Search

A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.

Objective: To explore and identify the non-coding RNAs related to tumors., Methods: We used RT-PCR and Northern blot to analyze non-coding RNAs in tumor tissues and cell lines., Results: Two predicted non-coding RNAs were confirmed to be expressed in cancer tissues and cell lines by RT-PCR and DNA sequencing. We detected the expression of two non-coding RNA transcripts by Northern blot. The length of NC28 was about 1800 nt, and that of NC119 was about 1200nt., Conclusions: NC28 and NC119 have a tumor-associated expression pattern. The non-coding RNAs may play a role in the development of tumors.

Inflammatory events may contribute to the pathogenesis of Parkinson's disease (PD) and interleukin 1 (IL-1) may exert both neurotoxic and neuroprotective effects. We conducted a case-control study in a cohort of 493 PD cases and 388 ethnically matched controls to investigate the association of IL-1alpha C-889T and IL-1beta C-511T polymorphisms with the risk of PD. No significant difference in the genotype distribution of the analyzed polymorphisms was found between PD and controls. However, after stratification by age, individuals over 70 years of age carrying IL-1alpha-889 C/T genotype demonstrated a significant decrease in risk of developing PD (OR = 0.44; 95% CI = 0.22-0.88, p = 0.021) and the decrease is strengthened by IL-1beta-511 T-carrying genotype (OR = 0.28; 95% CI = 0.11-0.71, p = 0.008). Our data suggest that IL-1alpha, acting synergistically with IL-1beta, plays role in PD susceptibility among Taiwanese people older than 70 years of age.

A microdevice is developed for RNA analysis that integrates one-step reverse transcription and 30 cycles of PCR (RT-PCR) amplification with capillary electrophoresis (CE) separation and fluorescence detection of the amplicons. The four-layer glass-PDMS-glass-glass hybrid microdevice integrates microvalves, on-chip heaters and temperature sensors, nanoliter reaction chambers (380 nL), and 5-cm-long CE separation channels. The direct integration of these processes results in attomolar detection sensitivity (<11 template RNA molecules or approximately 0.1 cellular equiv) and rapid 45-min analysis, while minimizing sample waste and eliminating contamination. Size-based electrophoretic product analysis provides definitive amplicon-size verification and multiplex analysis. Multiplexed differential gene expression analysis is demonstrated on mdh and gyrB E. coli transcripts. RNA splice variant analysis of the RBBP8 gene is used to identify tumorigenic tissue. RT-PCR microdevice analysis of normal breast tissue RNA generates the expected 202-bp normal splice isoform; tumor breast tissue RNA samples generate a 151-bp amplicon signifying the presence of the tumorigenic splice variant. The ability to perform RNA transcript and splice variant biomarker analysis establishes our RT-PCR microdevice as a versatile gene expression platform.

Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtypes caused enormous economical loss to poultry farms in China and Southeastern Asian countries. The vaccination program is a reliable strategy in controlling the prevalence of these disastrous diseases. The six internal genes of the high-yield influenza virus A/Goose/Dalian/3/01 (H9N2), the hemagglutinin (HA) gene of A/Goose/HLJ/QFY/04 (H5N1) strain, and the neuraminidase gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting peptide between HA1 and HA2. Eight gene expressing plasmids were constructed, and the recombinant virus rH5N3 was generated by cells transfection. The infection of chicken embryos and the challenge tests involving chickens demonstrated that the recombinant H5N3 (rH5N3) influenza virus is avirulent. The allantoic fluids of rH5N3-infected eggs contain high-titer influenza viruses with hemagglutination unit of 1:2048, which are eight times those of the parental H5N1 virus. The rH5N3 oil-emulsified vaccine could induce hemagglutination inhibition (HI) antibodies in chickens in 2 weeks post-vaccination, and maximum geometric mean HI-titer were observed 4 approximately 5 weeks post-vaccination and were kept under observation for 18 weeks. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04, which had 8 years expansion and differences among multiple amino acids in HA protein. The N3 neuraminidase protein marker makes it possible to distinguish between H5N1 infected- and H5N3 vaccinated animals.

We have developed a fully integrated multichannel polymerase chain reaction-capillary electrophoresis (PCR-CE) microdevice with nanoliter reactor volumes for highly parallel genetic analyses. Resistance temperature detectors and heaters made out of Ti/Pt are integrated on the microchip using a scalable radial design to provide precise temperature control of the four parallel PCR-CE reactor systems. Heating rates of >15 degrees C s(-1) and cooling rates of >10 degrees C s(-1) allow cycle times of 50 s and 30 complete PCR cycles in <27 min. PDMS membrane valves control and localize PCR reagents in the 380-nL reactors. By directly integrating PCR reactors with the CE separation system, efficient coupling of amplification with separation is achieved. The microdevice demonstrates good amplification uniformity and sensitivity down to 10 initial template copies in the 380-nL reactor (approximately 43 aM) with signal-to-noise ratio greater than 10. Parallel PCR-CE multiplex amplification and genetic analyses of four different samples with (1) both M13mp18 control template and E. coli K12 cells, (2) only M13mp18 template, (3) only E. coli K12 cells, and (4) negative control are completed in less than 30 min in a single run.

