Modification of fullerene nanocolumn structure by accelerated C60 ions.

Crystalline C60 and amorphous graphite-like films of nanocolumn arrays fabricated by glancing angle deposition of C60 fullerene at substrate temperatures of -425 K were studied by transmission electron microscopy (TEM) and atomic-force microscopy (AFM). Characteristic dimension of columns is 200-400 nm. We used co-deposition of C60 molecules and accelerated C60 ions to modify the structure and properties of nanocolumn arrays. Influence of incidence angle for C60 ions on formation of film morphology was revealed. Raman spectrum analysis showed that amorphous carbon nanocolumns consist of nanographite areas with average size of -1.5 nm. The films have high conductivity (close to graphite) and have no mechanical stresses. The carbon films were applied in all-solid-state rechargeable thin-film battery as an anode layer. The nanocolumn amorphous carbon film as anode electrode showed the discharge capacity of about 50 microAh cm(-2)microm(-1) and good cycling ability over 100 times in full cell system.

Synthesis, structure and properties of superhard nanostructured films deposited by the C60 ion beam.

In this work, we present results on study of DLC, nanocomposite and nanocrystal nanographite films synthesized utilizing mass-separated beam of C60-ions with energy in range from 2 to 6 keV (energy dispersions approximately 1 keV) and at Ts in the range of RT - 873 K. The dependence of the structure, mechanical and electrical properties from the ion energy and substrate temperature was revealed. We demonstrate a possibility to control the orientation of the base planes in the nanographite grains during the film growth. The dependence of mechanical properties of the films from the orientation of the base planes was defined. It is discussed a mechanisms of oriented growth for nanocrystal graphite. Possible applications of the textured nanocomposite and nanographite films are nanodevices, thin-filmed lithium batteries and field-emitter arrays.

c-JUN expression and apoptotic cell death in kainate-induced temporal lobe epilepsy.

Following kainate (KA)-induced epilepsy, rat hippocampal neurons strongly express immediate early gene (IEG) products, i.e., c-FOS and c-JUN, and neural stress protein, HSP72. Prolonged expression of c-JUN and c-FOS 48 hr after cerebral ischemia has been underwent delayed neuronal death. However, it is not yet clear whether IEGs actually assume the essential roles in the cell death process or simply as a by-product due to external stimuli because of the prolonged expression of c-FOS, more than one week, on intact CA2 neurons of the hippocampus in a KA-induced epilepsy model. This study investigated the relationships between prolonged expression of c-JUN and hippocampal neuronal apoptosis in a KA-induced epilepsy model. Epileptic seizure was induced in rats by a single microinjection of KA (1 microgram/microL) into the left amygdala. Characteristic seizures and hippocampal neuronal injury were developed. The expression of c-JUN was evaluated by immunohistochemistry, and neuronal apoptosis by in situ end labeling. The seizures were associated with c-JUN expression in the hippocampal neurons, of which the level showed a positive correlation with that of apoptosis. Losses of hippocampal neurons, especially in the CA3 region, were partly caused by apoptotic cell death via a c-JUN-mediated signaling pathway. This is thought to be an important component in the pathogenesis of hippocampal neuronal injury via KA-induced epilepsy.

Pathogenic significance of neuronal migration disorders in temporal lobe epilepsy.

To assess the epileptogenic lesions, a series of 202 cases with temporal lobectomy were analyzed histopathologically. The severity of hippocampal neuronal loss in patients with temporal lobe epilepsy was quantitatively analyzed and compared against autopsy controls of patients who died of nonneurologic disorders. For the histopathologic diagnosis of neuronal migration disorder (NMD), immunohistochemical stains for neurofilament protein (NF-M/H) and microtubule-associated protein 2 (MAP2) and Bielschowsky silver stains were routinely performed. Histopathology of NMD was classified by the 4-grade system. MAP2 immunoreactivity was useful in the identification of loss of normal polarization of dendrites in the abnormal neurons. NF-M/H immunohistochemistry and silver stains effectively labeled microscopic or occult lesions of NMD (grade II and III). Ammon hom sclerosis (AHS) was identified in 73.3% and NMD in 57.9%. There was more than 50% neuronal cell loss in 82.8% of AHS, and variable degrees of cell loss were observed in the dual-pathology groups. The frequency of dual pathology (both AHS and NMD) was 65.0% and showed relatively equal distributions in grades I, II, III, whereas the pure NMD group were classified predominantly as grades II and III. NMD might be a basic pathogenic substrate causing temporal lobe epilepsy. The dual pathology may indicate the presence of epileptogenic lesions in the neocortical and temporolimbic areas.

