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Scintigraphic measurement of tracheal mucus velocity in vivo.

Mucociliary clearance (MCC) is an important mechanism for removing inhaled particles, secretions and cellular debris from the respiratory tract. Here, a direct measurement of tracheal mucus velocity (TMV) for assessment of MCC, suitable for clinical and research use, is reported, and a comparison is made of TMV in normal subjects and patients with chronic obstructive pulmonary disease (COPD). A 0.1-mL bolus of radiolabelled (2-5 MBq), technetium-labelled macroaggregated human albumin (99mTc MAA) was injected through the cricothyroid membrane into the trachea of 20 young (< 50 yrs) and 12 older (> 50 yrs) normal subjects and 34 patients with COPD. Repeat studies were carried out in 13 normal subjects and 16 COPD patients. Movement of the bolus in the trachea was recorded (15 min) using a gamma camera interfaced to a computer. Data were analysed using specifically designed software. The test was well tolerated by subjects and patients, and no significant adverse events were reported. No significant differences were observed between data recorded from different regions of the bolus (leading edge, peak, trailing edge) by analysis of variance (ANOVA). Bland-Altman plots of the repeat studies indicated that data were more variable in normal subjects (coefficient of repeatability (COR) 10.3 mm.min-1) than in COPD patients (COR 5.5 mm.min-1). TMV (mean +/- SD) in young normal subjects (n = 20) was 10.7 +/- 3.5 mm.min-1. TMV was reduced in older normal subjects (n = 12; 6.5 +/- 2.6 mm.min-1) and further reduced in COPD (n = 34; 2.1 +/- 2.7 mm.min-1). In conclusion, this technique can be used to measure tracheal mucus velocity rapidly and safely in healthy subjects and patients with respiratory tract disease. This study has confirmed that tracheal mucus velocity declines with age and is further impaired in patients with chronic obstructive pulmonary disease.

Adult↗

Exogenous surfactant enhances mucociliary clearance in the anaesthetized dog.

Therapy with exogenous surfactants is currently used for the treatment of respiratory distress syndrome of the newborn (RDS) and is under investigation for treatments related to adult RDS. However, the possible use of exogenous surfactant as a means of enhancing mucus clearance in other respiratory diseases has not been addressed. We therefore studied the effects of an artificial surfactant (Curosurf) on in vivo tracheal mucus velocity in intubated pentobarbital-anaesthetized dogs. Five dogs were randomly administered, on separate occasions, either vehicle (saline) or 10 mg Curosurf by means of local instillation via a catheter into the right lung. Tracheal mucus was collected by inserting a soft-bristled cytology brush to the level of the carina, and analysed for viscoelasticity by microrheometry. Mucociliary clearability in vivo, tracheal mucus velocity (TMV) in mm.min-1, was determined by bronchoscopic observation of charcoal marker particle transit times. The initial placement of charcoal was at the same level of the lower trachea that mucus was collected from. The effect of ciliary beat frequency was assessed on the frog palate assay by a videoscopic technique. In the dog, TMV was significantly increased after administration of surfactant. The values of TMV in the vehicle- and surfactant-treated dogs were 6.3 +/- 4.0 vs 25.6 +/- 6.5 mm.min-1 (SD), respectively. There were no discernible differences between prevehicle and postvehicle TMV values, and no significant differences in any mucus viscoelastic parameter, as determined by magnetic rheometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, General↗

Hybridization capture increases on-target nanopore sequencing of plant RNA tobamovirus- derived cDNA libraries.

