Lake Tana large barbs diversity: Developmental and hormonal bases

Helerochronies caused by changes in the hormonal status are proposed to play a major role in the diversification of the Lake Tana barbs of Labeobarbus (=Barbus) genus (Dgebuadze and Mina, 2008). To test this hypothesis, we (i) compared temporal patterns of cranial development in L. intermedius, likely ancestral for Tana species flock, and L. brevicephalus and L. megastoma proposed to be paedomoiphic and peramoiphic, respectively; (ii) studied the role of thyroid hormone (TH) in the regulation of L. intermedius development; (iii) evaluated the effect of TH changes on the definitive morphology, and (iv) evaluated the possible role of TH in diversification of Tana barbs. Skull development was found to be heterochronicaily changed in L. brevicephalus and L. megastoma as compared to L. intermedius; revealed heteroclironies may account for differences in skull proportions distinguishing these barbs, TH was shown to play a major role in the regulation of ontogeny. Changes in TH level cause heterochronies in the development of a skull, axial skeleton, fins and fin girdles, squamation, and dentition. Heteroclironies were shown to influence adult morphology of these structures. These findings support the hypothesis of the involvement of developmental and hormonal factors in the diversification of the Lake Tana large barbs.     

Withaferin A Induces Proteasome Inhibition,Endoplasmic Reticulum Stress,the Heat Shock Response and Acquisition of Thermotolerance

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.     

Induction of stress resistance and extension of lifespan in Chaenorhabditis elegans serotonin-receptor knockout strains by withanolide A

IntroductionApproximately 300 million people worldwide suffer from depression. The COVID-19 crisis may dramatically increase these numbers. Severe side effects and resistance development limit the use of standard antidepressants. The steroidal lactone withanolide A (WA) from Withania somnifera may be a promising alternative. Caenorhabditis elegans was used as model to explore WA's anti-depressive and anti-stress potential.MethodsC. elegans wildtype (N2) and deficient strains (AQ866, DA1814, DA2100, DA2109 and MT9772) were used to assess oxidative, osmotic or heat stress as measured by generation of reactive oxygen species (ROS), determination of lifespan, and mRNA expression of serotonin receptor (ser-1, ser-4, ser-7) and serotonin transporter genes (mod-5). The protective effect of WA was compared to fluoxetine as clinically established antidepressant. Additionally, WA's effect on lifespan was determined. Furthermore, the binding affinities and pKi values of WA, fluoxetine and serotonin as natural ligand to Ser-1, Ser-4, Ser-7, Mod-5 and their human orthologues proteins were calculated by molecular docking.ResultsBaseline oxidative stress was higher in deficient than wildtype worms. WA and fluoxetine reduced ROS levels in all strains except MT9772. WA and fluoxetine prolonged survival times in wildtype and mutants under osmotic stress. WA but not fluoxetine increased lifespan of all heat-stressed C. elegans strains except DA2100. Furthermore, WA but not fluoxetine extended lifespan in all non-stressed C. elegans strains. WA also induced mRNA expression of serotonin receptors and transporters in wildtype and mutants. WA bound with higher affinity and lower pKi values to all C. elegans and human serotonin receptors and transporters than serotonin, indicating that WA may competitively displaced serotonin from the binding pockets of these proteins.ConclusionWA reduced stress and increased lifespan by ROS scavenging and interference with the serotonin system. Hence, WA may serve as promising candidate to treat depression.     

Growth Arrest by the Antitumor Steroidal Lactone Withaferin A in Human Breast Cancer Cells Is Associated with Down-regulation and Covalent Binding at Cysteine 303 of β-Tubulin

Withaferin A (WA), a C₅,C₆-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G₂ and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of β-tubulin. These effects were not observed with the naturally occurring C₆,C₇-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys³⁰³ of β-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.     

Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1

The rice (Oryza sativa L.) homeobox gene OSH1 causes morphological alterations when ectopically expressed in transgenic rice, Arabidopsis thaliana, and tobacco (Nicotiana tabacum L.) and is therefore believed to function as a morphological regulator gene. To determine the relationship between OSH1 expression and morphological alterations, we analyzed the changes in hormone levels in transgenic tobacco plants exhibiting abnormal morphology. Levels of the plant hormones indole-3-acetic acid, abscisic acid, gibberellin (GA), and cytokinin (zeatin and trans-zeatin [Z]) were measured in leaves of OSH1-transformed and wild-type tobacco. Altered plant morphology was found to correlate with changes in hormone levels. The more severe the alteration in phenotype of transgenic tobacco, the greater were the changes in endogenous hormone levels. Overall, GA₁ and GA₄ levels decreased and abscisic acid levels increased compared with wild-type plants. Moreover, in the transformants, Z (active form of cytokinin) levels were higher and the ratio of Z to Z riboside (inactive form) also increased. When GA₃ was supplied to the shoot apex of transformants, internode extension was restored and normal leaf morphology was also partially restored. However, such GA₃-treated plants still exhibited some morphological abnormalities compared with wild-type plants. Based on these data, we propose the hypothesis that OSH1 affects plant hormone metabolism either directly or indirectly and thereby causes changes in plant development.     

