Contaminating viral sequences in high-throughput sequencing viromics: a linkage study of 700 sequencing libraries.

OBJECTIVES: Sample preparation for high-throughput sequencing (HTS) includes treatment with various laboratory components, potentially carrying viral nucleic acids, the extent of which has not been thoroughly investigated. Our aim was to systematically examine a diverse repertoire of laboratory components used to prepare samples for HTS in order to identify contaminating viral sequences. METHODS: A total of 322 samples of mainly human origin were analysed using eight protocols, applying a wide variety of laboratory components. Several samples (60% of human specimens) were processed using different protocols. In total, 712 sequencing libraries were investigated for viral sequence contamination. RESULTS: Among sequences showing similarity to viruses, 493 were significantly associated with the use of laboratory components. Each of these viral sequences had sporadic appearance, only being identified in a subset of the samples treated with the linked laboratory component, and some were not identified in the non-template control samples. Remarkably, more than 65% of all viral sequences identified were within viral clusters linked to the use of laboratory components. CONCLUSIONS: We show that high prevalence of contaminating viral sequences can be expected in HTS-based virome data and provide an extensive list of novel contaminating viral sequences that can be used for evaluation of viral findings in future virome and metagenome studies. Moreover, we show that detection can be problematic due to stochastic appearance and limited non-template controls. Although the exact origin of these viral sequences requires further research, our results support laboratory-component-linked viral sequence contamination of both biological and synthetic origin.

Hip- and trunk-muscle activation patterns during perturbed gait.

CONTEXT: Selected muscles in the kinetic chain may help explain the body's ability to avert injury during unexpected perturbation. OBJECTIVE: To determine the activation of the ipsilateral rectus femoris (RF), gluteus maximus (MA), gluteus medius (ME), and contralateral external obliques (EO) during normal and perturbed gait. DESIGN: Single-factor, repeated measures. SETTING: University research laboratory. PARTICIPANTS: 32 physically active, college-age subjects. INTERVENTION: Subjects walked a total of 20 trials the length of a 6.1-m custom runway capable of releasing either side into 30° of unexpected inversion. During 5 trials, the platform released into inversion. MAIN OUTCOME MEASURES: Average, peak, and time to peak EMG were analyzed across the 4 muscles, and comparisons were made between the walking trials and perturbed trials. RESULTS: Significantly higher average and peak muscle activity were noted for the perturbed condition for RF, MA, and EO. Time to peak muscle activity was faster during the perturbed condition for the EO. CONCLUSION: Rapid contractions of selected postural muscles in the kinetic chain help explain the body's reaction to unexpected perturbation.

Autosomal SNP typing of forensic samples with the GenPlex™ HID System: results of a collaborative study.

The GenPlex™ HID System (Applied Biosystems - AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex™ HID System using 250-500pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex™ HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex™ HID System with the most commonly used STR kits, 500pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler™ kit (AB), GenPlex™ HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex™ HID showed a very low mean mach probability, while all STR kits except MiniFiler™ had very limited discriminatory power.

A task failure has no effect on the electromechanical delay of the peroneus longus.

OBJECTIVE: Ankle inversion injuries represent the most common trauma sustained by athletes. Muscle fatigue from activity may contribute to a delay in the response of the ankle proprioceptors and dynamic restraints during unexpected inversion. The purpose of this investigation was to determine if the electromechanical delay (EMD) of the peroneus longus is influenced by a task failure exercise. SUBJECTS: Sixteen subjects (age 20 +/- 1.1 y; mass 71.6 +/- 12.5 kg; height 173.0 +/- 8.7 cm; 9 male, 1 female) with no lower extremity injuries reported for data collection. MEASUREMENTS: Data were collected from each subject's dominant leg using surface electromyography (EMG). Electrodes were applied over the peroneus longus (PL) using a standard protocol. A stimulating electrode was applied to the common peroneal nerve. Subjects were placed in a monopedal stance on a force platform. A low amplitude, short duration stimulus was applied to the common peroneal nerve. The EMG was used to determine timing of the M wave and the force platform was used to determine the onset of foot pronation. Once 6 trials were recorded, subjects completed 2 sets of an isotonic activity that isolated the peroneals. The task was completed to failure for each set. Immediately following the task failure exercise, subjects returned to the force platform for 6 additional trials recorded as before. Analysis of data was performed by determining the onset of the M wave as the beginning of positive EMG activity following the end of the imposed stimulus response. This point was superimposed on the force platform curve and the point at which a 10 N.m force change occurred was used to calculate the EMD (time difference between the force platform indicator and the M wave indicator). RESULTS: Average EMD prior to the task failure exercise was 13.35 +/- 3.47 ms. Following the task failure exercise, the average EMD was 12.67 +/- 3.86 ms. A paired samples t test revealed no significant differences with regard to EMD between pre- and post-task failure exercise for the PL (p = 0.448). CONCLUSION: We concluded that the task failure exercise did not affect the electromechanical delay of the PL.