Aluminum coordination in the framework of USY and ZSM-5 zeolites containing charge-compensating cations (NH4+, H+, or Cu+) was investigated by Al K-edge EXAFS and XANES. This work was performed using a newly developed in-situ cell designed especially for acquiring soft X-ray absorption data. Both tetrahedrally and octahedrally coordinated Al were observed for hydrated H-USY and H-ZSM-5, in good agreement with 27Al NMR analyses. Upon dehydration, water desorbed from the zeolite, and octahedrally coordinated Al was converted progressively to tetrahedrally coordinated Al. These observations confirmed the hypothesis that the interaction of water with Brønsted acid protons can lead to octahedral coordination of Al without loss of Al from the zeolite lattice. When H+ is replaced with NH4+ or Cu+, charge compensating species that absorb less water, less octahedrally coordinated Al was observed. Analysis of Al K-edge EXAFS data indicates that the Al-O bond distance for tetrahedrally coordinated Al in dehydrated USY and ZSM-5 is 1.67 angstroms. Simulation of k3chi(k) for Cu+ exchanged ZSM-5 leads to an estimated distance between Cu+ and framework Al atoms of 2.79 angstroms.

The understanding of long-term landfill settlement is important for landfill design and rehabilitation. However, suitable models that can consider both the mechanical and biodecomposition mechanisms in predicting the long-term landfill settlement are generally not available. In this paper, a model based on unsaturated consolidation theory and considering the biodegradation process is introduced to simulate the landfill settlement behaviour. The details of problem formulations and the derivation of the solution for the formulated differential equation of gas pressure are presented. A step-by-step analytical procedure employing this approach for estimating settlement is proposed. The proposed model can generally model the typical features of short-term and long-term behaviour. The proposed model also yields results that are comparable with the field measurements.

Partial photoionization cross sections sigmaN(Egamma) and photoelectron angular distributions betaN(Egamma) were measured for the final ionic states He+ (N > 4) in the region between the N = 8 and N = 13 thresholds (Egamma > 78.155 eV) using the cold target recoil ion momentum spectroscopy technique (COLTRIMS). Comparison of the experimental data with two independent sets of theoretical predictions reveals disagreement for the branching ratios to the various HeN(+) states. The angular distributions just below the double ionization threshold suggest an excitation process for highly excited N states similar to the Wannier mechanism for double ionization.

This study investigated the activity of nitric oxide (NO) in the striatum (STR) for a further comprehension of the pathogenesis of Parkinson's disease (PD). Microiontophoresis was used to observe the effects of sodium nitroprusside (SNP), L-glutamic acid (GLU) and gamma-aminobutyric acid (GABA) on STR neurons' firing rates. It was observed that 77.27% (51/66) of the tested STR neurons were excited by SNP. This excitatory effect could be antagonized by the NO synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). During the microiontophoresis of GLU, the excitatory firing of STR neurons was also attenuated by addition of L-NAME while SNP application could enhance the excitation of the neurons. On the other hand, in the presence of GABA, SNP still excited the tested STR neurons. These results demonstrated that NOergic, GLUergic and GABAergic co-existed in the same STR neurons. NOergic and GLUergic were excitatory whereas GABAergic was inhibitory on the firing activity in STR neurons.

The aim of this study was to investigate the effects of urocortin (UCN) on oxidative stress and the mechanisms of urocortin on ischemia-reperfusion injury in vivo in the rat model. Thirty-six Sprague-Dawley rats were divided into 6 groups, including sham, control (normal saline solution), UCN1, UCN2, UCN3, and verapamil groups. The left anterior descending coronary artery of all rats except those in the sham group was treated with a 30-min occlusion followed by a 60-min reperfusion. Just before the occlusion, normal saline solution, UCN (5, 10, and 20 microg/kg body mass), or verapamil (1 mg/kg body mass) was administered. Heart rates, beating rhythm, and S-T segments were constantly monitored using an ECG. At the completion of the drug administration, blood samples were taken to measure the activity of superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-PX), and nitric oxide (NO) to evaluate the effects of UCN on oxidative stress. Finally, the size of infarction was measured. Arrhythmia rates were significantly lower, and the infarction size was significantly smaller (p < 0.01), in the UCN groups vs. the control group. Verapamil also significantly reduced arrhythmia rates and infarction size. The MDA activities were remarkably diminished, whereas the SOD, GSH-PX, and NO activities were significantly higher in the UCN and VER groups (p < 0.01). MDA, SOD, and NO activities were strongly correlated with UCN doses. These results suggest that UCN may play a protective role in ischemia-reperfusion injury in rat hearts against the oxidative stress by inhibiting free radicals' activities.

