Synthesis and characterization of Guar gum based biopolymeric hydrogels as carrier materials for controlled delivery of methotrexate to treat colon cancer

Guar Gum has been evaluated for its importance in food and pharmaceutical industry. A blended biopolymeric hydrogel was prepared by solution casting technique using guar gum (GG), chitosan (CS), polyvinyl alcohol (PVA), chemically crosslinked with tetra orthosilicate (TEOS) and impregnated with methotrexate (MTX) to assess its drug carrying capacity against colon cancer (HCT-116). The surface morphology, chemical bonding, hydrophilicity and water absorbing capacity were analyzed by atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), contact angle measurements and swelling properties in variable conditions. Furthermore, degradation, drug release kinetics, hemocompatibility, and cytotoxicity of MTX-loaded hydrogel was tested. The release of MTX from GG/CS/PVA biopolymeric blend occurred in sustained manner. Results displayed that in 7 h 25 min duration 96% of the drug was released in phosphate buffer saline (PBS) at pH 7.4. These blends were non-hemolytic, and antiproliferative against HCT-116. Furthermore, the MTT assay has revealed that MTX-loaded hydrogel had prominently decreased the cell viability (with IC50 11.7 µg/ml) as compared to free MTX (with IC50 21.57 µg/ml). Hence, these results suggest that guar gum based hydrogels are potential biomaterials for colon cancer treatment.     

Mitochondrial impairment observed in fibroblasts from South African Parkinson’s disease patients with parkin mutations

Parkinson’s disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients’ fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.     

Cellular iron homeostasis mediated by the Mrs4–Ccc1–Smf3 pathway is essential for mitochondrial function,morphogenesis and virulence in Candida albicans

Iron bioavailability is crucial for mitochondrial metabolism and biosynthesis. Dysregulation of cellular iron homeostasis affects multiple aspects of mitochondrial physiology and cellular processes. However, the intracellular iron trafficking pathway in Candida albicans remains unclear. In this study, we characterized the Mrs4–Ccc1–Smf3 pathway, and demonstrated its important role in maintaining cellular iron levels. Double deletion of vacuolar iron exporter SMF3 and mitochondrial iron transporter MRS4 further elevated cellular iron levels in comparison with the single MRS4 deletion. However, deletion of vacuolar iron importer CCC1 in the mrs4?/? mutant restored cellular iron homeostasis to normal wild-type levels, and also normalized most of the defective phenotypes in response to various environmental stresses. Our results also suggested that both Mrs4 and Ccc1 contributed to the maintenance of mitochondrial function. The mrs4?/? and mrs4?/?smf3?/? mutants exhibited an obvious decrease in aconitase activities and mitochondrial membrane potential, whereas deletion of CCC1 in the mrs4?/? mutant effectively rescued these defects. Furthermore, we also found that the Mrs4–Ccc1–Smf3 pathway was indispensable for cell-wall stability, antifungal drug tolerance, filamentous growth and virulence, supporting the novel viewpoint that mitochondria might be the promising target for better antifungal therapies. Interestingly, the addition of exogenous iron failed to rescue the defects on non-fermentable carbon sources or hyphae-inducing medium, indicating that the defects in mitochondrial respiration and filamentous development might result from the disturbance of cellular iron homeostasis rather than environmental iron deprivation. Taken together, our results propose the Mrs4–Ccc1–Smf3 pathway as a potentially attractive target for antifungal drug development.     

Can we optimise the exercise training prescription to maximise improvements in mitochondria function and content?

Background

While there is agreement that exercise is a powerful stimulus to increase both mitochondrial function and content, we do not know the optimal training stimulus to maximise improvements in mitochondrial biogenesis.

Scope of review

This review will focus predominantly on the effects of exercise on mitochondrial function and content, as there is a greater volume of published research on these adaptations and stronger conclusions can be made.

Major conclusions

The results of cross-sectional studies, as well as training studies involving rats and humans, suggest that training intensity may be an important determinant of improvements in mitochondrial function (as determined by mitochondrial respiration), but not mitochondrial content (as assessed by citrate synthase activity). In contrast, it appears that training volume, rather than training intensity, may be an important determinant of exercise-induced improvements in mitochondrial content. Exercise-induced mitochondrial adaptations are quickly reversed following a reduction or cessation of physical activity, highlighting that skeletal muscle is a remarkably plastic tissue. Due to the small number of studies, more research is required to verify the trends highlighted in this review, and further studies are required to investigate the effects of different types of training on the mitochondrial sub-populations and also mitochondrial adaptations in different fibre types. Further research is also required to better understand how genetic variants influence the large individual variability for exercise-induced changes in mitochondrial biogenesis.

General significance

The importance of mitochondria for both athletic performance and health underlines the importance of better understanding the factors that regulate exercise-induced changes in mitochondrial biogenesis. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Evaluation of the BH3-only Protein Puma as a Direct Bak Activator

Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as “direct activators” that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (K_D = 26 ± 5 nm) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator.     

