RESUMEN

Background: Sudden and unexpected deaths are increasing drastically. The main cause of sudden death is cardiovascular disease, out of which coronary artery disease predominates forming 80% of the cases. Most of the time, detecting early changes in myocardial infarction during the autopsy is challenging since gross infarct changes do not appear until after 24 to 48 hours of myocardial ischemia injury. So, the aim of this study was to compare two test to detect early changes of Myocardial Infarction one by using Triphenyl Tetrazolium Chloride (TTC) staining of the myocardial tissue, during autopsy and other by histopathological examination. Methods: The sample size of 60 hearts taken from all the sudden deaths cases brought to Mortuary with suspected cause of death due to cardiac origin. The heart was obtained from the deceased by standard post-mortem technique. Serial full-thickness transverse sections of the heart were taken at 2 cm intervals from the apex to the atrioventricular groove. All the serial slices of heart are taken for histochemical staining and TTC staining. Results: In histopathological examination 34 hearts were diagnosed with myocardial infarction and 26 hearts reported non myocardial infarction. With TTC 40 hearts remained unstained suggestive of myocardial infarction and 20 hearts were stained suggestive of non-infarcted hearts. TTC staining in our study shows an accuracy of 88.33%. Conclusion: The result of this study shows that the Triphenyl Tetrazolium Chloride test, a histochemical staining technique of heart, is reliable approach for forensic pathologists to diagnose early myocardial infarction during the post-mortem examination.

Asunto(s)

RESUMEN

Background Cardiovascular diseases, especially ischemic heart disease, are the most frequent cause of sudden and unexpected death that constitute a significant portion of the autopsies conducted in our country. Though these deaths may be natural as well as unnatural, they carry medico-legal importance because they occur in a person who has been apparently healthy before the supervening of death, and the cause of death is difficult to ascertain. An infarction can be missed by gross and histological examination within the first few hours of sudden death. 2,3,5-triphenyl-tetrazolium chloride (TTC) is a sensitive histochemical method for diagnosing myocardial infarction within four hours of sudden death. The use of such dyes, hence, can possibly aid in ascertaining the cause of death in such cases wherein there are no known preceding factors. Aim The aim of this article was to study the occurrence of myocardial ischemia by histochemical staining method - 2,3,5-triphenyl-tetrazolium chloride (TTC).  Methods This study involved patients who underwent postmortem examination in the Department of Forensic Medicine and Toxicology, Sri Ramachandra Medical College and Research Institute, Chennai. Results Of 62 cases, 31 cases were found to be positive for TTC staining, and those heart slices were subjected to histopathological examination. The maximum number of cases (77.4%) showed the age of infarction within zero to four hours, which was detected early by TTC staining compared to microscopic changes in the heart. Only seven cases were positive for myocardial infarction by histopathological examination, proving that it is difficult to detect acute infarction if the age of infarction is less than four hours. Conclusion This suggests that for all sudden death cases, 2,3,5-triphenyl tetrazolium chloride could be used as a better tool for the identification of early infarcts.

RESUMEN

Reduced blood flow (hypoxia) to the brain is thought to be the main cause of strokes because it deprives the brain of oxygen and nutrients. An increasing amount of evidence indicates that the Centella-Asiatica (HA-CA) hydroalcoholic extract has a variety of pharmacological benefits, such as antioxidant activity, neuroprotection, anti-inflammatory qualities, and angiogenesis promotion. Intermittent fasting (IF) has neurological benefits such as anti-inflammatory properties, neuroprotective effects, and the ability to enhance neuroplasticity. The current study evaluates the combined effect of IF (for 1, 6, and 12 days) along with HA-CA (daily up to 12 days) in adult zebrafish subjected to hypoxia every 5 min for 12 days followed by behavioral (novel tank and open-field tank test), biochemical (SOD, GSH-Px, and LPO), inflammatory (IL-10, IL-1ß, and TNF-α), mitochondrial enzyme activities (Complex-I, II, and IV), signaling molecules (AMPK, MAPK, GSK-3ß, Nrf2), and imaging/staining (H&E, TTC, and TEM) analysis. Results show that sub-acute hypoxia promotes the behavioral alterations, and production of radical species and alters the oxidative stress status in brain tissues of zebrafish, along with mitochondrial dysfunction, neuroinflammation, and alteration of signaling molecules. Nevertheless, HA-CA along with IF significantly ameliorates these defects in adult zebrafish as compared to their effects alone. Further, imaging analysis significantly provided evidence of infarct damage along with neuronal and mitochondrial damage which was significantly ameliorated by IF and HA-CA. The use of IF and HA-CA has been proven to enhance the physiological effects of hypoxia in all dimensions.