Chronic murine toxoplasmosis is defined by subtle changes in neuronal connectivity

Recent studies correlate chronic Toxoplasma gondii (T. gondii) infection with behavioral changes in rodents; additionally, seropositivity in humans is reported to be associated with behavioral and neuropsychiatric diseases. In this study we investigated whether the described behavioral changes in a murine model of chronic toxoplasmosis are associated with changes in synaptic plasticity and brain neuronal circuitry. In mice chronically infected with T. gondii, magnetic resonance imaging (MRI) data analysis displayed the presence of heterogeneous lesions scattered throughout all brain areas. However, a higher density of lesions was observed within specific regions such as the somatosensory cortex (SSC). Further histopathological examination of these brain areas indicated the presence of activated resident glia and recruited immune cells accompanied by limited alterations of neuronal viability. In vivo diffusion-tensor MRI analysis of neuronal fiber density within the infected regions revealed connectivity abnormalities in the SSC. Altered fiber density was confirmed by morphological analysis of individual, pyramidal and granule neurons, showing a reduction in dendritic arbor and spine density within the SSC, as well as in the hippocampus. Evaluation of synapse efficacy revealed diminished levels of two key synaptic proteins, PSD95 and synaptophysin, within the same brain areas, indicating deficits in functionality of the synaptic neurotransmission in infected mice. Our results demonstrate that persistent T. gondii infection in a murine model results in synaptic deficits within brain structures leading to disturbances in the morphology of noninfected neurons and modified brain connectivity, suggesting a potential explanation for the behavioral and neuropsychiatric alterations.KEY WORDS: Parasites, Behavioral manipulation, Neuronal connectivity     

Congruence between comparative morphology and molecular phylogenies: the final stage of evolution of the skeletal characters of male genitalia in the subtribe Polyommatina (Lepidoptera,Lycaenidae). Part 2. Uncus and subunci

The morphology of the male terminalia (uncus and subunci) was studied in 39 species of the subtribe Polyommatina (Lycaenidae). Traces of nine established characters were mapped on the molecular phylogeny of the subtribe proposed by Talavera et al. (2013). The ancestral character states for these morphological structures were established in Plebejus s. l. and Polyommatus s. l. According to the time calibration of the proposed molecular tree, the studied skeletal structures (uncus and subunci) evolved very slowly within the last million years. It was shown that the similar shape of the uncus and subuncus branches arose independently and underwent parallel morphological transformations. In particular, wide and, as a rule, ventrally directed uncus lobes arose independently in Polyommatus s. str., Alpherakya, Glabroculus, and Aricia. Yet, in various evolutionary lines of Polyommatina similar changes in the length ratio of the proximal and distal parts of the subunci as well as in the shape of their apical hooks were observed. The character states marking the groups Polyommatus s. str. and Aricia were established. The evolutionary changes in the uncus morphology were highly non-uniform in the various monophyletic lines of Polyommatina. As a result, representatives of some “old” lines have more specialized features of the uncus morphology in comparison with those belonging to “young” lines that retain ancestral character states. In Polyommatina, the transformation of the male terminalia was directed toward development of the most effective mechanism of female fixation during copulation. This goal was reached in similar ways in various evolutionary lines of Polyommatina, which explains numerous parallelisms in morphology of these structures.     

Complete Genomic Sequence and Comparative Analysis of the Genome Segments of Sweet Potato Chlorotic Stunt Virus in China

Background

Sweet potato chlorotic stunt virus (family Closteroviridae, genus Crinivirus) features a large bipartite, single-stranded, positive-sense RNA genome. To date, only three complete genomic sequences of SPCSV can be accessed through GenBank. SPCSV was first detected from China in 2011, only partial genomic sequences have been determined in the country. No report on the complete genomic sequence and genome structure of Chinese SPCSV isolates or the genetic relation between isolates from China and other countries is available.

