Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers.

Several complementary NMR approaches were used to study the interaction of mastoparan, a 14-residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using 1H-NMR spectroscopy in perdeuterated (SDS-d25) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic alpha-helix with high-order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3-4 residues. Secondly, solid-state 2H-NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup-deuterated dimyristoylglycerophosphocholine (DMPC-d4) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the alpha-segments and beta-segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG-rich phase and a DMPG-depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically 15N-labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid-state 15N-NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment. The two populations of 90% in-plane and 10% trans-membrane helices are characterized by a mosaic spread of +/- 30 degrees and +/- 10 degrees, respectively. The biological activity of mastoparan is discussed in terms of a pore-forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip-flop between the monolayers.

Orientational behavior of phospholipid membranes with mastoparan studied by 31P solid state NMR.

Solid state 31P NMR spectroscopy was used to study the perturbing effect of the wasp venom peptide mastoparan (MP) on lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). The 31P chemical shift anisotropy of multilamellar vesicles decreased with increasing peptide concentration, indicating that MP interacts strongly and selectively with the charged DMPG head group. Macroscopically oriented MP-lipid samples between glass plates were studied by 31P NMR as a function of tilt angle. These spectra showed the coexistence of orientation-dependent lamellar signals as well as an isotropic peak, suggesting that MP can induce non-lamellar phases in DMPC/DMPG membranes.

Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process.

Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1-binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.

Prognostic significance of Ki-67 expression in transitional cell bladder carcinoma after radical cystectomy.

We evaluated the prognostic significance of the Ki-67 labeling index (Ki-67 LI) in 75 patients with transitional cell carcinoma of the bladder who underwent radical cystectomy. Immunohistochemical staining of archival material was performed by the streptavidin-biotin method. Univariate survival analysis showed that Ki-67 LI (p < 0.001), histologic grade (p < 0.05), tumor stage (p < 0.001) and the number of positive lymph nodes (p < 0.001) significantly correlated with prognosis. Multivariate survival analysis indicated that the Ki-67 LI (p < 0.05), histologic grade (p < 0.01), tumor stage (p < 0.01), presence of lymph node metastases (p < 0.05) and use of neo-adjuvant therapy (p < 0.05) had independent prognostic value. The Ki-67 LI is an independent prognostic factor for patients with transitional cell bladder cancer treated by radical cystectomy.

Emotional and behavioral correlates of mediodorsal thalamic neurons during associative learning in rats.

Neuronal activity was recorded from the mediodorsal thalamic nucleus (MD) of behaving rats that were trained to lick a protruding spout just after a conditioned stimulus to obtain reward or to avoid shock. Conditioned stimuli included both elemental (auditory or visual stimuli) and configural (simultaneous presentation of auditory and visual stimuli predicting reward outcome opposite that predicted by each stimulus presented alone) stimuli. Of 122 MD neurons responding during the task, the activity of 13 increased just before licking only during the task, but not before spontaneous licking during the intertrial interval (conditioned behavior related). These conditioned behavior-related neurons were located mainly in the lateral MD, which has intimate anatomical connections with motor-related areas such as anterior cingulate and striatum. The activity of the other 109 neurons was related to conditioned stimulation (conditioned stimulus related). Most of these neurons responded differentially to both elemental and configural stimuli in terms of reward contingency, and also changed their responses during extinction and relearning trials. Conditioned stimulus-related neurons with latencies < 300 msec were located mainly in the rostromedial MD, which receives afferents from the basolateral nucleus of the amygdala in which sensory information from various sources converge. Furthermore, most differential neurons that were tested responded during the delay period in a reward task in which a delay was imposed between the conditioned stimulus and reward delivery. The present results, along with previous anatomical studies, suggest the existence of two limbic circuits: anterior cingulate-striatum-lateral MD (motor) and amygdala-medial MD-orbital prefrontal cortex (short-term memory/emotion).

Effects of chronic iron deficiency anemia on brain metabolism.

The effects of chronic iron deficiency anemia on brain (cortex) metabolism were estimated by 31P-nuclear magnetic resonance spectroscopy and biochemical analyses in male Wistar rats. Iron deficiency anemia was induced by supplying diet containing either approximately 2 or approximately 6 ppm Fe. Control diet was supplemented with 100 ppm Fe as ferric citrate. After 8-9 weeks, blood hemoglobin levels were approximately 13, 5, and 3 g/100 ml in the 100 ppm, 6 ppm, and 2 ppm Fe group, respectively. The blood lactate levels at rest in these groups were approximately 3, 5, and 6 mM. The blood glucose concentration also tended to be elevated in iron-deficient rats. The high-energy phosphate contents in brain were not affected by iron deficiency. The activities of succinate dehydrogenase and cytochrome oxidase per unit protein in the 2 ppm Fe group were significantly less than in the 100 ppm Fe group, but those activities were not significantly affected by feeding diet with 6 ppm Fe. The activities of lactate dehydrogenase in iron-deficient group tended to be elevated but not significantly. The activities of non-iron containing mitochondrial enzymes, citrate synthase and beta-hydroxyacyl CoA dehydrogenase, were unchanged. It is suggested that the brain has a higher tolerance to iron deficiency than skeletal muscle in terms of the metabolic characteristics, although this may be associated with a lower level of neural activity.