High-throughput sequencing (HTS) can support plant virus surveillance, but host nucleic acids often reduce on-target read recovery. We evaluated a targeted hybridization-capture workflow in which barcoded double-stranded cDNA (ds-cDNA) libraries generated from plant RNA extracts spiked with lyophilized tobamovirus-positive controls were enriched before Oxford Nanopore sequencing. Biotinylated probes targeted conserved regions of cucumber green mottle mosaic virus (CGMMV), species Tobamovirus viridimaculae; pepper mild mottle virus (PMMoV), species Tobamovirus capsici; and tobacco mosaic virus (TMV), species Tobamovirus tabaci. Across four pairs per virus, relative target-read abundance increased after capture from 0.76 &#xb1; 0.33% to 37.62 &#xb1; 15.72% for CGMMV, 8.16 &#xb1; 3.86% to 24.68 &#xb1; 12.34% for PMMoV, and 15.62 &#xb1; 10.40% to 36.83 &#xb1; 30.33% for TMV. Exact two-sided Wilcoxon signed-rank tests yielded P = 0.125 for each virus; with four nonzero differences in a common direction, this was the minimum attainable two-sided P value. Genome-coverage breadth was maintained. Retrospective duplex qPCR supported an increased virus-to-18S ratio for CGMMV, showed a variable PMMoV response, and showed a decreased virus-to-18S ratio for TMV because the 18S signal shifted earlier by as much as or more than the TMV signal. The findings provide proof-of-concept evidence for target-dependent library enrichment but do not establish analytical sensitivity, diagnostic performance, or field validity. Validation with naturally infected, low-titer, and mixed-infection samples and comparison with simpler targeted workflows are required.

biosecurity↗

Premedication with pronase or N-acetylcysteine improves visibility during gastroendoscopy: an endoscopist-blinded, prospective, randomized study.

AIM: To assess the efficacy of premedicaton with pronase or N-acetylcysteine (NAC) at 20 min before upper gastrointestinal (UGI) endoscopy and to determine whether pronase or NAC pretreatment influences the reliability of the rapid urease test. METHODS: A total of 146 patients were prospectively and randomly assigned into the study groups according to different premedications before endoscopy. One endoscopist assessed mucosal visibility (MV) with scores ranged from 1 to 4 at four sites in the stomach. The sum of the MV scores from these four locations was defined as the total mucosal visibility (TMV) score. Identification of H pylori was performed using CLO test, histology, and serology. RESULTS: The Group with pronase premedication had a significantly lower TMV score than did the groups with gascon and gascon water (P < 0.001 and P < 0.01, respectively). The group with NAC had a significantly lower TMV score than the group with gascon (P < 0.01) and a trend of a lower MV score than the group with gascon water (P = 0.06). The TMV score did not significantly differ between the group with pronase and the group with NAC (P = 0.39 and P = 0.14, respectively). The sensitivity and specificity of the CLO test were 92.5% and 93.9%, respectively, in groups premedicated with pronase and NAC together. CONCLUSION: Premedication with pronase or NAC at 20 min before UGI endoscopy improves the mucosal visibility of the stomach. Neither pronase nor NAC produces any obvious interference with the CLO test for the identification of H pylori infection.

Acetylcysteine↗

Mechanism of assembly of tobacco mosaic virus in vitro.

We previously proposed that the assembly reaction of TMV in vitro is initiated by 20S protein aggregate specifically interacting to the 5'-end of TMV-RNA, after which the helical rod grows by the addition of protein subunits. Other workers have reported that the source of protein for the growing helix is also the 20S protein aggregate, but not protein subunits. We now summarize the experimental results that confirm our previous hypothesis. 1) TMV-particle, as gauged by infectivity assay and sucrose gradient analysis, could be formed from PRR under conditions where the formation of 20S protein aggregate could not occur. 2) TMV-particle could be formed from PRR by stepwise addition of NBS-modified protein which is lacking the ability to form 20S protein aggregate. 3) The stable disk aggregate of CGMMV-protein are unable to grow the helical rod. 4) The process of rod elongation by protein subunits was observed directly in electron micrography.

Bromosuccinimide↗

Tobacco mosaic virus RNA as genetic determinant: genesis of a discovery.