Asunto(s)

RESUMEN

Current colorimetric methods for quantitative determination of seed viability (SV) with 2,3,5-triphenyl tetrazolium chloride (TTC) have been plagued by issues of being cumbersome and time-consuming during the experimental process, slow in extraction and staining, and exhibiting inconsistent results. In this work, we introduced a new approach that combines TTC-staining with high-temperature extraction using dimethyl sulfoxide (DMSO). The optimization of the germination stage, TTC-staining method, and 1,3,5-triphenylformazan (TTF) extraction method were meticulously carried out as follows: When the majority of wheat seeds had grown the radicle, and the length of radicles was approximately equal to the seed length (24 h-germination), 2 g germinating seeds were placed into a beaker (20 mL) containing 5 mL 10 g·L-1 TTC solution. The seeds were stained with TTC in the dark at 25 °C for 1 h. Following the staining, 1 mL 1 mol·L-1 H2SO4 was added to stop the reaction for 5 min. The H2SO4 solution was then removed, and the seeds were gently rinsed with deionized water. Subsequently, the TTF produced in the seeds was extracted directly with 5 mL DMSO solution at 55 °C for 1 h. The absorbance of the extract was measured at 483 nm, and the index of SV was calculated according to a predetermined TTC calibration curve and expressed by mg TTC·g-1 (seed)·h-1. The new method has been demonstrated to be rapid, stable, and highly sensitive, as evidenced by the accurate measurement of seed viability with different aging degrees.

Asunto(s)

RESUMEN

Cerebral ischemia-reperfusion injury induces multi-dimensional damage to neuronal cells through exacerbation of critical protective mechanisms. Targeting more than one mechanism simultaneously namely, inflammatory responses and metabolic energy homeostasis could provide additional benefits to restrict or manage cerebral injury. Being proven neuroprotective agents both, progesterone (PG) and trimetazidine (TMZ) has the potential to add on the individual therapeutic outcomes. We hypothesized the simultaneous administration of PG and TMZ could complement each other to synergize, or at least enhance neuroprotection in reperfusion injury. We investigated the combination of PG and TMZ on middle cerebral artery occlusion (MCAO) induced cerebral reperfusion injury in rats. Molecular docking on targets of energy homeostasis and apoptosis assessed the initial viability of PG and TMZ for neuroprotection. Animal experimentation with MCA induced ischemia-reperfusion (I/R) injury in rats was performed on five randomized groups. Sham operated control group received vehicle (saline) while the other four I-R groups were pre-treated with vehicle (saline), PG (8 â€‹mg/kg), TMZ treated (25 â€‹mg/kg), and PG â€‹+ â€‹TMZ (8 and 25 â€‹mg/kg) for 7 days by intraperitoneal route. Neurological deficit, infarct volume, and oxidative stress were evaluated to assess the extent of injury in rats. Inflammatory reactivity and apoptotic activity were determined with alterations in myeloperoxidase (MPO) activity, blood-brain barrier (BBB) permeability, and DNA fragments. Reperfusion injury inflicted cerebral infarct, neurological deficit, and shattered BBB integrity. The combination treatment of PG and TMZ restricted cellular damage indicated by significant (p â€‹< â€‹0.05) decrease in infarct volume and improvement in free radical scavenging ability (SOD activity and GSH level). MPO activity and LPO decreased which contributed in improved BBB integrity in treated rats. We speculate that inhibition of inflammatory and optimum energy utilization would critically contribute to observed neuroprotection with combined PG and TMZ treatment. Further exploration of this neuroprotective approach for post-recovery cognitive improvement is worth investigating.