Expression of cloned cDNA for the human mitochondrial RNA polymerase in Escherichia coli and purification.

A full-length cDNA for the mature, mitochondrial form of human mitochondrial RNA polymerase was cloned and expressed under the control of T5 or tac promoter in Escherichia coli. The cDNA was efficiently expressed at 37 degrees C, but virtually all the polymerase produced was insoluble, and renaturation of the inclusion bodies was unsuccessful. When the cells were grown at 25 degrees C, however, a portion of approximately 10% was soluble and active. The protein was purified 100-fold from the soluble lysates to homogeneity by two-step chromatography using Ni-nitrilotriacetic acid-Sepharose and heparin-agarose columns, as an N-terminal histidine tag attached and as the tag cleaved away. The purified polymerases with and without the histidine tag were both active in RNA polymerization in vitro as measured with poly(dA-dT) template, and specific activity was 140,000 units/mg. The purified enzyme has the same biochemical properties as the polymerase fraction partially purified from the human mitochondria, except for the promoter-specific activity that was not observed with the purified polymerase in the presence of mitochondrial transcription factor A. Additional factor(s) and/or mammalian-specific or regulatory modification(s) of the polymerase should be necessary for promoter-specific transcription.

Augmented expression of cardiac atrial natriuretic peptide system in hypertensive rats.

The present study was aimed at investigating the regulation of atrial natriuretic peptide (ANP) system in association with either enhanced or attenuated activity of the renin-angiotensin system (RAS). The cardiac tissue mRNA and peptide levels of ANP were measured in rats with two-kidney, one clip (2K1C) or deoxycorticosterone acetate (DOCA)-salt hypertension. Plasma renin concentration was increased in 2K1C hypertension along with increases of renin mRNA and protein contents in the clipped kidney. On the contrary, it was suppressed in DOCA-salt hypertension along with decreases of renin mRNA and protein contents in the remaining kidney. The plasma ANP concentration was similarly increased in both models of hypertension. The cardiac tissue ANP contents were not significantly changed, but the tissue ANP mRNA levels were upregulated in the hypertrophied heart in these two models of hypertension. It is suggested that the cardiac ANP system is transcriptionally enhanced by cardiac hypertrophy associated with hypertension, independent of the systemic RAS.

Toxicities of 166Holmium-chitosan in mice.

166Holmium (166Ho) is a radionuclide of rare earth chemical and is known to have antitumor activity. Several chemicals were complexed with 166Ho to facilitate the transport of this radionuclide to the site of action. In this study, 166Ho was complexed to chitosan (Chit) which decreases the distribution of Ho into other tissues when applied intrahepatically. To investigate the single dose toxicity, mice were administered intravenously with 1 mCi/kg body weight of 166Ho-Chit (DW-166HC), Chit or nothing. Organ weights, hematological and histopathological studies were performed in 6 animals per group at 1, 3 and 14 days after administration. In 166Ho-Chit treated animals, a slight decrease of erythrocyte number was observed at day 14 and increases of relative liver and lung weights were found at day 3. Although marked multiple necrotic foci in the white pulp and depletion of marginal zone in the spleen were noted at day 1, these findings were decreased in severity and fully recovered at day 3 and day 14, respectively. Slightly decreased kidney weights were observed both in Chit and in 166Ho-Chit treated groups without histological alterations. Thus it is suggested that most effects of 166Ho-Chit observed at an early stage after administration are limited to rapidly dividing cells and reversible within 14 days.