The newly isolated peptide, urocortin (UCN) has been found to have potent cardioprotective effects. In order to investigate the effect of UCN on L-type calcium currents (I(Ca,L)), exploring the mechanisms of UCN's cardioprotective effects, we directly measured the I(Ca,L) in the adult rat cardiac myocytes exposed to UCN using standard whole-cell patch-clamp recording technique. Our results showed that UCN exerted decreasing effects on the I(Ca,L) of the single adult rat cardiac myocytes. The current density was inhibited by about 35% after exposure of the cells to UCN (0.1 micromol L(-1)) for 10 min, from the control value of 7.19 +/- 1.44 pA/pF to 4.74 +/- 0.75 pA/pF (n = 5, P < 0.05). This I(Ca,L)-inhibiting action of UCN was concentration dependent. Moreover, no frequency dependence of UCN effects on I(Ca,L) was observed. In combination with previous reports, our results suggest that there might be a close relationship between the cardioprotective effects of UCN and L-type calcium channels.

Objective: To study influenza epidemic and analyze antigenic and genetic characterization of the predominant strains in Wuhan area in 2003., Methods: Epidemiological data and specimens from influenza patients were collected from surveillance sites weekly. Viruses were isolated from the specimens. Three H3 isolates were chosen to do antigenic analysis by hemagglutination inhibition (HI) test and their HA1 region was sequenced., Results: Totally 58 influenza viruses were isolated from 418 specimens, 57 of them were identified as H3 subtype and 1 of them was B subtype; both monthly positive rate and numbers of influenza like illness had two peaks of winter and summer, the highest peak appeared in July. The 3 new H3 isolates were antigenically different from vaccine strain A/Panama/2007/99, 14 amino acid changes have been found in HA1 domain of these 3 strains compared with A/Panama/2007/99, phylogenetic analysis also confirmed the difference in HA1 domain., Conclusions: Influenza epidemic had two peaks in Wuhan area in 2003. The activity of H3 virus was strengthened remarkably. And they are antigenically and genetically different from the vaccine strain.

Background and Purpose: The cost of health care in the last year of life is a major issue of health services research. The purposes of this study were to examine health care use and expenditure under National Health Insurance (NHI) by Taiwanese adults in the last year of life, and to compare their results with those of randomly selected survivors., Methods: A total of 9,369 adult decedent NHI beneficiaries were selected from NHI enrollee files and the Death Certificate data file for 1999. To compare expenses with those of patients without fatal illness, 10,000 randomly sampled adult beneficiaries of NHI who were alive on December 31, 1999, were selected as a survival group. NHI reimbursement and utilization information for these decedents and survivors were obtained by linking these samples to NHI claims files., Results: The total NHI expenditure in the last year of life for the 9,369 decedents was US$71.6 million. About 54.5% of all medical expenses in the last year of life were incurred in the last 3 months of life. Nephritis and cancer were the most expensive causes of death, with per capita expenses of US$15,220 and US$10,828, respectively. The average expenses for survivors increased with age, while that for decedents increased with age from the 20- to 44-year age group to the 45- to 64-year age group, then decreased for decedents aged 65 and over. The age-dependent pattern of decreasing expenses with increasing age was found in cancer deaths for all age groups and in elderly decedent groups for most causes of death., Conclusions: This study demonstrated the relatively large amount of health care resources used by Taiwanese NHI participants in their last year of life and suggests the importance of greater awareness of the implications of allocation of medical care resources for terminally ill patients by policymakers and health care providers.

Whole cell patch-clamp recordings were obtained from dissociated mouse lumbar dorsal root ganglion (DRG) neurons. Recordings were made from control neurons and neurons axotomized by transection of the corresponding spinal nerve 1-2 days prior to dissociation. Medium to large muscle and cutaneous afferent neurons were identified by retrograde transport of True Blue or Fluoro-Gold injected into the corresponding peripheral tissue. Action potentials were classified as non-inflected spikes (A(0)) and inflected spikes (A(inf)). High-frequency, low-amplitude subthreshold membrane potential oscillations were observed in 8% of control A(0) neurons, but their incidence increased to 31% in the nerve injury group. Fifty percent of axotomized muscle afferent A(0) cells displayed oscillations, while 26% of axotomized cutaneous afferents exhibited oscillations. Lower-frequency oscillations were also observed in a small fraction (4%) of A(inf) neurons on strong depolarization. Their numbers were increased after the nerve injury, but the difference was not statistically significant. The oscillations often triggered burst firing in distinct patterns of action potential activity. These results indicate that injury-induced membrane oscillations of DRG neurons, previously observed in whole DRG of rats, are present in dissociated DRG neurons of the adult mouse. Moreover, these observations indicate that both muscle and cutaneous afferents in the A(beta) size range give rise to injury-induced membrane oscillations, with muscle afferents being more prone to develop oscillations.

Ectopic spike activity, generated at low levels in intact sensory dorsal root ganglia and intensified following axotomy, is an important cause of neuropathic pain. The spikes are triggered by subthreshold membrane potential oscillations. The depolarizing phase of oscillation sinusoids is due to a phasic voltage-sensitive Na(+) conductance (gNa(+)). Here we examine the repolarizing phase for which K(+) conductance (gK(+)) is implicated. In vivo, gK(+) blockers have excitatory effects inconsistent with the elimination of oscillations. Indeed, using excised dorsal root ganglia in vitro, we found that gK(+) block does not eliminate oscillations; on the contrary, it has a variety of facilitatory effects. However, oscillations were eliminated by shifting the K(+) reversal potential so as to neutralize voltage-insensitive K(+) leak channels. Based on these data, we propose a novel oscillatory model: oscillation sinusoids are due to reciprocation between a phasically activating voltage-dependent, tetrodotoxin-sensitive Na(+) conductance and passive, voltage-independent K(+) leak. In drug-free media, voltage-sensitive K(+) channels act to suppress oscillations and increase their frequency. Numerical simulations support this model and account for the effects of gK(+) block. Oscillations in dorsal root ganglia neurones appear to be based on the simplest possible configuration of ionic conductances compatible with sustained high frequency oscillatory behaviour. The oscillatory mechanism might be exploited in the search for novel analgesic drugs.