RESUMEN

Background: Stroke is a neurological disorder characterized by brain tissue damage following a decrease in oxygen supply to brain due to blocked blood vessels. Reportedly, 80% of all stroke cases are classified as cerebral infarction, and the incidence rate of this condition increases with age. Herein, we compared the efficacies of Korean White ginseng (WG) and Korean Red Ginseng (RG) extracts (WGex and RGex, respectively) in an ischemic stroke mouse model and confirmed the underlying mechanisms of action. Methods: Mice were orally administered WGex or RGex 1 h before middle cerebral artery occlusion (MCAO), for 2 h; the size of the infarct area was measured 24 h after MCAO induction. Then, the neurological deficit score was evaluated and the efficacies of the two extracts were compared. Finally, their mechanisms of action were confirmed with tissue staining and protein quantification. Results: In the MCAO-induced ischemic stroke mouse model, WGex and RGex showed neuroprotective effects in the cortical region, with RGex demonstrating superior efficacy than WGex. Ginsenoside Rg1, a representative indicator substance, was not involved in mediating the effects of WGex and RGex. Conclusion: WGex and RGex could alleviate the brain injury caused by ischemia/reperfusion, with RGex showing a more potent effect. At 1,000 mg/kg body weight, only RGex reduced cerebral infarction and edema, and both anti-inflammatory and anti-apoptotic pathways were involved in mediating these effects.

RESUMEN

Bulk ultrasound ablation is a thermal therapy approach in which tissue is heated by unfocused or weakly focused sonication (average intensities on the order of 100 W/cm2) to achieve coagulative necrosis within a few minutes exposure time. Assessing the role of bubble activity, including acoustic cavitation and tissue vaporization, in bulk ultrasound ablation may help in making bulk ultrasound ablation safer and more effective for clinical applications. Here, two series of ex vivo ablation trials were conducted to investigate the role of bubble activity and tissue vaporization in bulk ultrasound ablation. Fresh bovine liver tissue was ablated with unfocused, continuous-wave ultrasound using ultrasound image-ablate arrays sonicating at 31 W/cm2 (0.9 MPa amplitude) for either 20 min at a frequency of 3.1 MHz or 10 min at 4.8 MHz. Tissue specimens were maintained at a static overpressure of either 0.52 or 1.2 MPa to suppress bubble activity and tissue vaporization or at atmospheric pressure for control groups. A passive cavitation detector was used to record subharmonic (1.55 or 2.4 MHz), broadband (1.2-1.5 MHz) and low-frequency (5-20 kHz) acoustic emissions. Treated tissue was stained with 2% triphenyl tetrazolium chloride to evaluate thermal lesion dimensions. Subharmonic emissions were significantly reduced in overpressure groups compared with control groups. Correlations observed between acoustic emissions and lesion dimensions were significant and positive for the 3.1-MHz series, but significant and negative for the 4.8-MHz series. The results indicate that for bulk ultrasound ablation, where both acoustic cavitation and tissue vaporization are possible, bubble activity can enhance ablation in the absence of tissue vaporization, but can reduce thermal lesion dimensions in the presence of vaporization.

Asunto(s)

RESUMEN

Objective To evaluate the effect of inhibition of ubiquitin carboxy terminal hydrolase LI (UCHL1) on cerebral ischemia/reperfusion injury in mice. Methods Male BALB/c mice were randomly divided into sham group, ischemia/reperfusion (I/R) group, UCHL1 small interfering RNA (siRNA)group and scramble siRNA (control) group, 10 mice in each group. I/R model was established by reperfusion 24 hours after middle cerebral artery occlusion (MCAO) 60 minutes. In the siRNA group and control group, 10 JJLI UCHL1 siRNA or scramble siRNA was injected into the brain through the lateral ventricle 24 hours before MCAO. The expression of UCHL1 was detected by RT-PCR and Western blotting; the volume of cerebral infarction and the rate of edema were assessed by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining; and the score of neurological symptoms was assessed by neurobehavioral scoring. Results Compared with the sham group, the level of UCHL1 mRNA and protein in ischemic penumbra of I/R group were significantly higher (P< 0.05), while the expression of UCHL1 protein and mRNA in siRNA group were significantly lower (P< 0.05); at the same time, the volume of cerebral infarction, edema rate and neurobehavioral damage in I/R group increased significantly, while the volume and edema rate of cerebral infarction and neurobehavioral damage in siRNA group further increased (P< 0.05). Conclusion Inhibition of UCHL1 can aggravate the cerebral ischemia/reperfusion injury in mice, suggesting that the induction of UCHL1 after MCAO has a protective effect on the cerebral ischemia/reperfusion injury in mice.