Analysis of spontaneous electrical activity in cerebellar Purkinje cells acutely isolated from postnatal rats.

Whole-cell patch recording techniques were used to analyze spontaneous electrical activity in cerebellar Purkinje cells acutely isolated from postnatal rats. Spontaneous activity was present in 65% of the cells examined, and it included simple and complex firing patterns which persisted under conditions that eliminated residual or reformed synaptic contacts. Under voltage clamp, both spontaneous and quiescent cells displayed similar voltage-dependent conductances. Inward current was carried by Na+ through tetrodotoxin (TTX)-sensitive channels and by Ca2+ through P-type and T-type Ca channels. P-type current was present in all cells examined. T-type current was found in <50%, and it did not correlate with spontaneous activity. We found no evidence of a transient (A-type) potassium current or hyperpolarization-activated cationic current in either spontaneous or quiescent cells. Spontaneous activity did correlate with a lower activation threshold of the Na current, resulting in substantial overlap of the activation and inactivation curves. TTX reduced the holding current of spontaneous cells clamped between -50 and -30 mV, consistent with the presence of a Na "window" current. We were unable, however, to measure a persistent component of the Na current using voltage steps, a result which may reflect the complex gating properties of Na channels. An Na window current could provide the driving force underlying spontaneous activity, as well as plateau potentials, in Purkinje cells.

Identification of acutely isolated cells from developing rat cerebellum.

Immunocytochemical staining was used to identify nerve and glial cells from postnatal rat cerebelli in situ and following tissue dissociation. Purkinje cells were identified using antibodies for the calcium-binding proteins calbindin and PEP19. Purkinje cells isolated during the second postnatal week were 15-20 microns in diameter and relatively abundant and displayed thin perisomatic processes. These features were used to identify Purkinje cells with scanning electron microscopy, which revealed extensive membrane infoldings. Golgi and nuclear cells were identified using antibodies against rat-303 antigen. Pale, nuclear, and Purkinje cells were identified using antibodies for rat-302 antigen. Although staining for rat-302 and rat-303 was weak during the second postnatal week, we were able to identify Golgi and pale cells even after tissue dissociation. Isolated Golgi cells were 8-10 microns in diameter and fewer in number than Purkinje cells and did not counterstain with calbindin antibodies. Isolated pale cells were 8-10 microns in diameter, rare, and resistant to calbindin antibodies. Isolated neurons from cerebellar nuclei were not located with either 302 or 303 staining, suggesting that they remained in the tissue. Golgi-Bergmann cells and astrocytes were identified using antibodies for glial fibrillary acidic protein. Isolated glial cells were 12-15 microns in diameter, more numerous than Purkinje cells, and unstained with calbindin antibodies. With phase-contrast optics, glial cells appeared flatter than neuronal cell types and had acentric nuclei. These results demonstrate that specific cell types in developing rat cerebellum can be identified after acute isolation, which should facilitate analysis of their endogenous properties.

High-voltage-activated calcium current in developing neurons is insensitive to nifedipine.

We have analyzed the effect of nifedipine on the macroscopic high-threshold, voltage-activated (HVA) calcium current in four cell types: postnatal rat Purkinje and dorsal root ganglion (DRG) neurons, embryonic chick DRG neurons, and adult cat ventricular myocytes. As is consistent with previous reports, nifedipine reduced HVA current in myocytes in a voltage-sensitive manner. Analysis of nifedipine actions on neurons, however, was compromised by slow inactivation of the current at holding potentials between -80 mV and -40 mV. The slow inactivation was voltage-dependent, irreversible after 5 min, and contributed to "rundown" of the current. At -40 mV, slow inactivation displayed two time constants: 12 +/- 8 s and 7 +/- 4 min. When slow inactivation was taken into account, we found no evidence for a nifedipine-sensitive component of the HVA current in these neurons. Consistent with previous studies, DRG neurons were reduced irreversibly by omega-conotoxin, whereas cardiac and Purkinje cells were unaffected. Our biophysical and pharmacological results are consistent with two types of neuronal HVA currents (N type and P type) in developing neurons that are distinct from cardiac HVA currents (L type).