Selection line rats congenitally high or low for autotomy in the neuroma model of neuropathic pain (HA and LA rats) were found to be correspondingly high and low in a second type of neuropathic pain, the Chung model, which employs an alternative phenotypic endpoint, tactile allodynia. It has been proposed that both phenotypes reflect ectopic hyperexcitability in axotomized primary sensory neurons. To test this hypothesis we made in vitro recordings from sensory neurons in the L4 and 5 dorsal root ganglia. Baseline excitability was similar in HA and LA rats, and axotomy caused an increase in both lines. However, in the one neuronal subclass previously linked to neuropathic pain in these models the increase was significantly greater in HA than LA rats, and only at the time when pain scores in the two lines were diverging. Heritable differences in electrical response to axotomy in a specific afferent cell type appear to be a fundamental determinant of neuropathic pain.

New features are revealed in the low-energy photoionization spectrum of Ar by critically combining high photon resolution and differential photoelectron spectroscopic techniques. Two LS-forbidden doubly excited resonances are seen in the 3p(-1)(3/2, 1/2) partial cross sections which exhibit mirroring profiles, resulting in complete cancellation in the total photoionization cross section, as was predicted by Liu and Starace [Phys. Rev. A 59, R1731 (1999)]. These results demonstrate that a new class of weakly spin-orbit induced, mirroring resonances should be observable in partial, but not in total, collisional cross sections involving atoms, molecules, and solids in general.

Primary sensory neurons with myelinated axons were examined in vitro in excised whole lumbar dorsal root ganglia (DRGs) taken from adult rats up to 9 days after tight ligation and transection of the L(5) spinal nerve (Chung model of neuropathic pain). Properties of subthreshold membrane potential oscillations, and of repetitive spike discharge, were examined. About 5% of the DRG neurons sampled in control DRGs exhibited high-frequency, subthreshold sinusoidal oscillations in their membrane potential at rest (V(r)), and an additional 4.4% developed such oscillations on depolarization. Virtually all had noninflected action potentials (A(0) neurons). Amplitude and frequency of subthreshold oscillations were voltage sensitive. A(0) neurons with oscillations at V(r) appear to constitute a population distinct from A(0) neurons that oscillate only on depolarization. Axotomy triggered a significant increase in the proportion of neurons exhibiting subthreshold oscillations both at V(r) and on depolarization. This change occurred within a narrow time window 16-24 h postoperative. Axotomy also shifted the membrane potential at which oscillation amplitude was maximal to more negative (hyperpolarized) values, and lowered oscillation frequency at any given membrane potential. Most neurons that had oscillations at V(r), or that developed them on depolarization, began to fire repetitively when further depolarized. Spikes were triggered by the depolarizing phase of oscillatory sinusoids. Neurons that did not develop subthreshold oscillations never discharged repetitively and rarely fired more than a single spike or a short burst, on step depolarization. The most prominent spike waveform parameters distinguishing neurons capable of generating subthreshold oscillations, and hence repetitive firing, was their brief postspike afterhyperpolarization (AHP) and their low single-spike threshold. Neurons that oscillated at V(r) tended to have a more prolonged spike, with slower rise- and fall-time kinetics, and lower spike threshold, than cells that oscillated only on depolarization. The main effects of axotomy were to increase spike duration, slow rise- and fall-time kinetics, and reduce single-spike threshold. Tactile allodynia following spinal nerve injury is thought to result from central amplification ("central sensitization") of afferent signals entering the spinal cord from residual intact afferents. The central sensitization, in turn, is thought to be triggered and maintained in the Chung model by ectopic firing originating in the axotomized afferent neurons. Axotomy by spinal nerve injury enhances subthreshold membrane potential oscillations in DRG neurons, augments ectopic discharge, and hence precipitates neuropathic pain.