RESUMEN

AIM: Assess performance of broth microdilution (BMD) as well as agar dilution methods for antimicrobial susceptibility testing of Staphylococci using tetrazolium salt. METHODS AND RESULTS: Minimum inhibitory concentration (MIC) of eight antimicrobials; vancomycin (VA), linezolid, oxacillin, gentamicin (CN), tetracycline, ciprofloxacin, erythromycin and clindamycin was investigated for 80 isolates of Staphylococci by BMD with the addition of dimethyl thiazole diphenyl tetrazolium bromide (MTT), agar dilution with the addition of MTT and triphenyl tetrazolium chloride at the standard bacterial concentration together with addition of MTT at an experimental bacterial concentration. BMD (MTT) showed the highest agreement in comparison with the standard BMD. CONCLUSIONS: Colorimetric BMD was rapid and easy to interpret. Colorimetric agar dilution (MTT) was less tedious than BMD. SIGNIFICANCE AND IMPACT OF THE STUDY: Colorimetric antibiotic susceptibility is a good option to provide rapid reliable results for critically ill patients. In addition, agar dilution (MTT) helps to investigate outbreaks of methicillin-resistant Staphylococcus aureus (MRSA), VISA or VRSA. BMD (MTT) can be performed routinely to detect VA MIC in MRSA blood stream infections and hospital acquired pneumonia, where, high VA MIC is associated with a higher mortality rate.

Asunto(s)

RESUMEN

Chitosan-alginate microspheres (MS) were developed for cefixime vaginal administration, to overcome problems associated with its oral administration. The effect of increasing drug-loading amount, by keeping the chitosan-alginate content constant, was investigated. Mucoadhesion studies indicated that all formulations assured in situ permanence longer than 2 h. Entrapment efficiency increased with drug loading concentration in the starting solution, reaching a plateau at 30 mg/mL indicative of the achievement of an optimal drug-to-polymer ratio. MS swelling properties increased with the entrapped drug amount, and, interestingly, water-uptake reached its maximum value at the same drug loading concentration of 30 mg/mL. The relationship found between MS water-uptake and drug release rate confirmed MS prepared with 30 mg/mL cefixime as the best formulation. Microbiological studies showed a relation between cefixime release rate from MS and Escherichia coli viability reduction, definitely indicating the selected MS formulation as the best for an effective local treatment of urogenital infections.

Asunto(s)

RESUMEN

OBJECTIVE: Mitochondrial dysfunction is known to be implicated in stroke, but the complex mechanisms of stroke have led to few stroke therapies. The present study to disrupted mitochondrial oxidative phosphorylation through a known electron transport chain (ETC) uncoupler, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP). Analyzing the resulting neurological deficits as well as infarct volume could help determine the role of mitochondria in stroke outcome and determine whether uncoupling the ETC could potentially be a strategy for new stroke therapies. The objective of this study was to determine the effects of uncoupling electron flow on mitochondrial oxidative phosphorylation and stroke infarction. METHODS: Cerebral endovascular cells (CECs) were treated with various concentrations of FCCP, and bioenergetics were measured. For the stroke mouse model, FCCP (1 mg/kg, i.p) or vehicle was administered followed by 1-hour transient middle cerebral artery occlusion (tMCAO). Infarct volume was measured after a 23-hour reperfusion, and triphenyl tetrazolium chloride (TTC) staining was used to assess infarct volume. RESULTS: FCCP significantly decreased basal respiration, ATP turnover, maximal respiration, and spare capacity when the concentration of FCCP was greater than 1000 nM. The mice pretreated with FCCP had a significantly increased infarct volume within the cortex, striatum, and total hemisphere. Mice receiving FCCP had a significantly increased neurological deficit score compared to the vehicle. CONCLUSIONS: FCCP compromised mitochondrial oxidative phosphorylation in CECs in a dose-dependent manner. Uncoupling the electron transport chain with FCCP prior to tMCAO exacerbated stroke infarction in mice.