Chronic effects of topical application of capsaicin to the sciatic nerve on responses of primate spinothalamic neurons.

The responses of 144 spinothalamic tract (STT) cells were recorded in 15 anesthetized macaque monkeys (Macaca fascicularis). Three to 4 weeks prior to the acute experiment, the sciatic nerve was surgically exposed on one or both sides so that capsaicin or vehicle could be applied. Responses of STT cells recorded in 3 experimental groups were compared: untreated (21 cells), vehicle-treated (40 cells), and capsaicin-treated (83 cells). The background activity of cells in the vehicle- and capsaicin-treated groups was the same as in the untreated group (that is, cells on the side contralateral to surgery). Responses to innocuous (BRUSH) and noxious (PINCH) mechanical stimuli were unchanged by vehicle or by capsaicin treatment. However, responses to other noxious (PRESSURE and SQUEEZE) mechanical stimuli were significantly increased in the vehicle-treated group. Compared with a large reference population, all experimental groups showed a significant increase in overall responsiveness to mechanical stimuli (as determined by cluster analysis), greatest in the vehicle-treated group. Responses to noxious heat stimuli were significantly reduced in the capsaicin-treated group for 45 degrees C and 47 degrees C stimuli. Volleys in A fibers, probably A delta fibers, evoked prolonged responses in many STT cells of all treatment groups. Electron microscopic counts of axons in the sciatic nerves of animals treated with capsaicin showed a reduced number of C fibers but no appreciable loss of myelinated axons. This loss of unmyelinated sensory fibers was presumably responsible for the reduction in the responses of the STT cells to noxious heat stimuli. Increased responses to some noxious mechanical stimuli and to A fiber volleys may have been the consequence of several factors, including surgical manipulation, a chemical action of vehicle and a contralateral action of capsaicin treatment.

Divalent ions released from stainless steel hypodermic needles reduce neuronal calcium currents.

We have investigated the effects of saline solutions exposed to stainless steel hypodermic needles on their ability to reduce voltage-dependent calcium currents in chick dorsal root ganglion neurons and rat cerebellar Purkinje cells. Salines exposed to needles with brass hubs, but not those with plastic hubs, for as little as 2-3 sec reduced calcium currents in both cell types. The amplitude of the response was exposure-dependent and reversible. Elemental analysis of the exposed salines using inductively-coupled-plasma atomic emission spectroscopy revealed that Cu and Zn (but not Cd, Cr, Co, Fe, Mn, Ni or Pb) were released from the brazed needles. The amount of Cu plus Zn released in 30 sec was estimated to be 12-26 microM, depending upon the specific needle examined. Longer exposures produced proportionately higher concentrations of the metals. Dose-response curves for Cu or Zn ions applied directly to cells confirmed that similar concentrations of these ions reduced neuronal calcium currents. Our results indicate that divalent ions released from stainless steel hypodermic needles with brass hubs can interfere with measurements of calcium currents. In addition the results contribute new information regarding potential physiological and pathological actions of copper and zinc ions in biological tissues.

Increased number of unmyelinated fibers in the ventral root after peripheral neurectomy in adult rat.

We examined the possibility that peripheral nerve injury in the adult rat triggers sprouting of unmyelinated ventral root afferent fibers. Three to 5 months after the sciatic nerve was sectioned on one side in the adult rat, myelinated and unmyelinated fibers were counted at 3 sites along the length of the ventral root. A sciatic nerve lesion resulted in about a 3-fold increase in the number of unmyelinated fibers in the L5 ventral root. Our data suggest that a peripheral nerve lesion in the adult rat triggers sprouting of unmyelinated afferent fibers in the ventral root. No evidence was found that dorsal rhizotomy triggers sprouting of afferent fibers.

