampal toxicity via overlapping but non-identical mechanisms.


Specific in vitro synaptogenesis between identified Lymnaea and Helisoma neurons. We tested the ability of identified neurons from two different families of pulmonate molluscs to form specific connections in vitro. The presynaptic neuron chosen for this study was the giant dopamine cell of Lymnaea stagnalis and Helisoma trivolvis which is known to synapse upon specific visceral and parietal ganglion neurons in both species. Here we show that the giant dopamine cells can reform specific connections in vitro on follower neurons from both species. Thus the mechanisms that determine synapse specificity are conserved between two different families of molluscs.


Circadian rhythms of vasopressin content in the suprachiasmatic nucleus of the rat. The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a circadian pacemaker in mammals. We determined the circadian profiles of arginine-vasopressin (AVP), a major peptide in the dorsomedial SCN, in rats under light-dark (LD), constant dark (DD) and constant light (LL) conditions. Under LD conditions, AVP levels in the SCN showed circadian rhythmicity with a peak at early light phase and a broad trough during the dark phase. This rhythm in the AVP contents was maintained even after 14 days of free-running under DD conditions and 3 days under LL conditions. These circadian patterns of AVP are similar to those of somatostatin, another peptide in the dorsomedial SCN. This indicates a common mode of regulation for peptides in this subfield of the SCN.


D600, a Ca2+ antagonist, prevents stimulation of nerve regeneration by magnetic fields. We have found that regeneration of the rat sciatic nerve is stimulated if the rats are exposed to a 50 Hz sinusoidal magnetic field. In this study we investigated the effect of a Ca2+ antagonist, methoxyverapamil (D600) on this response. D600 was administered to the regenerating segment of the nerve via implanted osmotic minipumps. Magnetic field exposure increased regeneration distances in vehicle perfused nerves. This enhancement was blocked by perfusion with D600 at a concentration which alone failed to affect outgrowth of nerve fibres, lending support to the idea that Ca2+ fluxes could be involved in mediating the biological actions of magnetic fields.


Site of initiation of replication of the ribosomal genes of pea (Pisum sativum) detected by two-dimensional gel electrophoresis. The time course of replication of the 9 kb ribosomal DNA repeats of synchronized root cells of pea was followed by two-dimensional gel electrophoresis. The temporal order of appearance of single-stranded replication intermediates shows that replication begins within the subrepeats located in the intergenic spacer region about 1.5 kb downstream from the 3' end of the 25S gene. Hybridization to specific probes indicated that this location is identical to that established earlier by a different method.


Biological activities of human interferon and 2'-5' oligoadenylates in plants. Exogenous human interferon 2 (IFN) and 2'-5' oligoadenylates (2-5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1-1 u/ml and 2-5A at 1-10 nM.


The wheat mitochondrial genome contains an ORF showing sequence homology to the gene encoding the subunit 6 of the NADH-ubiquinone oxidoreductase. A region of the mitochondrial (mt) DNA of wheat was studied because of its homology with other plant mtDNAs. Sequence analysis revealed an open reading frame encoding a polypeptide of 247 amino acids. Comparison of the sequence of the putative polypeptide with the protein sequence data of the Swiss-Prot library reveals homology with subunit 6 of the NADH-ubiquinone complex of mitochondria from Marchantia polymorpha, Podospora anserina, Chlamydomonas reinhardtii and of chloroplasts from M. polymorpha and Oryza sativa. No similarity was detected when compared with the subunit 6 of animal mitochondria, probably due to the rapid evolution of the sequence. A single 1.2 kb transcript appears in northern RNA blots. We found 15 edited sites of which only 13 give amino acid changes. This is the first report of a mt nad6 gene in higher plants.


The organization of the prosystemin gene. The organization of the gene encoding tomato prosystemin, a 200 amino acid protein precursor of the 18 amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato plants, is reported. The prosystemin sequence reveals that the gene, which is composed of five homologous pairs of exons plus a non-homologous exon at the C-terminus containing the systemin sequence, has evolved by several gene duplication-elongation events from a much smaller ancestral gene. The nucleotide and amino acid sequence homologies among the exons suggest that a small ancestral gene was duplicated to form at least two tandem repeats, followed by subsequent duplication-elongation events that resulted in five tandemly repeated nucleotide sequences and three duplicated amino acid sequence elements. Since the systemin nucleotide or amino acid sequence was not duplicated, it was either not part of the gene duplication-elongation events or its coding region evolved separately and may even have been added to the tandemly repeated part of the gene at a later time.