Capsaicin regulates voltage-dependent sodium channels by altering lipid bilayer elasticity.

At submicromolar concentrations, capsaicin specifically activates the TRPV1 receptor involved in nociception. At micro- to millimolar concentrations, commonly used in clinical and in vitro studies, capsaicin also modulates the function of a large number of seemingly unrelated membrane proteins, many of which are similarly modulated by the capsaicin antagonist capsazepine. The mechanism(s) underlying this widespread regulation of protein function are not understood. We investigated whether capsaicin could regulate membrane protein function by changing the elasticity of the host lipid bilayer. This was done by studying capsaicin's effects on lipid bilayer stiffness, measured using gramicidin A (gA) channels as molecular force-transducers, and on voltage-dependent sodium channels (VDSC) known to be regulated by bilayer elasticity. Capsaicin and capsazepine (10-100 microM) increase gA channel appearance rate and lifetime without measurably altering bilayer thickness or channel conductance, meaning that the changes in bilayer elasticity are sufficient to alter the conformation of an embedded protein. Capsaicin and capsazepine promote VDSC inactivation, similar to other amphiphiles that decrease bilayer stiffness, producing use-dependent current inhibition. For capsaicin, the quantitative relation between the decrease in bilayer stiffness and the hyperpolarizing shift in inactivation conforms to that previously found for other amphiphiles. Capsaicin's effects on gA channels and VDSC are similar to those of Triton X-100, although these amphiphiles promote opposite lipid monolayer curvature. We conclude that capsaicin can regulate VDSC function by altering bilayer elasticity. This mechanism may underlie the promiscuous regulation of membrane protein function by capsaicin and capsazepine-and by amphiphilic drugs generally.

Effects of a task failure exercise on the peroneus longus and brevis during perturbed gait.

Ankle inversion injuries represent the most common trauma sustained by athletes. Muscle fatigue from activity may contribute to a delay in the response of the ankle proprioceptors and dynamic restraints during unexpected inversion. The purpose of this investigation was to determine changes in peroneal average EMG, peak EMG, and time to peak EMG following a task failure exercise. Thirty-two subjects (age 20+/-1.43 yrs; 21 male, 11 female) with no lower extremity injuries reported for data collection. Data were collected from each subject's dominant leg using surface electromyography (EMG). EMG electrodes were applied over the peroneus longus (PL) and brevis (PB) using a standard protocol Subjects walked at a fixed pace on a 6.1 m runway with one section that could be unexpectedly dropped into 30 degrees of inversion upon foot contact. Trials with perturbed and unperturbed gait were randomized to reduce prediction of the unexpected inversion. Once 3 trials of perturbed gait were recorded, subjects completed an isotonic activity that isolated the peroneals. The task was completed to failure. Immediately following the task failure exercise, subjects walked on the perturbation runway once again until 3 trials of perturbed gait were recorded. Analysis revealed no significant differences with regard to average muscle activity between pre- and post-task failure exercise for the PL (F1,31 = 0.133; p = 0.718) or for the PB (F1,31 = 0.795; p = 0.380). There was also no significant difference in peak muscle activity pre- to post-task failure for the PL (F1,31 = 0.032; p = 0.859) or the PB (F1,31 = 0.156; p = 0.695). Finally, there was no significant difference in time-to-peak muscle activity pre- to post-task failure for the PL (F1,31 = 0.830; p = 0.369) or the PB (F1,31 = 1.037; p = 0.316). We concluded that the task failure exercise did not contribute to changes in peroneal activity during perturbed gait. These results indicate that peroneal fatigue does not play a significant role in the incidence of inversion ankle sprains.

Hand movements in laparoscopic suturing: a simple vector analysis.