Fiber counts at multiple sites along the rat ventral root after neonatal peripheral neurectomy or dorsal rhizotomy.

We hypothesized that the afferent fibers in the ventral root of the rat are the third branches of dorsal root ganglion cells; these afferent processes in the ventral root are of varying length and end bluntly along the length of the root. In the case of an injury at either the central or the peripheral processes of the dorsal root ganglion cells in the neonatal stage, these fibers sprout at the blunt endings along the length of the ventral root. We cut either the sciatic nerve or the dorsal root on one side in neonatal rats. After the rats were fully grown, the number of both myelinated and unmyelinated fibers was counted in electron photomicrographs at multiple sites along the length of the ventral root. We observed a greatly increased number of unmyelinated fibers in the ventral root after the sciatic nerve had been cut at the neonatal stage. The magnitude of increase was more at the distal than at the proximal portion of the ventral root, suggesting that added fibers originated from the distal side. Neonatal dorsal rhizotomy, however, did not produce the same result. These results are consistent with our hypothesis that peripheral nerve injury at the neonatal stage triggers sprouting of the third branches of the dorsal root ganglion cells which end bluntly along the length of the ventral root in the normal animal.

Different classes of cat spinal neurons display differential sensitivity to sodium pentobarbital.

The effect of graded doses of systemically injected sodium pentobarbital on several classes of spinal neurons was studied using spinal cats. Classes of spinal neurons included unidentified dorsal horn cells, ascending tract dorsal horn cells, and motoneurons. Single unit activity of spinal neurons was evoked by electrically stimulating a peripheral nerve with an intensity strong enough to excite both A and C fibers. The A- and C-fiber evoked activity was compared before and after intravenous injections of small incremental doses of sodium pentobarbital. The activity of different classes of spinal neurons showed different sensitivities to graded doses of systemically injected pentobarbital. The reflex activity of motoneurons elicited by stimulation of peripheral nerve was much more sensitive to pentobarbital than that of dorsal horn cells. In general, activity evoked by peripheral unmyelinated fibers was more susceptible to pentobarbital than was that evoked by myelinated fibers. However, intravenous injections of pentobarbital produced nondifferential suppression of dorsal horn cell activity evoked by noxious and innocuous mechanical stimuli applied to the peripheral receptive fields.

Transcription initiation site selection and abortive initiation cycling of phage SP6 RNA polymerase.

Effects of mutations around the phage SP6 transcription initiation site on SP6 RNA polymerase's selection of initiation site were studied. In the in vitro transcription reactions, the limiting concentration of a ribonucleotide causes the SP6 RNA polymerase to stall long enough only at the positions of the limited nucleotide and dissociate from the elongation complex. As a result, a series of RNA oligomers comprises a sequencing ladder, and abortive initiation cycling products up to 6-mer are made in high yield. Precise sizing of the product RNAs from the elongation pausings determined the initiation site of each mutant. When the wild-type +1 G is changed to C or A without change in the upstream sequence including TATA from -4 to -1, transcription still starts only at the +1 site. But, the mutant containing TATCC from -4 to +1 C. We propose that the phage SP6 RNA polymerase selects the initiation site precisely at a certain distance from a direct contact point in the upstream promoter sequence, regardless of the species of initiating nucleotide. It is also suggested that the sequence-dependent perturbations of DNA helical structure, for example D to B form, may shift the initiation site.

Proportion and location of spinal neurons receiving ventral root afferent inputs in the cat.