Salt stress alters A/T-rich DNA-binding factor interactions within the phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum. The common ice plant, Mesembryanthemum crystallinum, shifts from C3 to crassulacean acid metabolism (CAM) photosynthesis in response to osmotic stress. The expression of a number of genes encoding enzymes involved in the CAM pathway increases as a result of increased transcription rates. To begin to investigate the mechanisms responsible for the transcriptional activation, we have characterized the 5' control region of a specific isoform of phosphoenolpyruvate carboxylase gene (Ppc1) that plays a key role in CAM. We have determined the nucleotide sequence of the 5' flanking region of this gene. Ppc1 contains a long 5'-leader sequence with the transcriptional start site located 332/333 nucleotides 5' of the translational initiation codon. Multiple DNA interactions with nuclear factors are detectable within the 5'-flanking region of Ppc1. We have used copper orthophenanthroline footprinting to demonstrate that one particularly abundant factor (designated PCAT-1) binds the Ppc1 promoter at two distinct A/T-rich sites located -128 to -158 and -187 to -205 bp upstream of the transcriptional start site. These binding sites share a loose consensus motif having the sequence AARTAAC(T/A)A(G/T)TTTY. Gel retardation competition experiments with oligonucleotides containing these A/T-rich binding sites suggest that both sites bind the same factor, but with different affinities. Fractionation of crude nuclear extracts by heparin-agarose chromatography indicates that PCAT-1 is more prevalent in extracts prepared from salt-stressed leaf tissue. Additional binding activities that interact with the PCAT-1 binding sites have been detected that either increase or decrease in abundance or binding affinity in response to salt stress.


Identification and characterization of a full-length cDNA encoding for an auxin-induced 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments and expression of its mRNA in response to indole-3-acetic acid. 1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) is the key regulatory enzyme in the ethylene biosynthetic pathway. The identification and characterization of a full-length cDNA (pAIM-1) 1941 bp in length for indole-3-acetic acid (IAA)-induced ACC synthase is described in this paper. The pAIM-1 clone has an 87 bp leader and a 402 bp trailing sequence. The open reading frame is 1452 bp long encoding for a 54.6 kDa polypeptide (484 amino acids) which has a calculated isoelectric point of 6.0. In vitro transcription and translation experiments support the calculated molecular weight and show that the enzyme does not undergo processing. Eleven of the twelve amino acid residues which are conserved in aminotransferases are found in pAIM-1. The sequence for pMAC-1 which is one of the 5 genes we have identified in mung bean is contained in pAIM-1. pAIM-1 shares between 52 to 65% homology with previously reported sequences for ACC synthase at the protein level. There is little detectable pAIM-1 message found in untreated mung bean tissues; however, expression is apparent within 30 min following the addition of 10 microM IAA reaching a peak after approximately 5 h with a slight decrease in message after 12 h. These changes in message correlate with changes in ACC levels found in these tissues following treatment with 10 microM IAA.


Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules. Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N2 fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.


A major barley allergen associated with baker's asthma disease is a glycosylated monomeric inhibitor of insect alpha-amylase: cDNA cloning and chromosomal location of the gene. A 14.5 kDa barley endosperm protein that is a major allergen in baker's asthma disease, as previously shown by both in vitro (IgE binding) and in vivo tests, has been identified as a glycosylated monomeric member of the multigene family of inhibitors of alpha-amylase/trypsin from cereals. A cDNA encoding this allergen (renamed BMAI-1) has been isolated and characterized. The deduced sequence for the mature protein, which is 132 residues long, is identical in its N-terminal end to the 20 amino acid partial sequence previously determined from the purified allergen, and fully confirms that it is a member of the multigene family of cereal inhibitors. Southern-blot analysis of wheat/barley addition lines using the insert in the BMAI-1 cDNA clone as a probe, has led to the location of the allergen gene (Iam1) in barley chromosome 2, while another related member of this protein family, the barley dimeric alpha-amylase inhibitor BDAI-1 gene (Iad1) has been located in chromosome 6. Iam1 is the first member of this inhibitor family in cereals to be assigned to chromosome group 2, thus extending the dispersion of genes in the family to five out of the seven homology groups of chromosomes in wheat and barley (chromosomes 2, 3, 4, 6 and 7).