Methodology/Principal Findings

The complete genomic sequences of five isolates from different areas in China were characterized. This study is the first to report the complete genome sequences of SPCSV from whitefly vectors. Genome structure analysis showed that isolates of WA and EA strains from China have the same coding protein as isolates Can181-9 and m2-47, respectively. Twenty cp genes and four RNA1 partial segments were sequenced and analyzed, and the nucleotide identities of complete genomic, cp, and RNA1 partial sequences were determined. Results indicated high conservation among strains and significant differences between WA and EA strains. Genetic analysis demonstrated that, except for isolates from Guangdong Province, SPCSVs from other areas belong to the WA strain. Genome organization analysis showed that the isolates in this study lack the p22 gene.

Conclusions/Significance

We presented the complete genome sequences of SPCSV in China. Comparison of nucleotide identities and genome structures between these isolates and previously reported isolates showed slight differences. The nucleotide identities of different SPCSV isolates showed high conservation among strains and significant differences between strains. All nine isolates in this study lacked p22 gene. WA strains were more extensively distributed than EA strains in China. These data provide important insights into the molecular variation and genomic structure of SPCSV in China as well as genetic relationships among isolates from China and other countries.     

Peg viability and pod set in peanut: Response to impaired pegging and water deficit

Fertilized peanut (Arachis hypogaea L.) ovaries develop into aerial gynophores known as pegs, which are supposed to endure delayed penetration into the soil (pegging) caused by increased surface soil strength promoted by drought. There is no information, however, on the pattern of decay in peg viability in response to impaired pegging duration, which may affect seed yield severely. Two peanut cultivars (Florman and ASEM) were grown in pots under two contrasting water availability levels (WA) imposed at the R2 growth stage (start of peg formation). Pegs of ca. 5 mm were tagged at this stage, and WA extended for 10 different periods (between 7 and 41 days) of restriction to pegging (RP_n). Tagged pegs were used for analysis of histological changes and pod set evaluation. Reduced WA caused a significant (P ≤ 0.001) decrease in peg viability and pod set, but no negative effect was detected on these traits for at least 11 days of treatment. The extent of maximum peg viability (stage 1) was shorter for water deficit (11 days of RP) than for well-watered plants (15 days of RP), and was followed by a phase of linear decrease (maximum rate between −0.056 and −0.073 days⁻¹) in peg viability (stage 2). The latter finished at ca. 33 days of RP, with permanent loss in peg viability (stage 3). Tissue deterioration began at the start of stage 2, until complete atrophy was reached at the start of stage 3. This trend proceeded faster for water-deficit pots and cultivar Florman.     

Participation of Actin on Giardia lamblia Growth and Encystation

Background

Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia.

Methodology and Principal Findings

By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45%) and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of flourescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%).

Conclusions and Significance

All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression.     

Mitochondrial biogenesis in Neurospora crassa. I. An ultrastructural and biochemical investigation of the effects of anaerobiosis and chloramphenicol inhibition

The isolation of a new class of mutants permitting facultative anaerobiosis in Neurospora crassa is described. Backcross analyses to the obligate aerobe prototroph (An ^-) indicate single nuclear gene inheritance (An ^-/An ⁺). An ⁺ and An ^- are indistinguishable in morphology and growth rates under aerobic conditions. Anaerobic growth requires nutritional supplements that are dispensable for aerobic growth. Conidiogenesis, conidial germination, and vegetative growth rate are suppressed by anaerobiosis. An ⁺ mutants produce substantial quantities of ethanol under anaerobic conditions. Anaerobiosis and chloramphenicol both affect mitochondrial enzyme activity and morphology. Chloramphenicol inhibition leads to reduction in cytochrome oxidase and swollen mitochondria with few cristae. Anaerobiosis leads to reduction in both cytochrome oxidase and malate dehydrogenase activities, enlarged mitochondria with fewer cristae, enlarged nuclei, and other alterations in cellular morphology. The fine structure of anaerobically grown cells changes with the time of anaerobic growth. We conclude that either inhibition of mitochondrial membrane synthesis or inhibition of respiration might lead to the observed alterations in mitochondria.     