RESUMEN

Objective: To detect and evaluate the hypothalamic infarction in middle cerebral artery occlusion (MCAO) model rat. Methods: For 15 Sprague-Dawley rats weighed 200-250 g, aged 6-8 months, their right middle cerebral artery was occluded for 90 min by a silicon-coated 4-0 nylon filament and reperfused. Sprague-Dawley rats underwent diffusion weighted MR imaging (DWI) scanning (at 1 h and 24 h after reperfusion) and 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining (at 24 h after reperfusion) to determine the hypothalamic and cerebral infarct volume. The relationship between hypothalamic infarct volume and cerebral infarction volume was analyzed by DWI scanning. The results of TTC staining were compared with those of 24 h DWI scanning. Results: Fifteen Sprague-Dawley rats successfully received intraluminal MCAO/reperfusion procedures. The incidences of hypothalamic infarction on brain DWI scanning and TTC staining were 100% and 40% at 24 h after reperfusion, respectively. Therefore, DWI scanning was more sensitive than TTC staining to detect hypothalamic injury (P=0.001). The hypothalamic infarct volume on DWI scanning was (8.59±2.89) mm3 and (11.65±3.19) mm3 at 1 h and 24 h after reperfusion, respectively. On DWI scanning, hypothalamic and cerebral infarct volume at 24 h after reperfusion were correlated with each other significantly (r=0.573, P=0.025), so were the increases of hypothalamic and cerebral infarct volume (r=0.554, P=0.032) from 1 h to 24 h. Conclusion: DWI scanning was more sensitive than TTC staining to detect hypothalamic injury in intraluminal transient MCAO model. Hypothalamic and cerebral infarct volume were correlated with each other.

RESUMEN

BACKGROUND: We have found that infarcted brain regions exhibit green channel autofluorescence (GCAF). Here, we compare ex vivo GCAF-imaging with 2,3,5-triphenylteterazolium chloride (TTC)-staining. NEW METHOD: C57BL/6 mice (n=120) underwent GCAF-imaging after transient or permanent middle cerebral artery occlusion (tMCAO or pMCAO). COMPARISON WITH EXISTING METHODS: TTC-staining may not reflect subtle ischemic injury. TTC-stained tissues, when reused, are prone to processing artifacts related to prior TTC-staining. GCAF imaging requires little experimental manipulation of animals and brain tissues, and allows for more consistent measurements of infarct volume and reliable reuse of the fresh unstained tissues. RESULTS: Lesion volumes measured at 24-h after 1-h tMCAO by using GCAF-images were similar to those using TTC-staining: 87.6±13.6mm3 vs. 83.8±12.8mm3 in 1mm-thick sections (n=9 mice, 10 slices/mouse, p=0.88; Pearson's r=0.91, p<0.001) and 75.1±7.6mm3 vs. 73.6±6.7mm3 in 2 mm-thick sections (n=9 mice, 5 slices/mouse, p=0.99; Pearson's r=0.87, p<0.001), respectively. In serial ex vivo imaging performed at 1, 2, 3, 6, 12, and 24-h after tMCAO, GCAF-imaging correlated well with TTC-staining at all time-points. In the pMCAO model however, the correlation was strong at later time-points (6-24-h); but at time points up to 3-h, GCAF-imaging was more sensitive than TTC-staining to detect ischemic areas, as verified by histology, where ischemic damage was observed in the GCAF-positive areas of the cerebral cortex and striatum, even in the face of normal TTC-staining. CONCLUSION: GCAF-imaging is a reliable alternative to TTC-staining in the qualitative and quantitative assessments of focal brain ischemia, and more sensitive for detecting early ischemic damage in pMCAO.