Purification and characterization of seven chloroplast ribosomal proteins: evidence that organelle ribosomal protein genes are functional and that NH2-terminal processing occurs via multiple pathways in chloroplasts. Putative genes for 21 ribosomal proteins (RPs) have been identified in the chloroplast DNA of four plants by nucleotide sequencing and homology comparison but few of the gene products have been characterized. Here we report the purification and N-terminal sequencing of seven proteins from the spinach chloroplast ribosome. The data show them to be the homologues of Escherichia coli RPs L20, L32, L33, L36, S12, S16 and S19, and thus support the view that their genes identified in the chloroplast DNA represent functional genes. The initiating methionine residue was not detected in the mature protein in most cases but it was present in S16, indicating that only the formyl group is removed in this case. This result and the previously reported finding of N-methyl alanine at the N-terminus of chloroplast L2 indicate the existence of multiple N-terminal processing pathways in the chloroplast.


The cruciferin gene family in radish.

The cruciferin gene family in radish. In order to analyse the cruciferin gene family in radish a cDNA library was screened either with heterologous rapeseed probes or by differential screening and sequencing. We could identify six partial cDNA clones belonging to two different groups of cruciferin genes which do not cross-hybridize, and probably three distinct subfamilies. One of these classes corresponds to the previously described cruciferin from rapeseed and Arabidopsis. A gene corresponding to the second group, as well as its border sequences, was isolated from a radish genomic library and analysed in more detail. The cruciferin gene (cruRS) contains three introns and encodes a 479 amino acid protein. The transcription initiation site was determined. The expression of the different group of genes was studied by northern blot analysis: genes of both classes are expressed simultaneously and roughly at the same level between 25 and 35 days after flowering. Cruciferin gene copy number was estimated by Southern blot analysis. There appear to be seven or eight genes in one class and three in the other, located at different loci.


Cloning and sequencing of the petBD operon from the cyanobacterium Synechococcus sp. PCC 7002. The genes encoding the photosynthetic cytochrome b6 (petB) and subunit 4 (petD) have been cloned and sequenced from the unicellular, photoheterotrophic, transformable cyanobacterium Synechococcus sp. PCC 7002, formerly designated Agmenellum quadruplicatum. The gene arrangement was found to be similar to that reported in the cyanobacterium Nostoc PCC 7906. The DNA and derived protein sequences were compared to chloroplast and the other cyanobacterial sequences. By pulsed-field electrophoresis, the petBD operon and the petCA operon, encoding the Rieske iron-sulfur protein and cytochrome f, were found to be located on separate, unlinked, Not I-digested DNA fragments. The petBD operon was found on the third largest Not I fragment (NC-325) while the petCA operon was found on the second largest Not I fragment (NB-370). These results suggest the two operons are not in proximity. The 1.35 kb transcript was shown to be light-regulated. Transcripts from cells grown under constant illumination showed a decrease in petB transcript levels to undetectable levels within 2 h after the cells were placed in the dark. Upon reillumination, transcript levels rose to three-fold over that seen initially under constant illumination.


Isolation and characterization of a tobacco gene with homology to pectate lyase which is specifically expressed during microsporogenesis. A genomic clone has been isolated which contains an open reading frame of 1191 bp interrupted by two small introns. The ORF has been sequenced and the transcriptional start determined. The predicted amino acid sequence shows homology to the deduced amino acid sequences of two pollen-specific pectate lyase genes identified in tomato. The genomic clone was isolated using a partial cDNA clone, TP10, which had been isolated from a Nicotiana tabacum pollen cDNA library by means of differential screening. TP10 has been fully sequenced and contains an open reading frame of 792 bp which shows 96% homology to the ORF in the genomic clone. The transcript corresponding to TP10 is maximally expressed late in pollen development, and has not been detected in vegetative tissues.