We examined the relation between ectopic afferent firing and tactile allodynia in the Chung model of neuropathic pain. Transection of the L5 spinal nerve in rats triggered a sharp, four- to six-fold increase in the spontaneous ectopic discharge recorded in vivo in sensory axons in the ipsilateral L5 dorsal root (DR). The increase, which was not yet apparent 16 h postoperatively, was complete by 24 h. This indicates rapid modification of the electrical properties of the neurons. Only A-neurons, primarily rapidly conducting A-neurons, contributed to the discharge. No spontaneously active C-neurons were encountered. Tactile allodynia in hindlimb skin emerged during precisely the same time window after spinal nerve section as the ectopia, suggesting that ectopic activity in injured myelinated afferents can trigger central sensitization, the mechanism believed to be responsible for tactile allodynia in the Chung model. Most of the spike activity originated in the somata of axotomized DRG neurons; the spinal nerve end neuroma accounted for only a quarter of the overall ectopic barrage. Intracellular recordings from afferent neuron somata in excised DRGs in vitro revealed changes in excitability that closely paralleled those seen in the DR axon recordings in vivo. Corresponding changes in biophysical characteristics of the axotomized neurons were catalogued. Axotomy carried out at a distance from the DRG, in the mid-portion of the sciatic nerve, also triggered increased afferent excitability. However, this increase occurred at a later time following axotomy, and the relative contribution of DRG neuronal somata, as opposed to neuroma endings, was smaller. Axotomy triggers a wide variety of changes in the neurochemistry and physiology of primary afferent neurons. Investigators studying DRG neurons in culture need to be alert to the rapidity with which axotomy, an inevitable consequence of DRG excision and dissociation, alters key properties of these neurons. Our identification of a specific population of neurons whose firing properties change suddenly and synchronously following axotomy, and whose activity is associated with tactile allodynia, provides a powerful vehicle for defining the specific cascade of cellular and molecular events that underlie neuropathic pain.

Spike activity in dorsal root ganglion (DRG) neurons depolarizes passive neighbors that share the same ganglion. We asked whether age or prior nerve injury affect this 'cross-depolarization' signal. Intracellular recordings made from excised DRGs in vitro revealed that the prevalence and duration of cross-depolarization were no greater in adult than in young rats, and that its amplitude was significantly smaller in adults. The amplitude of cross-depolarization was not affected by nerve injury. The decrease in membrane input resistance (R(in)) observed during cross-depolarization was less than that expected from equivalent depolarization alone. This affirms prior evidence that the neural process underlying cross-depolarization causes a net increase in R(in).

This study was designed to investigate the effect of nicotine on spontaneous and amphetamine (AMP)-induced motor activity in rats with or without tolerance to nicotine. Tolerance were induced by treating the rats with nicotine (0.3 mg/kg, s.c.) 2 hr before receiving challenge doses. Motor activity including locomotion and stereotypy was monitored automatically by videocamera every 15 min for 90 min. The results indicated that: (1) Nicotine increased spontaneous locomotion at 0.15 or 0.3 mg/kg (s.c.) in naive rats and at 0.6 mg/kg in tolerant rats. Nicotine also slightly affected AMP-induced locomotion at 0.15, 0.3 or 0.6 mg/kg in both naive and tolerant rats, and (2) Nicotine increased spontaneous stereotypy at 0.3 or 0.6 mg/kg in naive rats only and showed no effect on AMP-induced stereotypy in either naive or tolerant rats. Comparing the results of spontaneous motor activity between naive and tolerant rats, it revealed behavioral desensitization in locomotion at low doses (0.15 or 0.3 mg/kg) and hyperlocomotion at higher dose (0.6 mg/kg), and revealed desensitization in stereotypy at 0.3 or 0.6 mg/kg. Moreover, nicotine had temporary (at 0-15 min interval) attenuating effect on AMP-induced locomotion in naive rats but showed a potentiating effect on AMP-induced locomotion in tolerant rats. The present results indicated that acute tolerance modified the action of nicotine in both spontaneous and AMP-induced locomotion, while stereotypy was changed only in the spontaneous one but not in the AMP-induced one. In other words, acute tolerance modified the effect of nicotine on locomotion-related dopaminergic system, and it affected the stereotypy-related dopaminergic system only in the spontaneous one but not in the AMP-induced one.

Recent studies have shown that grass genomes have very similar gene compositions and regions of conserved gene order, as exemplified by collinear genetic maps of DNA markers. We have begun the detailed study of sequence organization in large (100-500 kb) segments of the nuclear genomes of maize, sorghum and rice. Our results indicate collinearity of genes in the regions homoeologous to the maize adh1 and sh2-a1 genes. Comparable genes were found to be physically closer to each other in grasses with small genomes (rice and sorghum) than they are in maize. In several instances, we have found evidence of tandem and 'distantly tandem' duplications of segments containing maize and sorghum genes. These duplications complicate characterizations of microcollinearity and could also interfere with some map-based approaches to gene isolation.

A cluster of five alpha-zein subfamily 4 (alpha-zein SF4) genes are present in a 56 kb region of the maize W22 genome. Two types of alpha-zein SF4 genes are in the cluster. One of the genes, termed a type 1 (T1) alpha-zein SF4 gene, contains no early in-frame stop codons. Four of the genes, termed type 2 (T2) alpha-zein SF4 genes, contain one or two early in-frame stop codons. The base sequence of the T1 alpha-zein SF4 gene is similar (> 90%) to the sequences of any of the four T2 alpha-zein SF4 genes. However, their sequences differ markedly at distances greater than -875 bp upstream from the translation initiation codon of the alpha-zein coding region. This region of dissimilarity is well inside the functional 5'-flanking region for the genes since a 1.8 kb transcript is initiated in this region and the sequences of the T2 alpha-zein SF4 genes are similar in this region. Two sizes of mRNA transcripts, 1.8 kb and 0.9 kb, were detected in a gene specific manner for 4 of the 5 genes in this alpha-zein SF4 gene cluster. One of the T2 alpha-zein SF4 genes had only the 0.9 kb transcript. The RNA level for the 0.9 kb transcript of the T1 alpha-zein SF4 gene was 5- to 10-fold higher than the transcript levels of any of the T2 alpha-zein SF4 genes. In each case, the amount of the 0.9 kb transcript detected was at least 5-fold higher than the amount of the 1.8 kb transcript. A cDNA clone with a sequence identical to a T2 alpha-zein SF4 gene was isolated, providing the first direct evidence for the transcription of T2 alpha-zein genes containing early in-frame stop codon(s) in maize endosperm.

Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/l 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical beta-glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.

Five genes of the alpha-zein subfamily four (SF4) are located in a 56 kb genomic region of the maize inbred line W22. Their nucleotide and deduced amino acid sequences have been determined. The sequences define two types of alpha-zein SF4 genes-type 1 (T1) and type 2 (T2). The single T1 alpha-zein SF4 gene codes for an alpha-zein protein with a M(r) of about 22,000. This is the first alpha-zein SF4 gene sequenced that contains no early in-frame stop codons in its coding sequence. The four T2 alpha-zein SF4 genes in this cluster contain one or two early in-frame stop codons. In addition, our T1 and T2 genes differ markedly in the base sequences of their distal 5' non-translated flanking regions. The nucleotide and the deduced amino acid sequences of these two types of alpha-zein SF4 genes are similar (greater than 90%) to one another and to all known alpha-zein SF4 genes and cDNAs. Of the known W22 alpha-zein SF4 genes, only one in six does not contain an early in-frame stop codon. If the number of alpha-zein SF4 genes is 15-20, then we estimate that only about 4 of the W22 alpha-zein SF4 genes are without in-frame early stop codons.

It is well recognized that meniscal tear in the avascular portion is difficult to heal. The purpose of this study is to evaluate the ability of periosteum and fibrin adhesive system (FAS) in promoting the meniscal repair in the avascular zone. Full-thickness defects of 1 x 3 mm were created in the avascular portion of lateral menisci in adult mongrels. The control menisci were left without any treatment. The experimental defects were either repaired with autogenous periosteum or the combination of periosteum and FAS. Histologic and ultrastructural evaluation of the reparative tissue were compared at 4, 8, and 16 weeks postoperatively. The reparative tissue in the control group remained fibrous even at week 16, whereas that in the grafted and graft/FAS groups resembled normal adjacent fibrocartilage. Moreover, the regenerated chondrocytes in the graft/FAS group appeared more mature than those in the grafted group. Our results suggest that periosteum is a potential source of primitive cells from which chondrocytes could be differentiated and developed during the reparative process of meniscal fibrocartilage. As to FAS, it not only acted as a hemostatic agent and a fixant, but also could indirectly accelerate the chondrogenesis of periosteum in the meniscal defects. It is sufficient to conclude that the combination of periosteum and FAS in promoting meniscal defects or severe tears in the avascular portion bears its potential in future clinical application.

The genes encoding the alpha-zein proteins of maize constitute a large multigene family of some 75 genes. This multigene family can be divided into four subfamilies based on the nucleotide sequences of their genes and the deduced amino acid sequences of their proteins. We describe for the first time evidence of a clustering of five alpha-zein subfamily 4 (SF4) genes that are members of one of the major alpha-zein subfamilies in a 56 kb region of the genome of the maize inbred line W22. None of the other three known alpha-zein gene subfamilies (SF1, SF2, or SF3) are present in this cluster. The genomic region was reconstructed using restriction endonuclease maps to identify and align three overlapping cosmid clones isolated from a genomic library. The alpha-zein genes are not evenly spaced; the minimum distance between genes is 3.5 kb; the maximum is 13 kb. All the alpha-zein genes in the cluster have the same transcriptional orientation. The location and sequences of some of the repetitive DNA elements in this gene cluster were determined. We estimate that there are a minimum of eight repetitive DNA elements in this region. The sequences of the repetitive elements (not functionally defined) are located between or among the alpha-zein genes. The regions containing two of these repetitive elements (Rep1 and Rep4) have been sequenced; they are about 15 kb apart in the genome. These repetitive elements have similar sequences for about 300 bp out of the 400 bp compared. The regions of sequence similarity, however, are in reverse orientation to one another.(ABSTRACT TRUNCATED AT 250 WORDS)

Eight monkeys, after complete spinal cord transection at the midthoracic level, were placed in a supine position and an exercise program was given to one hindlimb but not the other. Within 3 days, the exercised limb showed more muscle tone and more active cutaneous and proprioceptive reflexes. The difference increased progressively throughout the observation period of 11 to 36 days. These results suggest an important role of afferent influence on the recovery of spinal shock. The exercise program probably protects the neuromuscular apparatus from deterioration and thus maximize the recovery.