Asunto(s)

RESUMEN

Accumulation of electronic waste has increased catastrophically and out of that various plastic resins constitute one of the leading thrown out materials in the electronic machinery. Enrichment medium, containing high impact polystyrene (HIPS) with decabromodiphenyl oxide and antimony trioxide as sole carbon source, was used to isolate microbial cultures. The viability of these cultures in the e-plastic containing mineral medium was further confirmed by triphenyl tetrazolium chloride (TTC) reduction test. Four cultures were identified by 16S rRNA sequencing as Enterobacter sp., Citrobacter sedlakii, Alcaligenes sp. and Brevundimonas diminuta. Biodegradation experiments were carried out in flask level and gelatin supplementation (0.1% w/v) along with HIPS had increased the degradation rate to a maximum of 12.4% (w/w) within 30days. This is the first report for this kind of material. The comparison of FTIR, NMR, and TGA analysis of original and degraded e-plastic films revealed structural changes under microbial treatment. Polystyrene degradation intermediates in the culture supernatant were also detected using HPLC analysis. The gravity of biodegradation was validated by morphological changes under scanning electron microscope. All isolates displayed depolymerase activity to substantiate enzymatic degradation of e-plastic.

Asunto(s)

RESUMEN

The archetypal triphenyl tetrazolium chloride (tetrazolium red) has been used in spectrophotometric microplate assays for 2-hydroxy ketones and, by extension, for the activity of enzymes including aminotransferase, transketolase, and pyruvate decarboxylase. Better sensitivity, dynamic range, and reproducibility of this method may be achieved by (i) in situ solubilization of the colored formazan with Cremophor EL and (ii) use of the newer-generation tetrazolium salts tetrazolium violet, iodonitrotetrazolium, or WST-1 (water-soluble tetrazolium). Two to 125 µM hydroxy pyruvate, dihydroxy acetone, and indole pyruvate produced by serine:pyruvate, ß-alanine (ω-amino acid):pyruvate, and aromatic amino acid aminotransferase, respectively, could be detected with WST-1.

Asunto(s)

RESUMEN

Objective To explore the changes of MR diffusion imaging (DWI) appearance in newborn rats with hypoxic‐ischemic brain damage(HIBD) ,and its relationship with the changes of Triphenyl tetrazolium chloride (TTC) staining .Methods Using liga‐tion of the left carotid artery method to establish three different degrees of HIBD animal models ;DWI was performed at each time point(6-12 h ,12-24 h ,3 d ,7 d);Fresh brain tissue taken from another model groups of newborn rats in 12 h ,24 h ,3 d ,7 d were staining in TTC ,then we observed its relationship with DWI .Results The lesion location of three model groups mainly distributed in the left side of cortex and subcortical region ,with prolonged hypoxia time ,hippocampus ,lateral side white matter ,thalamus were also have varying degrees of involvement .The right side of the cortex and subcortical in some cases involved .TTC staining showed posi‐tive results in 3 d ,its loss stained area were consistent with DWI abnormal signal area .Conclusion DWI can be evaluation of HIBD lesions early .The early lesions of HIBD mainly distributed in the left side of cortex and subcortical region .

RESUMEN

This study was designed to detect the impact of Valerian Ligusticum Pill (VLP) on cerebral ischemia reperfusion injury in rats, and explore the mechanism of angiogenesis. Sixty SD male rats were randomly divided into five groups, including sham operation group, model group, VLP-low (30mg·kg-1) group, VLP-high (50mg·kg-1) group and nimodipine (10mg·kg-1) group. The ischemia reperfusion injury model was induced by occlusion of middle cerebral artery with suture embolus, reperfusion after 30 minutes' ischemia. When the rats were awake, the first neurological function scores was determined with modified neurological severity score (mNSS). The rats were given VLP (30mg·kg-1, 50mg·kg-1) and nimodipine (10mg·kg-1) through intragastric administration at 2 mL, once a day for a total of 7 days, while an equal amount of distilled water was used in the sham operation group and model control group. After 7 days, the rats were given second neurological function scores, and improvement of neurological function=[the first score]-[the second score]. The rats were sacrificed to investigate the infarction volume percentage with 2,3,5-triphenyl tetrazolium chloride method; do the qualitative and half quantitative analyses for protein vascular endothelial growth factor receptor 2 (VEGFR2) in the tissue of cortex infarction around by Western blot; detect the new blood vessels of cortex infarction around by ki67/lectin immunofluorescence double staining method. Results suggest that VLP could significantly improve the neurological function, reduce the percentage of infarct volume, increase the expression of VEGFR2 and number of new blood vessels in the cortex infarction around compared with model group. In conclusion, VLP may relive the acute cerebral ischemia reperfusion injury in rats by inducing angiogenesis.