A DNA library from an individual Beta patellaris chromosome conferring nematode resistance obtained by microdissection of meiotic metaphase chromosomes. We have used a standard protocol established for human chromosomes to create a chromosome-specific plasmid library from a Beta patellaris chromosome conferring nematode resistance. A monosomic addition line was chosen carrying 18 sugar-beet (Beta vulgaris L.) and one wild-beet (B. patellaris) chromosome. The wild-beet chromosome can readily be identified as a univalent during metaphase I of meiosis. Highly synchronized meiotic material was used to excise the univalents from four pollen mother cells. The chromatin was lysed in a 1 nl collection drop, the DNA purified and restricted with Rsa I, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. The amplified DNA was inserted into a standard plasmid vector and cloned. Approximately 23,000 recombinant plasmids were obtained of which 15,800 could be utilized. Their insert sizes ranged from 80 to 700 bp with an average of 130 bp. 61 clones were tested in more detail by genomic Southern hybridization with sugar-beet and wild-beet DNA. Of these 32 plasmids (52%) contained single-copy inserts, 11 (18%) were specific for wild-beet DNA indicating that the DNA cloned originates in the univalent chromosome. The application of this technique for establishing high-density RFLP maps for discrete regions of plant genomes is discussed.


Differential accumulation of mRNAs encoding extracellular and intracellular PR proteins in tomato induced by virulent and avirulent races of Cladosporium fulvum. Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a beta-1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa beta-1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic beta-1,3-glucanase and a basic 35 kDa beta-1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic beta-1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa beta-1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.


Regulation of the maize HRGP gene expression by ethylene and wounding. mRNA accumulation and qualitative expression analysis of the promoter by microprojectile bombardment. The expression of the maize gene coding for a hydroxyproline-rich glycoprotein (HRGP) has been studied by measuring the mRNA accumulation after wounding or ethylene treatment. RNA blot and in situ hybridization techniques have been used. The temporal and tissue-specific expression has been observed: the cells related to the vascular system show the more intense HRGP mRNA accumulation. Transcriptional constructions of the maize HRGP promoter have been tested on different maize tissues by microbombarding. A 582 bp promoter is able to direct the expression of the gus gene on calli and young leaves. Constructions having shorter promoter sequences lose this ability. The 582 bp construction retains the general specificity of expression observed for the HRGP gene.


Import and processing of the precursor of the delta subunit of tobacco chloroplast ATP synthase. A cDNA clone encoding the complete precursor of the delta subunit of chloroplast ATP synthase has been isolated from a tobacco (Nicotiana tabacum) leaf cDNA library in lambda gt11. The 880 bp insert encodes a polypeptide of 248 amino acid residues, of which 61 residues constitute an N-terminal presequence and 187 residues make up the mature delta subunit. Transcription and translation of the cDNA in vitro produced a protein of 29 kDa which was imported by isolated pea chloroplasts and processed to the mature 20 kDa subunit. The delta subunit precursor was processed to the mature size by a processing peptidase present in pea stromal extracts. Hybridisation of the cDNA to Southern blots of tobacco genomic DNA suggests the presence of two genes in the haploid genome.


Expression of desiccation-related proteins from the resurrection plant Craterostigma plantagineum in transgenic tobacco. Three cDNAs encoding desiccation-induced proteins from the resurrection plant Craterostigma plantagineum were each ligated to a triplicated CaMV 35S promoter and a nopaline synthase 3'-flanking region in an Agrobacterium vector and introduced into tobacco. Transgenic plants expressed the encoded Craterostigma proteins at high levels. This did not lead to changes in the phenotype, in the growth habit or in basic photosynthetic parameters. In tobacco, one protein was targeted to the chloroplast stroma which is its normal location in Craterostigma. These desiccation-related proteins are not sufficient per se to increase drought tolerance as measured by ion-leakage tests.


Characterization of a Brassica napus gene encoding a cruciferin subunit: estimation of sizes of cruciferin gene families. A gene encoding a subunit of the 12S storage globulin, cruciferin, in Brassica napus (oilseed rape) has been isolated and characterized. The gene consists of about 2200 bp including three short intervening sequences. Primer extension analysis showed that the major transcription start site is located 30 bp 5' of the predicted ATG start codon. This gene belongs to one of three different major families encoding cruciferin subunits. By use of gene-family-specific probes and Southern blotting analysis the number of genes of the three different cruciferin subtypes in B. napus was estimated.