Changes of contractile properties of extensor digitorum longus in response to creatine-analogue administration and/or hindlimb suspension in rats.

Changes of contractile properties of extensor digitorum longus in response to hindlimb suspension and/or altered high-energy phosphate contents were studied in rats. A reduction of high-energy phosphates, especially phosphocreatine, was seen in rats fed creatine analogue beta-guanidinopropionic acid (beta-GPA), but they were elevated after 10-d supplementation of creatine. The one-half relaxation time was increased by feeding beta-GPA, but was normalized by creatine supply. The fatigue resistance of creatine-depleted muscle was significantly improved, but tended to decrease by suspension and creatine supply, although it was still better than that in the control diet group. It is indicated that the contractile properties of muscle are influenced by the high-energy phosphate content. It is also suggested that the endurance capacity may be influenced by the mitochondrial respiratory capacity, but not necessarily by the levels of high-energy phosphates.

[Pre-operative autologous blood collection in neurosurgical patients].

Homologous transfusion has been known to cause viral infections and other complications. Recently, autologous transfusion has been adopted widely as a safer and more effective procedure to prevent these complications. The authors report the experiences of 29 patients who had been operated on after preparation of autologous blood. Furthermore, the authors report a study concerning maximum surgical blood order schedule (MSBOS) of these operations. 212 patients operated on between January, 1991 and June, 1993 were used for this study of MSBOS. Although intraoperative transfusion was performed on 14 of 29 patients, the need for homologous transfusion was avoided in 12 of these 14 patients by the use of autologous blood. The frequency of homologous transfusion was reduced significantly after the introduction of pre-operative autologous blood collection in our clinic. The patients' value of hemoglobin fell after the collection of blood but these levels were not so seriously low as to impede the performance of operations. 212 cases of operated patients were divided according to the operative methods and diagnosis for calculation of MSBOS. The results were as follows; Craniotomy and removal of glioma 5u, meningioma 11u, neurinoma 7u, AVM 5u, transsphenoidal surgery 3u, Moyamoya disease 2u and V-P or S-P shunt 0u. Pre-operative autologous blood collection is easy to achieve for scheduled neurosurgical operations, and autologous transfusion is a beneficial procedure which should be used more widely.

Responses of beta-adrenoceptor in rat soleus to phosphorus compound levels and/or unloading.

Responses of beta-adrenoceptor (beta-AR) in rat soleus to gravitational unloading and/or changes in the levels of phosphorus compounds by feeding either creatine or its analogue beta-guanidinopropionic acid (beta-GPA) were studied. A decrease in the density of beta-AR (about -35%) was induced by 10 days of hindlimb suspension, but the affinity of the receptor was unaffected. Suspension unloading tended to increase the levels of adenosine triphosphate and phosphocreatine and decrease inorganic phosphate. Even without unloading, the beta-AR density decreased after an oral creatine supplementation (about -20%), which also tended to elevate the high-energy phosphate levels in muscle. However, an elevation of beta-AR density was induced (about +36%) after chronic depletion of high-energy phosphates by feeding beta-GPA (about +125%). Data suggest that the density of beta-AR in muscle is elevated if the high-energy phosphate contents are chronically decreased and vice versa. However, it may not be directly related to the degree of muscle contractile activity.

Responses of contractile properties in rat soleus to high-energy phosphates and/or unloading.

Responses of contractile properties of soleus to unloading and/or changes in high-energy phosphate contents were studied in rats. Reduction of high-energy phosphates, especially phosphocreatine, in ankle extensors was induced by feeding beta-guanidinopropionic acid (beta-GPA). The major finding in the study was that the fatigability and speed-related contractile properties responded to unloading and creatine supplementation in a similar manner. The high-energy phosphate contents tended to be elevated after 10-d supplementation of creatine and hindlimb suspension. The shift toward slow-type, mainly due to an increased one-half relaxation time, was seen in rats fed beta-GPA. Such a shift was reversed by feeding creatine or by hindlimb suspension; however, the suspension-induced shift of contractile properties toward fast-type was not prevented completely by beta-GPA feeding. Although the muscle fatigue resistance did not change by beta-GPA feeding alone, the decrease in fatigue resistance following suspension and creatine supply was less in the beta-GPA group. It is suggested that the levels of high-energy phosphates and tension production play important roles in the regulation of contractile properties of the soleus muscle.

Early detection of cerebral infarction by 31P spectroscopic imaging.

Recent advances in magnetic resonance spectroscopy permit noninvasive study of brain metabolism in vivo, 31P spectroscopic imaging being the method for evaluation of localized phosphorous metabolism. Experimentally, an ischemic-hypoxic brain insult is characterized by depletion of high energy metabolites. These changes are seen immediately after an ischemic insult. We had the opportunity of carrying out 31P spectroscopic imaging of hyperacute cerebral infarction, while MRI and CT were negative. Cerebral infarction of the middle cerebral artery territory was suggested by 31P spectroscopic imaging, which was closely consistent with a later-developing region of low density on CT. In cerebral infarction, early detection of the lesion is a useful pointer to the patient's prognosis, making 31P spectroscopic imaging a potential tool.

