The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vescicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells

Spectrofluorometric analysis of autofluorescing components of crude serum from a rat liver model of ischemia and reperfusion

Autofluorescence (AF) of crude serum was investigated with reference to the potential of its intrinsic AF biomarkers for the noninvasive diagnosis of liver injury. Spectral parameters of pure compounds representing retinol (vitamin A) and fluorescing free fatty acids were characterized by spectrofluorometry, to assess spectral parameters for the subsequent AF analysis of serum, collected from rats undergoing liver ischemia/reperfusion (I/R). Differences in AF spectral profiles detected between control and I/R were due to the increase in the AF components representing fatty acids in I/R serum samples. No significant changes occurred for retinol levels, consistently with the literature reporting that constant retinol levels are commonly observed in the blood, except for malnutrition or chronic severe liver disease. Conversely, fatty acids, in particular arachidonic and linoleic acid and their derivatives, act as modulating agents in inflammation, representing both a protective and damaging response to stress stimuli. The biometabolic and pathophysiological meaning of serum components and the possibility of their direct detection by AF spectrofluorometry open up interesting perspectives for the development of AF serum analysis, as a direct, cost effective, supportive tool to assess liver injury and related systemic metabolic alterations, for applications in experimental biomedicine and foreseen translation to the clinics

Fluorescence properties of the Na+/H+ exchanger inhibitor HMA (5-(N,N-hexamethylene) amiloride) are modulated by intracellular Ph

HMA (5-(N,N-hexamethylene)amiloride), which belongs to a family of novel amiloride derivatives, is one of the most effective inhibitors of Na+/H+ exchangers, while uneffective against Na+ channels and Na+/Ca2+ exchangers. In this study, we provided evidence that HMA can act as a fluorescent probe. In fact, human retinal ARPE19 cells incubated with HMA show an intense bluish fluorescence in the cytoplasm when observed at microscope under conventional UV-excitation conditions. Interestingly, a prolonged observation under continuous exposure to excitation lightdoes not induce great changes in cells incubated with HMA for times up to about 5 min, while an unexpected rapid increase in fluorescence signal is observed in cells incubated for longer times. The latter phenomenon is particularly evident in the perinuclear region and in discrete spots in the cytoplasm. Since HMA modulates intracellular acidity, the dependence of its fluorescence properties on medium pH and response upon irradiation have been investigated in solution, at pH 5.0 and pH 7.2. The changes in both spectral shape and amplitude emission indicate a marked pH influence on HMA fluorescence properties, making HMA exploitable as a self biomarker of pH alterations in cell studies, in the absence of perturbations induced by the administration of other exogenous dyes

Stable hybrid myotubes: a new model for studying re-expression of enzymatic activities in vitro

Heterokaryons represent a stable and reproducible model system for the study of biochemical and molecular aspects responsible for muscle gene activation. Previous experiments have used this fusion system to demonstrate human gene activation in hybrids formed between human and non-human cells. The aim of this research was to apply this experimental model to the correction of a cytoplasmic activity, namely glucose-6-phosphate dehydrogenase (G6PD), in vitro, in hybrid myotubes formed between G6PD-negative and positive myoblasts. Different identification methods were used (Hoechst stain and Fluorescent Latex Microspheres, FLMs) to identify hybrid myotubes formed. We demonstrated the restoration of G6PD activity in all hybrid myotubes formed; we then tried to elucidate the mechanisms underlying the restoration of this specific activity and apply the results obtained to the understanding of more complex mechanisms involved in muscle gene activation

Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy

Rose Bengal acetate photodynamic therapy (RBAc–PDT) induced multiple cell death pathways in HeLa cells through ROS and ER stress. Indeed, apoptosis was the first preferred mechanism of death, and it was triggered by at least four different pathways, whose independent temporal activation ensures cell killing when one or several of the pathways are inactivated. Apoptosis occurred as early as 1 h after PDT through activation of intrinsic pathways, followed by activation of extrinsic, caspase-12-dependent and caspase-independent pathways, and by autophagy. The onset of the different apoptotic pathways and autophagy, that in our system had a pro-death role, was timed by determining the levels of caspases 9, 8, 3 and 12; Bcl-2 family; Hsp70; LC3B; GRP78 and phospho-eIF2α proteins. Interestingly, inhibition of one pathway, that is, caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting that the independent onset of the different apoptotic pathways and autophagy did not occur in a subordinated manner. Altogether, our data indicate RBAc as a powerful photosensitiser that induces a prolonged cytotoxicity and time-related cell death onset by signals originating from or converging on almost all intracellular organelles. The fact that cancer cells can die through different mechanisms is a relevant clue in the choice and design of anticancer PDT