RESUMEN

The study was taken up to assess if the media constituents played any role in governing the variable colony characteristics or pathogenicity of the bacterial wilt pathogen, Ralstonia solanacearum cultured on the widely employed Kelman medium. The effects due to the constituents 2,3,5-triphenyl tetrazolium chloride (TTC), peptone, casein hydrolysate and glucose on colony characteristics were investigated using -80°C stored culture of strain 'NH-Av01' (race 1, biovar 3) isolated from tomato. Comparing the pigment inducing TTC from two brands, its source or mode of storage/incorporation did not impart any significant effects. The source of peptone, on the other hand, displayed striking effects on the extent of colony growth, fluidity and red pigmentation depending on type, brand or batch / lot of manufacture as documented with 20 different formulations. Significant differences in the pathogenicity of isolate derived from different peptone sources in seedling-challenge assay on tomato were observed. The observations on peptone effects were endorsed with four other isolates belonging to distinct geographic locations, crops (eggplant, chilli, ginger) or races (race 1 or 4). The peptone source did not influence the pathogen-responses in biovar tests but notably altered the pattern of lawn formation and inhibition zone development during antagonistic assays. Casein hydrolysate displayed some variable effects while glucose source had no effect. This study brings to light the significant modifying effects by the peptone-constituent in Kelman medium on the physiology of R. solanacearum and the virulence of isolate and the need to consider the source of media components during culture maintenance, host-pathogen interaction studies or microbe-microbe interaction investigations.

RESUMEN

It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50-100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed 'Brownian motion'-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as 'Cytobacts' and 'Peribacts', respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host.

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Baicalin is one of the principal flavonoids isolated from the dried root of Scutellaria baicalensis Georgi that has long been used to treat ischemic stroke. However, its neuroprotective mechanisms against cerebral ischemia injury are poorly understood. AIM OF THE STUDY: To explore the neuroprotective mechanisms of baicalin against cerebral ischemia reperfusion injury. MATERIAL AND METHODS: In chemical systems, we conducted electron paramagnetic resonance (EPR) spin trapping experiments to evaluate the scavenging effects of baicalin on superoxide and nitric oxide, and mass spectrometry (MS) studies on the reaction of baicalin and peroxynitrite. In cellular experiments, we investigated the effects of baicalin against extraneous and endogenous peroxynitrite mediated neurotoxicity in SH-SY5Y cells treated with peroxynitrite donor, synthesized peroxynitrite and exposed to oxygen glucose deprivation and reoxygenation (OGD/RO) in vitro. Moreover, we studied the neuroprotective effects of baicalin by using a rat model of middle cerebral artery occlusion in vivo. FeTMPyP, a peroxynitrite decomposition catalyst, was used as positive control. Cell viability and apoptotic cell death was accessed by MTT assay and TUNEL assay respectively; 3-nitrotyrosine formation and infarction volume were detected by immunostaining experiments and TTC staining respectively. RESULTS: Baicalin revealed strong antioxidant ability by directly scavenging superoxide and reacting with peroxynitrite. Baicalin protected the neuronal cells from extraneous and endogenous peroxynitrite-induced neurotoxicity. In ischemia-reperfused brains, baicalin inhibited the formation of 3-nitrotyrosine, reduced infarct size and attenuated apoptotic cell death, whose effects were similar to FeTMPyP. CONCLUSIONS: Baicalin can directly scavenge peroxynitrite and the peroxynitrite-scavenging ability contributes to its neuroprotective mechanisms against cerebral ischemia reperfusion injury.

Asunto(s)