Changes in proton T1 in dog brains due to the administration of haloperidol.

Changes in proton T1 in dog brains due to the administration of haloperidol were determined by the intravenous administration of a single dose of 20 mg of haloperidol to mongrel dogs. The MRI used was the Aberdeen type with the static magnetic field of 0.1 T. A coil made exclusively for these animals (bore diameter 120 mm) was used. There was a significant increase in the T1 value in the striate body 30 minutes and more (within two hours) after the administration of haloperidol. Subtraction images were also obtained by subtracting the image of the pre-treatment (control) T1 values from the image of the post-treatment values (2 hours after the injection). The subtraction images also revealed increases in the T1 values of the striate body.

[Application of renal subcapsular assay to rat malignant brain tumor].

As a rapid screening method for testing chemotherapeutic agents against human tumor explants, renal subcapsular assay has been established by Bogden et al (1978). The authors investigated the effectiveness of the technique of Bogden for rat glioma. Preselected 1 mm3 fragments of viable tumor tissue of rat (CDF) were implanted under the renal capsule of normal immunocompetent rats. Then initial and final tumor sizes (formula; see text) were measured in situ with an oculometer inserted into a surgical microscope, and evaluated the changes of tumor sizes. The experiments were divided into three groups. The first group was that tumor pieces of rat (CDF) were implanted under the renal capsule of the syngenetic rats (CDF). Non-irradiated and preirradiated (400 rad) heterogenetic rats (Wistar) were used as the second and third groups. The explanted tumor pieces grew rapidly in the first group (syngenetic rats). On the other hand, poor growth of the second group (non-irradiated heterogenetic rats) was seen within 7 days after implantation, and rejected completely until 15 days after implantation. Explanted tumor pieces in the third group (pre-irradiated heterogenetic rats), however, grew enough big, though the change of tumor sizes was less than that of the first group. Histological examination was also performed. In the first and third group, proliferation of glioma cells was remarkable under the renal capsule, and neovascularization in the explanted tumor tissue was provided from the capsule, first, and then parenchym of the kidney. Even necrosis and hemorrhage were seen in the tumor tissue 15 days after implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisickling effect of tellurite: a potent membrane-acting agent in vitro.

Potassium tellurite (K2TeO3) was found to be a potent antisickling agent that inhibited red cell sickling at concentrations less than 10 mumol/L. The inhibitory effect depended on the incubation time, with the effect increasing with longer incubation periods. Because tellurite causes swelling of red cells, and because the antisickling effect of tellurite correlated with the degree of red cell swelling, the antisickling effect of tellurite is assumed to be due to the decreased mean cell hemoglobin concentration. Swelling of red cells by tellurite was accelerated by the addition of reduced glutathione. Tellurite appears to be a new type of antisickling agent that interacts with the red cell membrane.

[Effects of solcoseryl on the cerebral blood flow, intracranial pressure, systemic blood pressure and EEG in acute intracranial hypertensive cats (author's transl)].

The experiment was performed on 86 cases under intraperitoneal pentobarbital anesthesia. One balloon was placed in the extradural space of right frontal region, and the other balloon was placed in the left extradural space and the intracranial pressure was measured. A needle was stereotaxically inserted into the subcortical area in order to measure the cerebral blood flow. Systemic blood pressure was recorded by inserting a catheter into the femoral artery, and electrocorticogram was also recorded. An expanding intracranial lesion was made by inflating the extradural balloon with physiological saline. The animals were arbitrarily divided into two groups.: 1) light or moderate groups which intracranial pressure before the injection of drug was below 400 mmH2O. 2) severe groups above 400 mmH2O. After the maintenance of the pressure, Solcoseryl was infused intravenously. The investigation was focused to observe whether Solcoseryl reveales any potent effect on cerebral blood flow, intracranial pressure, systemic blood pressure and on electroencephalogram in acute intracranial hypertension. Results 1) Intravenous injection of Solcoseryl had the effect of lowering intracranial pressure in the light or moderate and severe groups. Particularly, dose of 80 mg/kg showed the marked effect, though with a rebound phenomenon in the light or moderate groups. Furthermore, the effect was more marked and lasting by drip infusion of Solcoseryl and also by intravenous injection of Solcoseryl after pretreatment with hydrocortisone, and at this time no rebound phenomenon was recognized. 2) Solcoseryl had the effect of increasing the cerebral blood flow accompained with the lowering of intracranial pressure. 3) Systemic blood pressure was transiently lowered by the injection of Solcoseryl 20 mg/kg or 80 mg/kg and recovered immediately. 4) Solcoseryl had no effect on electroencephalogram in the severe groups. Conclusion On the basis of these results, it is rational to conclude that Solcoseryl could be superior agent render to lower intracranial pressure and to improve cerebral blood flow in acute intracranial hypertension.