The effects of multi-domain versus single-domain cognitive training in non-demented older people: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Whether healthy older people can benefit from cognitive training (CogTr) remains controversial. This study explored the benefits of CogTr in community dwelling, healthy, older adults and compared the effects of single-domain with multi-domain CogTr interventions.</p> <p>Methods</p> <p>A randomized, controlled, 3-month trial of CogTr with double-blind assessments at baseline and immediate, 6-month and 12-month follow-up after training completion was conducted. A total of 270 healthy Chinese older people, 65 to 75 years old, were recruited from the Ganquan-area community in Shanghai. Participants were randomly assigned to three groups: multi-domain CogTr, single-domain CogTr, and a wait-list control group. Twenty-four sessions of CogTr were administrated to the intervention groups over a three-month period. Six months later, three booster training sessions were offered to 60% of the initial training participants. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS, Form A), the Color Word Stroop test (CWST), the Visual Reasoning test and the Trail Making test (TMT) were used to assess cognitive function.</p> <p>Results</p> <p>Multi-domain CogTr produced statistically significant training effects on RBANS, visual reasoning, and immediate and delayed memory, while single-domain CogTr showed training effects on RBANS, visual reasoning, word interference, and visuospatial/constructional score (all <it>P </it>< 0.05). At the 12-month posttest, the multi-domain CogTr showed training effects on RBANS, delayed memory and visual reasoning, while single-domain CogTr only showed effects on word interference. Booster training resulted in effects on RBANS, visual reasoning, time of trail making test, and visuospatial/constructional index score.</p> <p>Conclusions</p> <p>Cognitive training can improve memory, visual reasoning, visuospatial construction, attention and neuropsychological status in community-living older people and can help maintain their functioning over time. Multi-domain CogTr enhanced memory proficiency, while single-domain CogTr augmented visuospatial/constructional and attention abilities. Multi-domain CogTr had more advantages in training effect maintenance.</p> <p>Clinical Trial Registration</p> <p>Chinese Clinical Trial Registry. Registration number: ChiCTR-TRC-09000732.</p

In vivo fluorescence imaging of the transport of charged chlorine6 conjugates in a rat orthotopic prostate tumour

Polymeric drug conjugates are used in cancer therapy and, varying their molecular size and charge, will affect their in vivo transport and extravasation in tumours. Partitioning between tumour vasculature and tumour tissue will be of particular significance in the case of photosensitizer conjugates used in photodynamic therapy, where this partitioning can lead to different therapeutic effects. Poly-l-lysine chlorine6 conjugates (derived from polymers of averageMr 5000 and 25 000) were prepared both in a cationic state and by poly-succinylation in an anionic state. A fluorescence scanning laser microscope was used to follow the pharmacokinetics of these conjugates in vivo in an orthotopic rat prostate cancer model obtained with MatLyLu cells. Fluorescence was excited with the 454–528 nm group of lines of an argon laser and a 570 nm long pass filter used to isolate the emission. Results showed that the conjugates initially bound to the walls of the vasculature, before extravasating into the tissue, and eventually increasing in fluorescence. The anionic conjugates produced tissue fluorescence faster than the cationic ones, and surprisingly, the largerMr conjugates produced tissue fluorescence faster than the smaller ones with the same charge. These results are consistent with differences in aggregation state between conjugates. © 1999 Cancer Research Campaig

The Edinburgh Social Cognition Test (ESCoT):Examining the effects of age on a new measure of theory of mind and social norm understanding

<div><p>Current measures of social cognition have shown inconsistent findings regarding the effects of healthy aging. Moreover, no tests are currently available that allow clinicians and researchers to examine cognitive and affective theory of mind (ToM) and understanding of social norms within the same test. To address these limitations, we present the Edinburgh Social Cognition Test (ESCoT) which assesses cognitive and affective ToM and inter- and intrapersonal understanding of social norms. We examined the effects of age, measures of intelligence and the Broader Autism Phenotype (BAP) on the ESCoT and established tests of social cognition. Additionally, we investigated the convergent validity of the ESCoT based on traditional social cognition measures. The ESCoT was administered alongside Reading the Mind in Films (RMF), Reading the Mind in Eyes (RME), Judgement of Preference and Social Norm Questionnaire to 91 participants (30 aged 18–35 years, 30 aged 45–60 years and 31 aged 65–85 years). Poorer performance on the cognitive and affective ToM ESCoT subtests were predicted by increasing age. The affective ToM ESCoT subtest and RMF were predicted by gender, where being female predicted better performance. Unlike the ESCoT, better performance on the RMF was predicted by higher verbal comprehension and perceptual reasoning abilities, while better performance on the RME was predicted by higher verbal comprehension scores. Lower scores on inter-and intrapersonal understanding of social norms were both predicted by the presence of more autism-like traits while poorer interpersonal understanding of social norms performance was predicted by increasing age. These findings show that the ESCoT is a useful measure of social cognition and, unlike established tests of social cognition, performance is not predicted by measures of verbal comprehension and perceptual reasoning. This is particularly valuable to obtain an accurate assessment of the influence of age on our social cognitive abilities.</p></div