It is generally held that the American geneticists Alfred Hershey and Martha Chase were the first to elucidate, in 1952, the genetic functions of phage DNA. The discovery of the genetic functions of RNA in a plant virus (Tobacco mosaic virus, TMV) is commonly attributed to the American plant virologist Heinz Fraenkel-Conrat, and to the Germans Alfred Gierer and Gerhard Schramm, who came to the same conclusion independently in 1956. In reality, the first understandings dated back to about 1940, when several scientists discovered that TMV infectivity was closely related to the presence of undamaged RNA in the virus particles. A very important but underestimated contribution came from the English group of Roy Markham, Kenneth Smith and Richard Matthews in 1948. This group purified and characterized an isometric plant virus, Turnip yellow mosaic virus, and first showed that virus infectivity depended on the presence of the RNA, concluding that nucleic acid was essential for virus multiplication. This finding was confirmed by the same group one year later but it laid neglected. After a five year period, in which several groups attempted to solve the question of the function of TMV RNA, the American electron microscopist Roger Hart offered, in 1955, further direct evidence which correlated RNA to TMV infectivity. One year later, three research groups (Fraenkel-Conrat; Gierer and Schramm; Max Lauffer, David Trkule and Anne Buzzell) obtained evidence that put an end to the question, which was (and is) fundamental to molecular Genetics because it demonstrated that RNA can function independently of DNA.

RNA, Viral↗

A hot pepper cDNA encoding a pathogenesis-related protein 4 is induced during the resistance response to tobacco mosaic virus.

Hot pepper (Capsicum annuum) plants exhibit a hypersensitive response (HR) against infection by many tobamoviruses. A clone (CaPR-4) encoding a putative pathogenesis-related protein 4 was isolated by differential screening of a cDNA library prepared from resistant pepper plant leaves inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaPR-4 is very similar to those of other plant PR-4s. Southern blot analysis showed that small gene families of PR-4-related sequences were present in the pepper genome. Hot pepper cultivar Bugang, resistant to TMV-P0 and susceptible to TMV-P1.2, induced CaPR-4 expression by pathotype P0 inoculation in inoculated and systemic leaves, but not by pathotype P1.2. Effects of exogenously applied abiotic elicitors upon the CaPR-4 expression were also examined. The expression of the CaPR-4 gene was stimulated by methyl jasmonate (MeJA), ethephon and wounding treatment. However, application of salicylic acid (SA) did not trigger the expression. Evidence is emerging that jasmonic acid and ethylene play key roles in the SA-independent pathways of plant-pathogen interaction. Taken together, these results suggest that the CaPR-4 gene is one of the defense-related genes conferring resistance on pepper plants by the SA-independent pathway and the cross-talk between signaling compounds, jasmonic acid and ethylene could have a great regulatory potential in a plant's defense against TMV.

Acetates↗

Importance of short-lived lymphocytes in the immune response.

Lymphocytes are heterogeneous with respect to their life-span. Typical B cells, bearing on their membranes immunoglobulin receptors, easily detectable by immunofluorescence, belong mainly to the long-lived population: this can be observed using combined autoradiography and immunofluorescence. However, when primed mice receive (-3H) thymidine before a boosting injection of tobacco mosaic virus (TMV), many plasma cells appearing in the spleen during the secondary response are labelled. In irradiated recipients repopulated with spleen cells from donors primed with TMV and injected with tritiated thymidine 2 hours before killing, the majority of plasms cells appearing in the spleen after antigen injection were labelled. If irradiated mice were repopulated simultaneously with spleen cells from donors primed with TMV and injected with (-3H) thymidine, and from donors primed with haemocyanin, most of the anti-TMV plasms cells were labelled, while most of the anti-haemocyanin plasma cells were unlabelled. These results allowed us to exclude non-specific reutilization of labelled thymidine as the main reason of our observations. It is concluded that either plasma cells derive from shortlived precursors or they receive material from a labelled cell able to co-operate specifically with plasma cell precursors.

Animals↗

[Study of the process of thermal aggregation of several representative tobamovirus coat proteins].