BACKGROUND: Laparoscopic suturing is a complex task that is vital to the performance of many advanced laparoscopic procedures. Mastery can be difficult and problematic for surgical trainees. METHODS: We present a description of hand movements in laparoscopic suturing. Complex maneuvers are simplified into linear motions using vectors. The analysis is intended to be a tool for training in the art of laparoscopic surgery. RESULTS: Linear hand movements in the x and y axes produce opposite motions at the instrument tip. Position along the z axis influences the extent of hand movement relative to the instrument tip. Rotational movements of the hand produce an equal rotation of the instrument tip. Revolution is a complex motion that combines movements in x and y axes. Vector analysis reveals that the arc of revolution must be reversed to produce the desired needle motion. CONCLUSIONS: A conceptual understanding of hand-movement vectors facilitates the efficient mastery of the complex skills required for laparoscopic suturing.

EMG analysis of peroneal and tibialis anterior muscle activity prior to foot contact during functional activities.

The purpose of this investigation was to determine the pre-activity of the tibialis anterior (TA), peroneus longus (PL), and peroneus brevis (PB) prior to foot contact during three conditions. Twenty-six subjects (age 22 +/- 2 yrs; 15 male, 11 female) with no lower extremity injuries reported for data collection. Data were collected from each subject's dominant leg using surface electromyography (EMG). EMG electrodes were applied over the test muscles using a standard protocol. A heel-toe strike transducer was affixed to the bottom of the subject's shoe. The subject completed two randomized trials of walking on a treadmill (5.6 kph), jogging on a treadmill (9.3 kph) and drop landing from a 38 cm box. Isometric reference positions (IRPs) were recorded for the TA, PL, and PB. Muscle data were normalized to IRPs and the average processed EMG for the 200 ms prior to heel strike during walking and jogging and prior to toe strike when dropping from the box was used for analysis. A one-way repeated measures MANOVA was used to detect differences in pre-activity of the muscles between the three conditions. Univariate tests were used to determine differences for each muscle and Tukey's was applied post hoc to determine individual effect differences. The MANOVA revealed significant differences among the three conditions (F2.50 = 10.770; P < .0005). Average TA activity was significantly higher during jogging (Tukey's; P < .0005). Significant differences existed between each condition for the TA. Average PL and PB activity was significantly higher when drop landing (Tukey's; P < .0005). There was no significant difference between walking and jogging for the PL and PB. The amount of muscle pre-activity occurring before heel or toe strike provides useful information for the examination of reaction times to unexpected inversion during dynamic activities.

Nociceptin is a potent inhibitor of N-type Ca(2+) channels in rat sympathetic ganglion neurons.

Nociceptin, an endogenous agonist of the opioid receptor-like(1) (ORL(1)) receptor, is implicated in a wide range of physiological functions including cardiovascular control. However, the effect of nociceptin on peripheral sympathetic ganglion neurons has not been studied. Whole-cell voltage clamp was used to study Ca(2+) currents on freshly dissociated sympathetic superior cervical ganglion neurons from juvenile rats. Nociceptin (1 microM) caused a fast inhibition of the peak currents by 69+/-3% in all neurons. Strong positive prepulses counteracted the inhibition of the peak current by 64% and no effect of nociceptin was observed when the cells were pre-incubated with Pertussis toxin. The inhibition was reversible and dose-dependent with an EC(50) of 508+/-50 pM. Blockade of N-type channels by 1 microM omega-conotoxin GVIA reduced the peak currents by 83+/-1% and abolished the action of nociceptin. Naloxone could not prevent the inhibition by nociceptin and [D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin (DAMGO) only depressed a small proportion of the current in 1/7 neurons. These data suggests that nociceptin inhibits transmitter release from sympathetic neurons by a selective blockade of N-type channels, which may be of importance for its depressive effect on the cardiovascular system.

Correlation between N-acetylaspartate levels and histopathologic changes in cortical infarcts of mice after middle cerebral artery occlusion.