Spinal neurons receiving ventral root afferent inputs were investigated in anesthetized and paralyzed cats. We were concerned with the afferent fibers in the ventral root that travel distally and then enter the spinal cord through the dorsal root. The questions to be answered included the proportion and distribution of spinal neurons receiving ventral root afferent inputs and their peripheral input characteristics. The 1.7 ventral root was cut near the spinal cord and the distal stump was stimulated while making a systematic search for neurons in the entire gray matter of the ipsilateral spinal cord that responded to the stimulation. The following conclusions were made: (i) the afferent fibers in the cat ventral root enter the spinal cord through the dorsal root and evoke a variety of responses (excitation, inhibition, or mixed) in a large proportion of spinal neurons (about 20%): (ii) these responses seem to be mediated largely by spinal mechanisms: (iii) spinal neurons receiving ventral root afferent inputs are situated in a wide region of the ventral spinal cord: (iv) ventral root fibers in a single root enter the spinal cord and exert their responses over a large region of the spinal cord (at least two spinal segments rostrally and caudally): (v) some of the spinal neurons that responded to ventral root stimulation were found to be ascending tract cells, suggesting that ventral root afferent inputs can reach supraspinal structures: (vi) ventral root afferent fibers converge onto spinal neurons that have a variety of peripheral receptive field characteristics: and (vii) with some exceptions, most neurons receiving ventral root inputs were excited best by mechanical and/or thermal noxious stimuli applied to the periphery.

Differential inhibition produced by peripheral conditioning stimulation on noxious mechanical and thermal responses of different classes of spinal neurons in the cat.

The effect of conditioning stimulation of a peripheral nerve on responses of spinal neurons (dorsal horn cells and motoneurons) was studied in 16 decerebrate-spinal cats. The activity of dorsal horn cells was recorded with a microelectrode at the lumbosacral spinal cord and the single-unit activity of motoneurons was recorded from a filament of ventral rootlet divided from either the L7 or S1 ventral root. The responses of spinal neurons were evoked by noxious and innocuous mechanical stimuli and by noxious thermal stimuli applied to the receptive fields. The peripheral conditioning stimulation was applied to the tibial nerve with repetitive electrical pulses (2 Hz) at an intensity either suprathreshold for A delta or C fibers for 5 min. Applying conditioning stimulation to a peripheral nerve produced a powerful inhibition of the responses elicited by noxious stimuli, suggesting this inhibition is an antinociceptive effect. The inhibition produced by peripheral conditioning stimulation was differentially greater on the responses to noxious than to innocuous stimuli. Based on the results obtained from conditioning stimulation with graded strengths, afferent inputs from both myelinated and unmyelinated fibers seem to contribute to the production of the antinociceptive effect. The magnitude of the antinociceptive effect is bigger for the responses to noxious thermal than to mechanical stimuli. Furthermore, the reflex activity recorded in motor axons seemed to be more sensitive than in dorsal horn cells to the antinociceptive effect.

Spinal entry route for ventral root afferent fibers in the cat.

Twelve anesthetized and paralyzed cats were used to study the spinal entry routes of ventral root afferent fibers. In all animals, the spinal cord was transected at two different levels, L5 and S2. The L5 through S2 dorsal roots were cut bilaterally, making spinal cord segments L5-S2 neurally isolated from the body except for the L5-S2 ventral roots. From this preparation, a powerful excitation of the discharge rate of motor neurons and dorsal horn cells within the isolated spinal segments was observed after intraarterial injection of bradykinin (50 micrograms in 0.5 ml saline). This excitation of the spinal neurons can be considered the most convincing evidence of the potential physiologic role of the ventral root afferent fibers entering the spinal cord directly through the ventral root, because the apparent route of neuronal input from the periphery is through the ventral roots. However, additional control experiments conducted in the present study showed that the excitation persisted even after cutting all ventral roots within the isolated spinal segments, indicating that excitation was not mediated by the ventral roots. Furthermore, direct application of bradykinin on the dorsal surface of the spinal cord also increased the motoneuronal discharge rate, suggesting that excitation of spinal neurons produced by intraarterial injection of bradykinin is due to a direct action of bradykinin on the spinal cord. Thus, we provided an alternate explanation for the most convincing evidence indicating that physiologically important ventral root afferent fibers enter the spinal cord directly through the ventral root. Based on existing experimental evidence, it is likely that the majority of physiologically active ventral root afferent fibers travel distally toward the dorsal root ganglion and then enter the spinal cord through the dorsal root.