The role of the specific region of the tobacco mosaic virus (TMV) coat protein (CP) molecule (called "70A degree-region") in the regulation of ordered and unordered CP aggregation was investigated. CPs of the wild type TMV (strain U1), of temperature sensitive mutant with two amino acid substitutions in the "70A degree-region", and of cucumber virus 3 which is related to TMV but has a completely different structure in the "70A degree-region" were used. With the help of two different tests the processes of temperature-induced unordered aggregation of these three CPs were compared in solutions of different ionic strength and pH. On the basis of the data obtained it was concluded that the "70A-region" represents the most thermolabile region in the TMV CP molecule and that local thermal denaturation of this region results in unordered aggregation, when solution conditions (ionic strength and pH) favor formation of relatively large ordered aggregates (20S-"disks" or helical repolymerized protein).

Capsid↗

Detection of antiviral and antitumoral fractions of Chenopodium amaranticolor leaf extract.

Based on the fact that Chenopodium amaranticolor extracts showed inhibitory activity against tobacco mosaic virus (TMV) and Ehrlich tumour (EA), tests were carried out to investigate whether the antiviral and antitumoral activity were caused by the same compounds. When the extract was purified by CM Sephadex C-25 column, after precipitation with 90% ammonium sulphate, twenty active fractions against TMV and two pools of fractions active against EA were obtained. Only one fraction with high absorbance values at 260 and 280 nm was able to inhibit both TMV and EA. When the extract was purified by Bio Gel P-60 column two active fractions against TMV and EA were obtained, suggesting that they were contained in the 0.01 M fraction of the CM Sephadex column. It is suggested that C. amaranticolor leaf extract contained at least two protein-like substances manifesting antiviral and antitumoral activity.

Animals↗

Extremely low frequency weak magnetic fields enhance resistance of NN tobacco plants to tobacco mosaic virus and elicit stress-related biochemical activities.

Increasing evidence has accumulated concerning the biological effects of extremely low frequency magnetic fields (ELF-MFs) in different plant models. In the present study, effects of ELF-MFs in tobacco plants reacting to tobacco mosaic virus (TMV) with a hypersensitive response (HR) were evaluated. Plants were exposed for 8 or 24 h (either before or after TMV inoculation) to a static MF, at either -17 or 13 microT, combined with a 10 Hz sinusoidal MF with different intensities (25.6 or 28.9 microT). The working variables were the area and number of hypersensitive lesions in leaves. Following ELF-MFs exposure, an increased resistance was detected, particularly after an 8-h treatment, as shown by the decrease in lesion area and number. Moreover, two enzyme activities involved in resistance mechanisms were analyzed: ornithine decarboxylase (ODC) and phenylalanine ammonia-lyase (PAL). Uninoculated leaves previously exposed to ELF-MFs in general showed a significant increase relative to controls in ODC and PAL activities, in particular for 13 microT static MF plus 28.9 microT, 10 Hz sinusoidal MF (24 h) treatment. In conclusion, ELF-MFs seem to influence the HR of tobacco to TMV, as shown by the increased resistance and changes in ODC and PAL activities, indicating the reliability of the present plant model in the study of bioelectromagnetic interactions.

Electromagnetic Fields↗

Morphological analysis of the tuberomammillary nucleus in the rat brain: delineation of subgroups with antibody against L-histidine decarboxylase as a marker.