Mitochondrial alterations and apoptosis in smooth muscle from aged rats

We studied changes in mitochondrial morphology and function in the smooth muscle of rat colon. Under confocal microscopy, tissues loaded with potentiometric dye displayed rapid and spontaneous depolarization. Cyclosporin A (CsA), inhibitor of the permeability transition pore (PTP), caused an increase in mitochondrial membrane potential (ΔΨ_m) in tissues from adult young animals. In aged rats these changes were not observed. This suggests that physiological activation of PTP in aged rats is reduced. Electron microscopy showed alterations of the mitochondrial ultrastructure in tissues from aged rats involving a decreased definition of the cristae and fragmentation of the mitochondrial membranes. We also detected an increase in apoptotic cells in the smooth muscle from aged animals. Our results show that the aging process changes PTP activity, the ability to maintain ΔΨ_m and mitochondrial morphology. It is suggested that these can be associated with mitochondrial damage and cell death.     

Angiostrongylus cantonensis: Scanning Electron Microscopic Observations on the Cuticle of Moulting Larvae

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.     

Non-Specific Manipulation of Gammarid Behaviour by P. minutus Parasite Enhances Their Predation by Definitive Bird Hosts

Trophically-transmitted parasites often change the phenotype of their intermediate hosts in ways that increase their vulnerability to definitive hosts, hence favouring transmission. As a “collateral damage”, manipulated hosts can also become easy prey for non-host predators that are dead ends for the parasite, and which are supposed to play no role in transmission strategies. Interestingly, infection with the acanthocephalan parasite Polymorphus minutus has been shown to reduce the vulnerability of its gammarid intermediate hosts to non-host predators, whose presence triggered the behavioural alterations expected to favour trophic transmission to bird definitive hosts. Whilst the behavioural response of infected gammarids to the presence of definitive hosts remains to be investigated, this suggests that trophic transmission might be promoted by non-host predation risk. We conducted microcosm experiments to test whether the behaviour of P. minutus-infected gammarids was specific to the type of predator (i.e. mallard as definitive host and fish as non-host), and mesocosm experiments to test whether trophic transmission to bird hosts was influenced by non-host predation risk. Based on the behaviours we investigated (predator avoidance, activity, geotaxis, conspecific attraction), we found no evidence for a specific fine-tuned response in infected gammarids, which behaved similarly whatever the type of predator (mallard or fish). During predation tests, fish predation risk did not influence the differential predation of mallards that over-consumed infected gammarids compared to uninfected individuals. Overall, our results bring support for a less sophisticated scenario of manipulation than previously expected, combining chronic behavioural alterations with phasic behavioural alterations triggered by the chemical and physical cues coming from any type of predator. Given the wide dispersal range of waterbirds (the definitive hosts of P. minutus), such a manipulation whose efficiency does not depend on the biotic context is likely to facilitate its trophic transmission in a wide range of aquatic environments.     

Indicating Soil Acidity Using Vegetation Relevés in Spatially Limited Areas – Case Study from the Považský Inovec, Slovakia

A fine-scaled approach for predicting soil acidity using plant species in a spatially limited area (?epú?ky Nature Reserve, Slovakia) is presented here. This approach copes with some specific limitations: i) a limited pool of vegetation data may make the predictions too sensitive to the lack of species information, and ii) the predictions may be sensitive to the narrow pH gradient. Vegetation relevés and soil reaction (pH-H₂O and pH-CaCl₂) were systematically recorded. A set of species indicator values and amplitudes was calibrated with physical pH data using the Weighted Averaging (WA), HOF modelling and Non-Metric Multidimensional Scaling (NMDS) methods, along with Ellenberg indicator values. Two prediction methods were tested: i) WA and ii) Amplitude Overlap (AO). WA prediction with Ellenberg’s and WA-calibrated species indicator values were the most powerful technique (R ²?=?68.4–68.7% and 53.4–59.1% for pH-CaCl₂ and pH-H₂O, respectively). WA-prediction with HOF-based indicator values was less effective (R ²?=?61.7% and 50.7%) due to the decrease in species’ information because with HOF modelling many species are assumed indifferent or too rare. The NMDS method does not bring any significant gain to the calibration, though it avoids the lack of species information. The AO method was proven to be less powerful under studied circumstances, because it is sensitive both to the lack of species’ information and to the truncation of species responses. The results prove that a spatially explicit approach can provide significant indices to estimate changes in soil acidity – pH-CaCl₂ better than pH-H₂O.     