A PCR-based toolbox for the culture-independent quantification of total bacterial abundances in plant environments

A major obstacle in the culture-independent estimation of the abundance of bacteria associated with plants is contamination with plant organelles, which precludes the use of universal rRNA bacterial primers in quantitative PCR applications. We present here a PCR-based method that allows a priori determination of the degree of chloroplast and mitochondrial contamination in DNA samples from plant environments. It is based on differential digestibility of chloroplast, mitochondrial and bacterial small subunit rRNA gene amplicons with the restriction enzymes AfeI and BbvCI. Using this method, we demonstrated for field-grown lettuce plants that even a gentle washing protocol, designed to recover the microbial community and its metagenome from the leaf surface, resulted in substantial contamination with chloroplast DNA. This finding cautions against the use of universal primer pairs that do not exclude chloroplast DNA from amplification, because they risk overestimation of bacterial population sizes. In contrast, contamination with mitochondrial 18S rRNA was minor in the lettuce phyllosphere. These findings were confirmed by real-time PCR using primer sets specific for small subunit rRNA genes from bacteria, chloroplasts, and mitochondria. Based on these results, we propose two primer pairs (534f/783r and mito1345f/mito1430r) which between them offer an indirect means of faithfully estimating bacterial abundances on plants, by deduction of the mito1345f/mito1430r-based mitochondrial count from that obtained with 534f/783r, which amplifies both bacterial and mitochondrial DNA but excludes chloroplast. In this manner, we estimated the number of total bacteria on most leaves of field-grown lettuce to be between 10⁵ and 10⁶ g^− 1 of leaf, which was 1-3 orders of magnitudes higher than the number of colony-forming units that were retrieved from the same leaf surfaces on agar plates.     

Dynamics of auxin-dependent Ca2+ and pH signaling in root growth revealed by integrating high-resolution imaging with automated computer vision-based analysis

Plants adapt to a changing environment by entraining their growth and development to prevailing conditions. Such 'plastic' development requires a highly dynamic integration of growth phenomena with signal perception and transduction systems, such as occurs during tropic growth. The plant hormone auxin has been shown to play a key role in regulating these directional growth responses of plant organs to environmental cues. However, we are still lacking a cellular and molecular understanding of how auxin-dependent signaling cascades link stimulus perception to the rapid modulation of growth patterns. Here, we report that in root gravitropism of Arabidopsis thaliana, auxin regulates root curvature and associated apoplastic, growth-related pH changes through a Ca2+-dependent signaling pathway. Using an approach that integrates confocal microscopy and automated computer vision-based image analysis, we demonstrate highly dynamic root surface pH patterns during vertical growth and after gravistimulation. These pH dynamics are shown to be dependent on auxin, and specifically on auxin transport mediated by the auxin influx carrier AUX1 in cells of the lateral root cap and root epidermis. Our results further indicate that these pH responses require auxin-dependent changes in cytosolic Ca2+ levels that operate independently of the TIR1 auxin perception system. These results demonstrate a methodology that can be used to visualize vectorial auxin responses in a manner that can be integrated with the rapid plant growth responses to environmental stimuli.     

Ploidy and strain differences in seed germination of Glycine wightii at different pH levels

Summary Seeds from 27 wild strains (18 tetraploids and 9 diploids) of Glycine weightii were germinated at a pH range of 5 to 8. The differences in germination (%) between all the strains were highly significant but between pH levels they were only nearly significant (P=0.067) with no interaction between pH levels and strains. Mean germination (%) for all tetraploids seems to be slightly higher ( 2%) than that for all diploids, especially at pH's 5, 7 and 8 but this may be due to the significantly longer time ( one day) it took tetraploids to complete germination. The apparent inverse relationship between seed weight and germination (%) was not significant.Mean germination time was highly significant for strains, pH's and their interaction. Increasing mean germination (%) resulted in decreasing mean germination time among strains. Large seeds took less time to germinate especially those from some of the tetraploid strains. This indicates that it is possible to produce a variety with high germination (%), fast germination rate and possibly large seeds. If the marked difference in pH tolerance among strains will prove to be mainly hereditary, then it will be also possible to select for either specific pH tolerance or tolerance at a wide range of pH.     

Phosphate uptake and polyphosphate metabolism of mycorrhizal and nonmycorrhizal roots of pine and of Suillus bovinus at varying external pH measured by in vivo 31P-NMR

 Comparative in vivo ³¹P-NMR analyses of mycorrhizal and nonmycorrhizal roots of Pinus sylvestris and the fungus of Suillus bovinus in pure culture were used to investigate alterations in phosphate metabolism due to changes in external pH in the range 3.5–8.5. All control samples maintained a constant pH in both cytoplasm and vacuole. Mycorrhizal roots and pure fungus, but not nonmycorrhizal roots, transformed accumulated inorganic phosphate into mobile polyphosphate with a medium chain length. Phosphate uptake rates and polyphosphate accumulation responded differently to external pH. In all cases, maximal phosphate uptake occurred at an external pH close to 5.5. At an external pH of 8.5, both roots and fungus showed a distinct lag in phosphate uptake, which was abolished when the external pH was lowered to 7.5. An irreversible effect on phosphate uptake as a consequence of variation in external pH was also observed. The central role of the fungus in regulating mycorrhizal phosphate metabolism is discussed. Accepted: 15 April 1997