With an antibody specific for L-histidine decarboxylase (HD) in combination with immunohistochemical techniques and retrograde fluorescent tracing, the morphology, distribution, and projections of the histaminergic neurons of the posterior hypothalamus were studied in the adult male rat. Magnocellular neurons, situated on both sides of the mammillary recess and close to the ventral surface of the brain rostral and caudal to the mammillary bodies, were found to contain HD-immunoreactivity (HD-i). In addition to these magnocellular neurons, a substantial number of small and medium-sized neurons were immunostained, as were strands of cells of all sizes bridging the HD-i cell groups. A detailed mapping of the HD-i cells in frontal, sagittal, and horizontal sections showed that these neurons make up one continuous cell group, defined as the tuberomammillary nucleus (TM). This nucleus can be divided into several subgroups. Thus, approximately 600 HD-i neurons situated on each side of the mammillary recess compose the medial subgroup of the TM (TMM). The ventral subgroup of the TM (TMV) consists of some 1,500 neurons situated at the ventral surface of the brain, rostral (TMVr) and caudal (TMVc) to the mammillary bodies. The TMM contains a greater proportion of parvicellular neurons compared to the TMV. About 100 HD-i cells are scattered within the lateral hypothalamic area, the posterior hypothalamic region, the perifornical area, the supramammillary nucleus, and the dorsomedial hypothalamic nucleus. These cells are collectively named the diffuse part of the TM (TMdiff). The morphological differences between the TMM and the TMV did not signal differences in the efferent connections of these subgroups. Thus, single injections of the fluorescent tracer Fast Blue into different regions of the brain, including the spinal cord, resulted in retrograde labeling of HD-i neurons, which were distributed throughout the TM with no discernible topographic pattern. More specifically, each subgroup of the TM contributed projections innervating or passing through a large number of brain regions, including the olfactory bulb, hippocampus, caudate nucleus, paraventricular and supraoptic nuclei of the hypothalamus, cerebellum, tectum, medulla, and spinal cord. The widespread projections of the HD-i neurons contrasted to the more specific projections observed from non HD-i neurons in cell groups situated around the TM. Taken together, these findings suggest that the HD-i cells of the posterior hypothalamus constitute one major nucleus, the TM, and that this nucleus may be subdivided into three components, each of which has diffuse projections throughout the neuraxis.

Animals↗

Dielectrophoretic investigation of plant virus particles: Cow Pea Mosaic Virus and Tobacco Mosaic Virus.

This paper reports experimental results on the dielectrophoretic (DEP) behaviour on two nonenveloped plant viruses of different geometrical shapes, namely Cow Pea Mosaic Virus (CPMV) and Tobacco Mosaic Virus (TMV). The DEP properties of carboxy-modified latex beads of the same size are also reported. The DEP properties of single particles were obtained from measurement of the frequency at which the DEP force on a particle goes to zero (the crossover frequency). The DEP behaviour of particle ensembles was also measured using image processing. The dielectric properties of the particles were evaluated from the DEP data. The surface conductance was found to be 0.3 nS for CPMV, 0.38 nS for TMV, and 0.52 nS for 27 nm diameter carboxy-latex beads. Data analysis has shown that the optimal condition for separation of TMV and CPMV is a low-conductivity suspending medium - below 1 mS/m.

Comovirus↗

Virus-induced synthesis of messenger RNAs for precursors of pathogenesis-related proteins in tobacco.

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) induces a number of host-encoded, so-called pathogenesis-related (PR-) proteins, which are found in the intercellular space of the leaf and are associated with induced resistance. By immunoprecipitation of their in vitro translation products we were able to detect the mRNAs corresponding to a number of PR-proteins in TMV-infected tobacco, but not in healthy plants. Analysis by the Northern blot technique using cloned cDNA of PR1-mRNAs as probe showed that the mRNAs for the closely related proteins PR1a, 1b and 1c occur at a low level in healthy tobacco; upon TMV infection this level is increased > 100-fold. The PR1-specific probe did no hybridize to mRNAs corresponding to other PR-proteins. Sequencing of the 5'-terminal region of PR1-mRNAs showed that PR1-proteins are derived from precursors by removal of an N-terminal signal peptide of 30 amino acids.

Amino Acid Sequence↗

Rapid determination of methyl salicylate, a plant-signaling compound, in tomato leaves by direct sample introduction and thermal desorption followed by GC-MS.

Many plants infested by herbivores or viruses can rapidly produce and accumulate a plant-signaling compound, methyl salicylate (MeSA), in their leaves to activate disease resistance. In the present work, a simple, rapid, and sensitive method was developed for the determination of MeSA in tomato leaves by direct sample introduction and thermal desorption followed by GC-MS. Results show that the proposed method has a low detection limit (0.08 ng mg(-1)) and good precision (RSD = 8.9%). The present method was applied to the investigation of tomato plant defense response to tobacco mosaic virus (TMV) by rapid analysis of volatile compounds in plant leaves. It was found that tomato plants can produce large amounts of MeSA as a defense response to TMV. This indicates that MeSA may be a plant-signaling compound in tomato plant defense response to TMV.