Analysis of Multiple Brachyspira hyodysenteriae Genomes Confirms That the Species Is Relatively Conserved but Has Potentially Important Strain Variation

The intestinal spirochete Brachyspira hyodysenteriae is an important pathogen in swine, causing mucohemorrhagic colitis in a disease known as swine dysentery. Based on the detection of significant linkage disequilibrium in multilocus sequence data, the species is considered to be clonal. An analysis of the genome sequence of Western Australian B. hyodysenteriae strain WA1 has been published, and in the current study 19 further strains from countries around the world were sequenced with Illumina technology. The genomes were assembled and aligned to over 97.5% of the reference WA1 genome at a percentage sequence identity better than 80%. Strain regions not aligned to the reference ranged between 0.2 and 2.5%. Clustering of the strain genes found on average 2,354 (88%) core genes, 255 (8.6%) ancillary genes and 77 (2.9%) unique genes per strain. Depending on the strain the proportion of genes with 100% sequence identity to WA1 ranged from 85% to 20%. The result is a global comparative genomic analysis of B. hyodysenteriae genomes revealing potential differential phenotypic markers for numerous strains. Despite the differences found, the genomes were less varied than those of the related pathogenic species Brachyspira pilosicoli, and the analysis supports the clonal nature of the species. From this study, a public genome resource has been created that will serve as a repository for further genetic and phenotypic studies of these important porcine bacteria. This is the first intra-species B. hyodysenteriae comparative genomic analysis.     

Alterations in the enzyme activity and polypeptide composition of rat hepatic endoplasmic reticulum during acute exposure to 2-acetylaminofluorene

Studies have been made of the morphology, enzyme activity and protein composition of liver endoplasmic reticulum in rats exposed to acute doses of the carcinogen, 2-acetylaminofluorene (2-AAF). Electron microscopic examination revealed numerous ultrastructural changes in the hepatocyte; most consistent alterations were the disorganisation of endoplasmic reticulum system with apparent increase of smooth endoplasmic reticulum. Administration of 2-AAF to rats immediately depressed microsomal glucose-6-phosphatase activity and eventually induced epoxide hydratase activity 6–7-fold over control activity. The induction was time-dependent and maximal rates of induction were observed at dosages greater than 40 mg/kg body wt. The treatment also induced cytochrome b₅ content, NADH and NADPH cytochrome c reductase activities (1.0–1.5-fold). Only very small changes in the total content of cytochrome P-450 were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of microsomal proteins from 2-AAF pretreated animals showed time-dependent induction of two polypeptides which differed slightly in migration, in the region of M_r = 48 000; the faster-migrating induced polypeptide has been identified as epoxide hydratase. Two-dimensional PAGE analysis of microsomal proteins from 2-AAF exposed rats showed a reproducible deletion of a protein with molecular weight in the region of 67 000. The basis for the alterations in the protein composition of endoplasmic reticulum in response to 2-AAF treatment is discussed.     

Phenotypic Variants of Staphylococci and Their Underlying Population Distributions Following Exposure to Stress

This study investigated whether alterations in environmental conditions would induce the formation of small colony variant phenotypes (SCV) with associated changes in cell morphology and ultra-structure in S. aureus, s. epidermidis, and S. lugdunensis. Wild-type clinical isolates were exposed to low temperature (4°C), antibiotic stress (penicillin G and vancomycin; 0-10,000 µg mL^-1), pH stress (pH 3-9) and osmotic challenge (NaCl concentrations of 0-20%). Changes in cell diameter, cell-wall thickness, and population distribution changes (n ≥ 300) were assessed via scanning and transmission electron microscopy (SEM and TEM), and compared to control populations. Our analyses found that prolonged exposure to all treatments resulted in the subsequent formation of SCV phenotypes. Observed SCVs manifested as minute colonies with reduced haemolysis and pigmentation (NaCl, pH and 4°C treatments), or complete lack thereof (antibiotic treatments). SEM comparison analyses revealed significantly smaller cell sizes for SCV populations except in S. aureus and S. epidermidis 10% NaCl, and S. epidermidis 4°C (p<0.05). Shifts in population distribution patterns were also observed with distinct sub-populations of smaller cells appearing for S. epidermidis, and S. lugdunensis. TEM analyses revealed significantly thicker cell-walls in all treatments and species except S. lugdunensis exposed to 4°C. These findings suggest that staphylococci adapted to environmental stresses by altering their cell size and wall thickness which could represent the formation of altered phenotypes which facilitate survival under harsh conditions. The phenotypic response was governed by the type of prevailing environmental stress regime leading to appropriate alterations in ultra-structure and size, suggesting downstream changes in gene expression, the proteome, and metabolome.     

In vitro propagation and withaferin A production in Withania ashwagandha,a rare medicinal plant of India

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15 mg/g of DW) production was obtained on MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0 μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90 % survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0 μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.