Import of the precursor of the chloroplast Rieske iron-sulphur protein by pea chloroplasts. cDNA clones encoding the precursor of the Rieske FeS protein of the chloroplast thylakoid membrane have been isolated from a pea leaf cDNA library in lambda gt 11, following screening with antibodies to purified pea chloroplast Rieske FeS protein. The longest cDNA insert of 880 bp encodes a polypeptide of 230 amino acid residues, of which 50 residues constitute an N-terminal cleavable presequence and 180 residues make up the mature protein. Transcription and translation of the cDNA in vitro produced a polypeptide of 26 kDa which was efficiently imported by isolated pea chloroplasts and processed to the mature 20 kDa protein. Southern hybridisation to pea genomic DNA indicated the presence of a single gene encoding the Rieske FeS protein in the haploid genome.


Ras in signal transduction.

Ras in signal transduction. Ras protein is a GTP-binding protein, and acts as a signal transducer in fibroblast, lymphoid, myeloid, and neuronal cells. In all cases, tyrosine kinases, intrinsic to or associated with receptors, seem to play an important role for the activation of Ras in response to extracellular stimulations. A GDP/GTP exchange regulator and a GTPase stimulatory protein are thought to mediate signals from the kinases. The active Ras.GTP can cause different phenotypes, that is, proliferation, transformation, activation, or differentiation, depending on cell types, although it is not yet clear what is the primary target of the active Ras-GTP or how the various phenotypes are determined downstream of Ras protein.


Prenoids and palmitate: lipids that control the biological activity of Ras proteins. Ras proteins can be modified by two types of lipids--an isoprenoid and the fatty acid palmitate. These lipids help the otherwise cytoplasmic Ras protein to interact with the plasma membrane of a cell. The biological consequences of this association between Ras and membranes are dramatic, and can alter a cell's behavior from normal growth into malignancy. The scope and limits of our knowledge of the steps, structures and enzymes involved in this molecular transformation from soluble inactivity to membrane-bound potency are offered below. The prospects of regulating Ras function by controlling its intracellular location provides a tantalizing opportunity to translate research into a novel therapeutic reality.


Three-dimensional structure of p21H-ras and its implications. The three-dimensional structure of the H-ras oncogene product p21 has been determined in both its active, GTP-bound and its inactive, GDP-bound forms. This has supplied a wealth of information on the mode of binding of guanine nucleotides, on the mechanism of the GTPase reaction and on the conformational change of the protein which accompanies GTP hydrolysis. The structural analysis has also given clues to the interaction of p21 with the regulatory proteins GAP (GTPase Activating Protein) and nucleotide exchange factor. The three-dimensional structures of oncogenic mutants of p21 have also been determined and can nicely explain different biochemical and biological behaviour of these mutant proteins.


GTPase activating proteins.

GTPase activating proteins. Since Ras proteins negotiate many signalling pathways leading to cell growth or differentiation, the regulation of Ras activity is vital to cellular health. Ras activity, which derives from a collaboration between Ras and GTP, is terminated by the GTPase activating protein (GAP)-catalyzed hydrolysis of the GTP. Hence, a simple regulatory scheme emerges: extracellular signals control Ras activity via membrane receptors and GAPs. However, the signalling scenario is probably not so simple. In looking for factors which interpret Ras activity, researchers have been led to the same factors which also regulate Ras activity, namely the GAPs. Therefore, it may be that Ras proteins are actually regulators of GAPs.


Genetic analysis of ras homologs in yeasts. Ras proteins with extensive structural homology to mammalian p21ras have been studied in the two yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Comparative analysis of these two yeasts has revealed significant differences in the biochemical and physiological functions that are controlled by this subgroup of eukaryotic GTP-binding regulatory proteins. Despite such divergence of cellular functions, proteins and mechanisms involved in the regulation and modification of Ras proteins are highly conserved in yeasts and other eukaryotes. Genetic analysis of the function of yeast proteins that regulate or modify Ras proteins has provided important information about Ras proteins in general.