The aim of the present study was to evaluate the use of the endogenous neuronal compound N-acetylaspartate (NAA) as a marker of neuronal damage after focal cerebral ischemia in mice. After occlusion of the middle cerebral artery (MCAO) the ischemic cortex was sampled, guided by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and the NAA concentration was measured by high-pressure liquid chromatography (HPLC). Conventional histology and immunohistological methods using antibodies against neuron-specific enolase (NSE), neurofilaments (NF), synaptophysin, glial fibrillary acidic protein (GFAP), and carbodiamide-linked NAA and N-acetylaspartylglutamate (NAAG). The level of NAA rapidly declined to 50% and 20% of control levels in infarcted tissue after 6 hours and 24 hours, respectively. No further decrease was observed during the observation period of 1 week. Within the first 6 hours the number of normal-appearing neurons in the infarcted cortical tissue decreased to 70% of control, of which the majority were eosinophilic. After 24 hours almost no normal-appearing neurons were seen. The number of eosinophilic neurons decreased steadily to virtually zero after 7 days. The number of immunopositive cells in the NSE, NF, and synaptophysin staining within the infarct was progressively reduced, and after 3 to 7 days the immunoreactions were confined to discrete granulomatous structures in the center of the infarct, which otherwise was infested with macrophages. This granulomatous material also stained positive for NAA. The number of cells with positive GFAP immunoreactions progressively increased in the circumference of the infarct. They also showed increased immunoreaction against NAA and NSE. The study shows that the level of NAA 7 days after ischemia does not decline to zero but remains at 10% to 20% of control values. The fact NAA is trapped in cell debris and NAA immunoreactivity is observed in the peri-infarct areas restricts its use as a marker of neuronal density.

Octamer transcription factor-1 enhances hepatic nuclear factor-1alpha-mediated activation of the human UDP glucuronosyltransferase 2B7 promoter.

The human UDP glucuronosyltransferase, UGT2B7, is expressed in the liver and gastrointestinal tract, where it catalyzes the glucuronidation of steroids and bile acids. In this study, the UGT2B7 gene was isolated and its proximal promoter was analyzed. The UGT2B7 gene consists of 6 exons and extends over 16 kilobases (kb). It does not contain a canonical TATA box but has a region (-2 to -40) adjacent to the transcription start site that binds nuclear proteins. This region contains a consensus hepatic nuclear factor-1alpha (HNF1alpha)-binding site and an overlapping AT-rich segment. Varying lengths of the UGT2B7 gene promoter, with and without these sites, were fused to the firefly luciferase reporter gene and transfected into HepG2 cells. UGT2B7 promoter activity with the HNF1/AT-rich element was stimulated by cotransfection with HNF1alpha. Additional activation was observed when HNF1alpha and octamer transcription factor-1 (Oct-1) were cotransfected simultaneously. However, Oct-1 alone did not stimulate promoter activity and did not bind to the promoter in the absence of HNF1alpha. Deletion of the HNF1/AT-rich region, or mutations in this region, abolished UGT2B7 gene promoter activity and prevented HNF1alpha-mediated increases in promoter activity. The presence of HNF1alpha and octamer transcription factor-1 (Oct-1) in the protein complex that bound to the HNF1/AT-rich region was demonstrated by gel shift analyses with antibodies specific to HNF1alpha and Oct-1 protein. These results strongly suggest that the liver-enriched factor HNF1alpha binds to, and activates, the UGT2B7 gene promoter and that the ubiquitous transcription factor, Oct-1, enhances this activation by directly interacting with HNF1alpha. This interaction between HNF1alpha and Oct-1 may fine-tune UGT2B7 expression.

Tissue specific differences in the regulation of the UDP glucuronosyltransferase 2B17 gene promoter.

The human UDP glucuronosyltransferase UGT2B17, glucuronidates androgens and is expressed in the liver and the prostate. Although evidence suggests that variations in UGT2B17 expression between tissues may be a critical determinant of androgen response, the factors that regulate UGT2B17 expression in the liver and prostate are unknown. In this study, we have isolated a 596 bp promoter of the UGT2B17 gene and studied its regulation in the liver cell line, HepG2 and the prostate cell line, LNCaP. The transcription start site of UGT2B17 was mapped and proteins that bound to the proximal promoter were detected by DNase1 footprint analysis. A region (-40 to -52 bp) which resembled a hepatocyte nuclear factor 1 (HNF1) binding site bound proteins in nuclear extracts from HepG2 cells, but did not bind proteins from LNCaP nuclear extracts. In HepG2 cells, HNF1alpha bound to this region and activated the UGT2B17 promoter, as assessed by functional and gel shift assays. HNF1alpha activation of the promoter was prevented by mutation or deletion of the putative HNF1 site. The related transcription factor HNF1beta, which is present in HepG2 cells, did not activate the promoter. The UGT2B17 promoter could also be activated by exogenous HNF1alpha in LNCaP cells. However, because these cells do not contain HNF1alpha, other transcription factors must regulate the UGT2B17 promoter. Cotransfection experiments showed that HNF1beta, elevates promoter activity in LNCaP cells. This activation did not involve the putative HNF1 region (-40 to -52 bp) since mutation of this region did not affect promoter activation by HNF1beta. These results suggest that the UGT2B17 promoter is regulated by different factors in liver-derived HepG2 and prostate-derived LNCaP cells.