Transection of the spinal cord of the cat at a thoracic or lumbar level results, after as short a period as 12 days, in a preparation with such altered excitability that repeated natural stimulation of the dermatome just caudal to the transection site will induce, in as short a time as 3 days, seizure discharges. The trigger zone for the seizure spreads to caudal dermatomes when these caudal regions are repeatedly stimulated. The 'typical' T4-T7 seizure is a scratch reflex followed by the tonic-clonic seizure lasting for 20-30 s and ending with a scratch afterdischarge lasting for several minutes. Lower thoracic and upper lumbar seizures consist of tonic-clonic co-contractions of the muscles of the hindlegs, followed by rhythmical stepping movements lasting less than 1 min. Partial dorsal rhizotomy or local Cobalt application to the spinal cord may reduce the threshold for induction of seizure by natural stimulation and local Penicillin application to spinal cord induces seizure discharges similar to those induced by natural stimulation. Retransection of the spinal cord caudally, with elimination of the primary trigger zone, does not abolish the secondarily acquired triggers. The findings suggest that spinal circuits possess the ability to acquire new neuronal patterns of discharge and to transfer them to other more caudal segments.

Learning and interhemispheric transfer of visual flux, pattern and form discriminations were studied in the cat after selected exposure of one suprasylvian cortex to 6-hydroxydopamine (6-OHDA). Biochemical assay using High Performance Liquid Chromatography (HPLC) two weeks after 6-OHDA revealed no discernible norepinephrine or dopamine in the treated cortex, but elevated concentrations of these transmitters in the cortex of the opposite hemisphere. Visual discriminations learned before treatment with 6-OHDA were retained at a high level using either the eye on the side of chemical lesion or the eye on the untreated side. An asymmetric deficit in learning new form discriminations was present, however, when the eye on the untreated side was used, in contrast to normal learning using the eye on the side of the hemisphere with depleted adrenergic nerve supply. Once learning was achieved using the lesioned hemisphere transfer of the engram was found to the untreated hemisphere. Thus, the unlesioned hemisphere was unable to learn normally using direct retinal input from the ipsilateral eye, but showed good capacity for learning using indirect visual input from the contralateral eye. This suggests a powerful influence of the callosum on the learning abilities of the two hemispheres, an influence proved by sectioning the callosum. Callosotomy resulted in a reversal of the discriminative capacities seen after 6-OHDA, i.e. the lesioned hemisphere was defective relative to the unlesioned hemisphere.

Thirty specimens from 30 patients with urologic and male genital system tumors were cultured. Eighteen (60%) showed initial cell growth. Four (13%) were successful in primary culture. In a renal cell carcinoma and a bladder transitional cell carcinoma, the subcultures were successful after the primary culture. In eight months, having transferred the former for twelve generations and the latter for twenty-seven generations, these two cell lines are established. The morphological characters of the cultured cells, eradication of fibroblasts, conditions of culture and prevention of contamination are discussed.

The role of dorsal column (DC)-medial lemniscus and spinocervicothalamic systems on tactile reactions, placing and tactile localization (turning to localize a point touched on the body) was studied in cats with spinal and bulbar lesions. The cats with vision occluded were trained preoperately on horizontal bars for tactile localization. The bulbar lesions of cervicothalamic tract (CTT), which anatomically overlaps with gracile nuclear efferent projection, resulted in permanent loss of tactile placing and localization in contralateral hindlimb. Section of dorsal half of lateral funiculus at C3, which interrupts afferents to lateral cervical nucleus but also involves descending motor pathways, gave loss of tactile placing without impairment of tactile localization. However, combined lesions of both systems abolished tactile placing and localization permanently. Results suggest that tactile placing and localization persists after CTT lesion alone; temporarily loss after DC section but permanently abolished after destruction of both systems. It. is also noted that cats after lesions of both systems without involving motor pathways could walk on bars without vision. The deficits following combined lesions are suggested to be primarily sensory in nature.

In monkeys, cerebellar vermal cortical or fastigial nuclear lesion resulted in no significant postural asymmetry. Combined decerebration (but not bulbar pyramid section) and unilateral vermal cortical or fastigial nuclear lesion gave marked ipsilateral hyperextension and contralateral hyperflexion of limbs. Unilateral eighth nerve section resulted in only ipsilateral head tilt but combined unilateral eighth nerve section and decerebration or bilateral or contralateral cerebral cortical areas 4 and 6 lesion gave also ipsilateral flexion and contralateral extension of limbs. Cervical deafferentation or postbrachial spinal cord transection did not alter these results. This study indicates a powerful cerebral influence on postural effects of cerebellar vermal zonal lesion or eighth nerve section in monkeys. Possible mechanisms mediating these effects in monkeys as compared to cats were discussed.

