Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

<Emphasis Type="Italic">In vitro</Emphasis> sucrose concentration influences microtuber production and diosgenin content in white yam (<Emphasis Type="Italic">Dioscorea rotundata</Emphasis> Poir)

Dioscorea spp. is an important food crop in many countries and the source of the phytochemical diosgenin. Efficient microtuber production could provide source materials for farm-planting stock, for food markets, and for the production of high-diosgenin-producing cultivars. The first step in this study was optimizing the plant growth regulators for plantlet production, followed by a study of the effects of sucrose concentration on microtuber induction and diosgenin production. Significantly, more shoots (3.5) were produced at 4.65 μM (1 mg L^?1) kinetin (KIN), longer shoots (4.1 cm) were obtained at 2.46 μM (0.5 mg L^?1) indole-3-butyric acid (IBA), and root number (3.9) was significantly higher at 5.38 μM (1 mg L^?1) naphthalene acetic acid (NAA) than in other treatments. Increased sucrose concentrations in the optimized growth medium with 4.65 μM KIN and 5.38 μM NAA had significant effects on microtuber production (p < 0.01) and diosgenin content (p < 0.05). The most microtubers (6.2) were obtained with 100 g L^?1 sucrose, while those on 80 g L^?1 sucrose were the heaviest (0.7 g) and longest (7.4 mm). Microtubers formed in medium with 80 g L^?1 sucrose had significantly higher diosgenin content (3.64% [w/w]) than those in other sucrose treatments (< 2%) and was similar to that of field-grown parent tubers (3.79%). This result indicates an important role for sucrose in both microtuber growth and diosgenin production. Medium containing 4.65 μM KIN and 5.38 μM NAA is recommended for plantlet production, and medium containing 80 g L^?1 sucrose is recommended for microtuber and diosgenin production.     

Antioxidant and anti-stress compounds improve regrowth of cryopreserved <Emphasis Type="Italic">Rubus</Emphasis> shoot tips

Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB), and polyvinylpyrrolidone (PVP) were tested during cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro-grown blackberry cultivars were cold acclimated and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading, and rinsing for ‘Chehalem’ and at all steps for ‘Hull Thornless’. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH, and GB improved regrowth by ∼25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols.     

Improving the clinical assessment of consciousness with advances in electrophysiological and neuroimaging techniques

In clinical neurology, a comprehensive understanding of consciousness has been regarded as an abstract concept - best left to philosophers. However, times are changing and the need to clinically assess consciousness is increasingly becoming a real-world, practical challenge. Current methods for evaluating altered levels of consciousness are highly reliant on either behavioural measures or anatomical imaging. While these methods have some utility, estimates of misdiagnosis are worrisome (as high as 43%) - clearly this is a major clinical problem. The solution must involve objective, physiologically based measures that do not rely on behaviour. This paper reviews recent advances in physiologically based measures that enable better evaluation of consciousness states (coma, vegetative state, minimally conscious state, and locked in syndrome). Based on the evidence to-date, electroencephalographic and neuroimaging based assessments of consciousness provide valuable information for evaluation of residual function, formation of differential diagnoses, and estimation of prognosis.     

Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and β-Catenin Signaling

Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of β-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-β inhibitor SB431542 and the Wnt/β-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and β-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Renal Carcinogenesis After Uninephrectomy

Nephrectomized rats have widely been used to study chronic renal failure. Interestingly, renal cell carcinoma occurred in the remnant kidney after uninephrectomy (UNX). In this study, we probed insulin-like growth factor (IGF)-1 signaling pathway in UNX-induced renal cancer. Adult male Sprague-Dawley rats were randomized into two groups: UNX rats (n = 22) and sham-operated rats (n = 12). Rats were killed at 3, 7, and 10 months. After 7 months after nephrectomy, the UNX rats developed renal cell carcinoma with increased expression of proliferating cell nuclear antigen, and 68.2% (15/22) of the animals exhibited invasive carcinoma. Western blot demonstrated significant down-regulation of IGF binding protein 3 contrasting with the up-regulation of protein kinase Cζ and Akt/protein kinase B in the renal cancer tissues. These findings indicate a unique rat model of UNX-induced renal cancer associated with enhanced IGF-1 signaling pathway.     

Application of magnetic techniques in the field of drug discovery and biomedicine

Magnetic separation technology, using magnetic particles, is quick and easy method for sensitive and reliable capture of specific proteins, genetic material and other biomolecules. The technique offers an advantage in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization. Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and SNP analysis, ultimately leading to identification of novel drug targets. In this post-genomic era, technologies based on magnetic separation are becoming an integral part of todays biology laboratory. This article briefly reviews the selected applications of magnetic separation techniques in the field of biotechnology, biomedicine and drug discovery.     

Vitamins C and E improve regrowth and reduce lipid peroxidation of blackberry shoot tips following cryopreservation

Oxidative processes involved in cryopreservation protocols may be responsible for the reduced viability of tissues after liquid nitrogen exposure. Antioxidants that counteract these reactions should improve recovery. This study focused on oxidative lipid injury and the effects of exogenous vitamin E (tocopherol, Vit E) and vitamin C (ascorbic acid, Vit C) treatments on regrowth at four critical steps of the plant vitrification solution number 2 (PVS2) vitrification cryopreservation technique; pretreatment, loading, rinsing, and regrowth. Initial experiments showed that Vit E at 11–15 mM significantly increased regrowth (P < 0.001) when added at any of the four steps. There was significantly more malondialdehyde (MDA), a lipid peroxidation product, at each of the steps than in fresh untreated shoot tips. Vit E uptake was assayed at each step and showed significantly more α- and γ-tocopherols in treated shoots than those without Vit E. Vit E added at each step significantly reduced MDA formation and improved shoot regrowth. Vit C (0.14–0.58 mM) also significantly improved regrowth of shoot tips at each step compared to the controls. Regrowth medium with high iron concentrations and Vit C decreased recovery. However, in iron-free medium, Vit C significantly improved recovery. Treatments with Vit E (11 mM) and Vit C (0.14 mM) combined were not significantly better than Vit C alone. We recommend adding Vit C (0.28 mM) to the pretreatment medium, the loading solution or the rinse solution in the PVS2 vitrification protocol. This is the first report of the application of vitamins for improving cryopreservation of plant tissues by minimizing oxidative damage.     

The use of invertebrate functional groups to characterize ecosystem attributes in selected streams and rivers in south Brazil

An analysis conducted at nine stream/river sites in the Atlantic Forest region in the State of Paraná, Brazil used macroinvertebrate functional feeding group (FFG) assessments to evaluate ecological condition of the sites. The FFG approach categorizes qualitative macroinvertebrate collections according to their morphological-behavioral adaptations for food acquisition (e.g. scrapers that harvest non-filamentous, attached algae from stable surfaces in flowing water). FFG ratios were employed as surrogates for stream/river ecosystem attributes: balance between autotrophy and heterotrophy; linkage between riparian inputs of coarse particulate organic matter and in-stream food webs; relative dominance of fine particulate organic matter in transport (suspended load) compared to that deposited in the sediments; and geomorphic stability of the channel. The analyses indicated that all nine sites were heterotrophic, six of the nine carried expected levels of suspended organic load and showed below expected linkage with riparian inputs, and in only two were stable substrates limiting. The implications of the findings and recommendations for further analysis and modifications of the protocol are discussed.