Release of calcitonin gene-related peptide (CGRP) from guinea pig dura mater in vitro is inhibited by sumatriptan but unaffected by nitric oxide.

Migraine attacks can be provoked by administration of nitroglycerin, suggesting a role for nitric oxide (NO). The fact that release of the neuropeptide CGRP from trigeminal sensory nerves occurs during the pain phase of migraine and that NO can augment transmitter release prompted us to study CGRP release from the in situ dura mater in guinea pig skulls. Release of CGRP by capsaicin or by high potassium concentration was concentration-dependent and counteracted in calcium-free medium. The anti-migraine compound, sumatriptan, inhibited CGRP release via the 5-HT1-receptor. The NO donors, nitroglycerin, sodium nitroprusside and S-nitroso-N-acetylpenicillamine did not influence CGRP release, alone or together with the stimulants. We concluded that the skull preparation is well suited for scrutinizing CGRP release from dura mater. The fact that sumatriptan inhibits CGRP release as in migraine patients suggests a use for the present preparation in headache research.

N-substituted adenosines as novel neuroprotective A(1) agonists with diminished hypotensive effects.

The synthesis and pharmacological profile of a series of neuroprotective adenosine agonists are described. Novel A(1) agonists with potent central nervous system effects and diminished influence on the cardiovascular system are reported and compared to selected reference adenosine agonists. The novel compounds featured are derived structurally from two key lead structures: 2-chloro-N-(1-phenoxy-2-propyl)adenosine (NNC 21-0041, 9) and 2-chloro-N-(1-piperidinyl)adenosine (NNC 90-1515, 4). The agonists are characterized in terms of their in vitro profiles, both binding and functional, and in vivo activity in relevant animal models. Neuroprotective properties assessed after postischemic dosing in a Mongolian gerbil severe temporary forebrain ischemia paradigm, using hippocampal CA1 damage endpoints, and the efficacy of these agonists in an A(1) functional assay show similarities to some reference adenosine agonists. However, the new compounds we describe exhibit diminished cardiovascular effects in both anesthetized and awake rats when compared to reference A(1) agonists such as (R)-phenylisopropyladenosine (R-PIA, 5), N-cyclopentyladenosine (CPA, 2), 4, N-[(1S,trans)-2-hydroxycyclopentyl]adenosine (GR 79236, 26), N-cyclohexyl-2'-O-methyladenosine (SDZ WAG 994, 27), and N-[(2-methylphenyl)methyl]adenosine (Metrifudil, 28). In mouse permanent middle cerebral artery occlusion focal ischemia, 2-chloro-N-[(R)-[(2-benzothiazolyl)thio]-2-propyl]adenosine (NNC 21-0136, 12) exhibited significant neuroprotection at the remarkably low total intraperitoneal dose of 0.1 mg/kg, a dose at which no cardiovascular effects are observed in conscious rats. The novel agonists described inhibit 6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate-induced seizures, and in mouse locomotor activity higher doses are required to reach ED(50) values than for reference A(1) agonists. We conclude that two of the novel adenosine derivatives revealed herein, 12 and 5'-deoxy-5'-chloro-N-[4-(phenylthio)-1-piperidinyl]adenosine (NNC 21-0147, 13), representatives of a new series of P(1) ligands, reinforce the fact that novel selective adenosine A(1) agonists have potential in the treatment of cerebral ischemia in humans.

Astroglia contain a specific transport mechanism for N-acetyl-L-aspartate.