We examined the effect of complement depletion and leukocyte depletion on an experimental model of membranous nephropathy. Nephrosis was induced in 200-gm male Sprague-Dawley rats by priming with cationic bovine gamma-globulin in adjuvant on day 1 followed by intravenous challenge with antigen starting on day 10. No naive control rats had immunofluorescent deposits in glomeruli; urine protein was less than 10 mg/24 hour and glomerular thromboxane synthesis was 658 +/- 64 ng/mg glomerular dry weight. In contrast, all rats primed and challenged with cationic bovine gamma-globulin had intense granular capillary wall deposits of rats IgG, bovine gamma-globulin, C3 and C5; severe proteinuria (183 +/- 24 mg/24 hour) was observed, associated with a 3-fold increase in glomerular thromboxane (2,393 +/- 574 ng/mg, all p less than 0.01 versus naive controls). In some rats, administration of cobra venom factor and antiserum to rat C3, starting on day 8 was used to deplete complement; hemolytic C3 and C5 were less than 2% of normal at sacrifice. These rats had IgG and bovine gamma-globulin deposits, whereas they lacked glomerular C3 or C5. Proteinuria (209 +/- 28 mg/24 hour) and glomerular thromboxane (2,087 +/- 394 ng/mg) were markedly increased compared with control, but no different from normocomplementemic rats primed and challenged with cationic bovine gamma-globulin. In other rats, depletion of leukocytes was achieved by 1,000 R x-irradiation on day 7; at sacrifice, irradiated rats had 1,270 +/- 462 wbc/microliter versus 10,375 +/- 1,652 in nephrotic rats given no other treatment, with unaltered differentials. These rats had glomerular deposits of rat IgG, bovine gamma-globulin, C3 and C5 indistinguishable from nephrotic rats with normal leukocyte counts in intensity and distribution. Proteinuria (202 +/- 30) and glomerular thromboxane (2,358 +/- 189 ng/mg) were markedly elevated compared with naive controls, but were not different from the normocomplementemic or complement-depleted groups primed and challenged with antigen. An additional control group included rats primed with ovalbumin on day 1, irradiated with 1,000 R on day 7, and challenged with cationic bovine gamma-globulin starting on day 10. This group had granular capillary wall deposits of bovine gamma-globulin, but not deposits of IgG, C3, or C5; urine protein excretion (less than 10 mg/24 hours) and glomerular thromboxane synthesis (613 +/- 90) were not different from naive controls. Glomerular prostaglandin E2 synthesis did not differ among the five groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult cats were trained to insert a forelimb into horizontally mounted transparent cylinders and then either depress a barrier or pull a sliding tray against resistance in order to receive food reward. Unilateral lesions of both bulbar pyramid and red nucleus resulted in severe neurological deficits. However, the instrumental tasks were soon accomplished at preoperative levels of rapidity and strength, although movements of the digits and wrist were reduced and adjustive movements occurred chiefly at more proximal joints. These results support our hypothesis that conditioned and unconditioned behaviors are mediated through different mechanisms.

Cats were trained to reach with their fore limbs for food placed in horizontally mounted cylinders of various diameters. The chain instrumental reflex (entering cylinder, reaching for, grasping and bringing food toward the mouth) survived cerebellar paravermal cortical ablation or interposite-dentate nuclear lesion, with some exaggerated flexor or extensor responses respectively. Movements improved with time. Cats were then subjected to ablation of cerebral sensory cortex, SI and SII. SI or compined SI and SII lesion resulted in an initial period of decompensation of cerebellar symptoms. Sensory loss was also noted for 30-40 days after SI lesion and throughout the observation period up to 53 days after combined SI and SII lesion. The conditioned responses were soon accomplished at preoperative level. Furthermore, visual occlusion did not change the performance in conditioning situation. The result indicates that the compensation of cerebellar symptoms does not depend on an intact cerebral sensory cortex. It also suggests that functional accomplishment can be obtained through training despite neurological deficits following certain brain damage.

The hopping response reappears in the hind limb of the cat after complete spinal cord transection at postoperative day means = 11.6. This response is hypermetric and slow compared with that in the normal cat and only forward, medial, and lateral hopping can be elicited. Anatomical and physiological changes in the spinal cord which may account for the reappearance of the hopping response are discussed.

Cats after unilateral bulbar pyramid section showed permanent loss of tactile placing in the contralateral limbs. However, the cats could be trained to place the forelimb following light touch in a conditioning situation with food as a reward. This conditioned response survived subsequent unilateral red nucleus and cerebellar interposed nucleus lesions. Dorsal quadrant spinal cord lesion at the cervical level produced initial loss of the conditioned tactile placing but is recovered without retraining. Since those lesions interrupted subcortical afferent and efferent pathways essential to reflex tactile placing, these results indicate that reflex and conditioned responses involve different pathways and mechanisms. Training recruits or activates additional pathways for conditioned responses rather than strengthening those subserving the reflex responses.

Digital processing that increases resolution by spatial deconvolution and histogram-based amplitude mapping has been used to improve ultrasonic abdominal image quality. The processing was applied to pulse-echo ultrasound data obtained from clinical imaging instrumentation modified to permit digital recording of signals in either RF or video forms for subsequent off-line analysis. Spatial deconvolution was accomplished both along the axis and across the width of the ultrasonic beam. Axial deconvolution was carried out on RF data with a point spread function derived from the echo of a wire target. Lateral deconvolution was performed on the video envelope placed in a matrix by an inverse filter with parameters that adjust themselves to the spatial frequency content of the image being processed. Resultant image amplitudes were mapped into a hyperbolic distribution to increase image contrast for improved demonstration of low amplitudes. The combination of processing produced resolution improvements to show boundaries more sharply and contrast changes to demonstrate more detail in the images.