Gas Chromatography-Mass Spectrometry↗

Structure determination of cucumber green mottle mosaic virus by X-ray fiber diffraction. Significance for the evolution of tobamoviruses.

Cucumber green mottle mosaic virus (CGMMV) is a rod-shaped virus of the tobacco mosaic virus (TMV) group. The structure of cucumber green mottle mosaic virus has been determined by fiber diffraction methods at 3.4 A resolution, and refined by molecular dynamics methods to an R factor of 0.093. Disassembly of TMV is driven by the mutual repulsion of intersubunit carboxyl-carboxylate pairs, but one of these pairs is not conserved in CGMMV. An alternative pair, located about 5 A from the site of the TMV pair, has been found in CGMMV. Comparison of the two structures suggests that the carboxylate groups are free to migrate in the subunit interfaces during evolution.

Biological Evolution↗

Movement of a barley stripe mosaic virus chimera with a tobacco mosaic virus movement protein.

The tobacco mosaic virus (TMV) 30K movement protein (MP) gene was inserted into a full-length cDNA clone of barley stripe mosaic virus (BSMV) RNA beta replacing the triple gene block (TGB). The resulting recombinant ND-MPT genome, consisting of infectious wt transcripts of BSMV RNAs alpha and gamma, together with the hybrid RNA beta transcript, was inoculated onto test plants to study the functional compatibility between the BSMV TGB-adapted genetic system and the tobamovirus transport gene. ND-MPT infected the inoculated leaves of Nicotiana benthamiana and Chenopodium amaranticolor, which are common hosts for the parental viruses; the size, growth rate, and morphology of local lesions on C. amaranticolor were influenced by the foreign MP gene. However, the hybrid virus failed to infect barley, N. tabacum (var. Samsun), and N. clevelandii, the selective hosts. Thus, the TMV MP was able to functionally substitute for the BSMV TGB-coded MPs, i.e., the 30K MP functioned independently of any other BSMV sequences. However, the TMV MP gene promoted the cell-to-cell movement in a host-dependent manner.

Amino Acid Sequence↗

Second-site reversion of a dysfunctional mutation in a conserved region of the tobacco mosaic tobamovirus movement protein.

The N-terminal two-thirds of tobamovirus movement proteins (MPs) contain two well conserved regions. Within region I (amino acids 56-96) is an area predicted by computer analysis to have loop secondary structure (amino acids 76-87). A single or two double amino acid mutations were introduced into the loop in region I of the TMV MP to destabilize the structure. The three mutant MPs were defective in movement function. The single amino acid mutation resulted in a Pro81-->Ser substitution. The mutant virus, TP81S, containing the Pro81-->Ser substitution, was propagated on a transgenic line of Nicotiana tabacum that expresses the sunn-hemp mosaic tobamovirus MP. Inoculation of virus progeny from the transgenic plants onto hypersensitive N. tabacum indicated the presence of infectious virus at a low frequency. Necrotic lesions were detected at 4 days postinoculation, 2 days later than those induced by wild-type TMV. Inoculation of virus extracted from necrotic lesions onto N. tabacum resulted in a delayed and attenuated systemic infection relative to that induced by TMV, indicating that a second-site mutation restored movement function rather than a reversion of the original mutation. Sequence analysis revealed that the revertant MP gene had two additional amino acid substitutions, a Thr104-->Ile and a Arg167-->Lys. Introduction of the amino acid substitutions individually or in combination into the MP of TP81S indicated that both substitutions were required for the revertant phenotype. The data indicate that structure within region I is important in maintaining an active conformation for functional MP, that changes outside region I can compensate for alterations within the region, and suggest that region I may interact with a distal portion of the protein.

Amino Acid Sequence↗