N-Acetylaspartate (NAA) is the second most abundant amino acid in the adult brain. It is located and synthesized in neurons and probably degraded in the glia compartment, but the transport mechanisms are unknown. Rat primary neuron and astrocyte cell cultures were exposed to the L isomer of [3H]NAA and demonstrated concentration-dependent uptake of [3H]NAA with a Km approximately 80 microM. However, Vmax was 23+/-6.4 pmol/mg of protein/min in astrocytes but only 1.13+/-0.4 pmol/mg of protein/min in neurons. The fact that neuron cultures contain 3-5% astrocytes suggests that the uptake mechanism is expressed only in glial cells. The astrocyte uptake was temperature and sodium chloride dependent and specific for L-NAA. The affinity for structural analogues was (IC50 in mM) as follows: L-NAA (0.12) > N-acetylaspartylglutamate (0.4) > N-acetylglutamate (0.42) > L-aspartate (>1) > L-glutamate (>1) > or = DL-threo-beta-hydroxyaspartate > N-acetyl-L-histidine. The naturally occurring amino acids showed no inhibitory effect at 1 mM. The glutamate transport blocker trans-pyrrolidine-2,4-dicarboxylate exhibited an IC50 of 0.57 mM, whereas another specific glutamate transport inhibitor, DL-threo-beta-hydroxyaspartate, had an IC50 of >1 mM. The experiments suggest that NAA transport in brain parenchyma occurs by a novel type of sodium-dependent carrier that is present only in glial cells.

Diversity of Holocene life forms in fossil glacier ice.

Studies of biotic remains of polar ice caps have been limited to morphological identification of plant pollen and spores. By using sensitive molecular techniques, we now demonstrate a much greater range of detectable organisms; from 2000- and 4000-year-old ice-core samples, we obtained and characterized 120 clones that represent at least 57 distinct taxa and reveal a diversity of fungi, plants, algae, and protists. The organisms derive from distant sources as well as from the local arctic environment. Our results suggest that additional taxa may soon be readily identified, providing a plank for future studies of deep ice cores and yielding valuable information about ancient communities and their change over time.

N-Acetylaspartate distribution in rat brain striatum during acute brain ischemia.

Brain N-acetylaspartate (NAA) can be quantified by in vivo proton magnetic resonance spectroscopy (1H-MRS) and is used in clinical settings as a marker of neuronal density. It is, however, uncertain whether the change in brain NAA content in acute stroke is reliably measured by 1H-MRS and how NAA is distributed within the ischemic area. Rats were exposed to middle cerebral artery occlusion. Preischemic values of [NAA] in striatum were 11 mmol/L by 1H-MRS and 8 mmol/kg by HPLC. The methods showed a comparable reduction during the 8 hours of ischemia. The interstitial level of [NAA] ([NAA]e) was determined by microdialysis using [3H]NAA to assess in vivo recovery. After induction of ischemia, [NAA]e increased linearly from 70 micromol/L to a peak level of 2 mmol/L after 2 to 3 hours before declining to 0.7 mmol/L at 7 hours. For comparison, [NAA]e was measured in striatum during global ischemia, revealing that [NAA]e increased linearly to 4 mmol/L after 3 hours and this level was maintained for the next 4 h. From the change in in vivo recovery of the interstitial space volume marker [14C]mannitol, the relative amount of NAA distributed in the interstitial space was calculated to be 0.2% of the total brain NAA during normal conditions and only 2 to 6% during ischemia. It was concluded that the majority of brain NAA is intracellularly located during ischemia despite large increases of interstitial [NAA]. Thus, MR quantification of NAA during acute ischemia reflects primarily changes in intracellular levels of NAA.

Electron Microscopic Detection of Novel, Coiled Viruslike Particles Associated with Graft-Inoculation of Prunus Species.

Coiled, viruslike particles (spirions) were detected by electron microscopic examination of crude extracts from flowers, leaves, and/or roots of infected Prunus avium, P. mume, and P. serrulata. The particles were observed in ultrathin sections of lower epidermis, palisade, and spongy mesophyll cells of leaves of P. avium and P. mume. Spring (March and April) appears to be optimal for detection of the particles in both screenhouse and field-grown plants. The particles were successfully graft-transmitted to P. armeniaca (cvs. Luizet and Tilton), P. avium (cvs. Bing, F12/1, Mazzard, and Sam), P. mahaleb, and P. persica (cv. Elberta). Individual spirions measured 132 × 34 nm. The particles appear to be coiled forms of a filamentous virus. Filaments extending from some coiled particles were approximately 13 nm wide, with striations at a pitch of 3.24 nm. No disease symptoms or cytopathological abnormalities were associated with the presence of the particles in the Prunus species studied. The particles were not detected in virus-free control plants.