Inducing Ni sensitivity in the Ni hyperaccumulator plant <Emphasis Type="Italic">Alyssum inflatum</Emphasis> Nyárády (Brassicaceae) by transforming with <Emphasis Type="Italic">CAX1</Emphasis>, a vacuolar membrane calcium transporter

The importance of calcium in nickel tolerance was studied in the nickel hyperaccumulator plant Alyssum inflatum by gene transformation of CAX1, a vacuolar membrane transporter that reduces cytosolic calcium. CAX1 from Arabidopsis thaliana with a CaMV35S promoter accompanying a kanamycin resistance gene was transferred into A. inflatum using Agrobacterium tumefaciens. Transformed calli were sub-cultured three times on kanamycin-rich media and transformation was confirmed by PCR using a specific primer for CAX1. At least 10 callus lines were used as a pool of transformed material. Both transformed and untransformed calli were treated with varying concentrations of either calcium (1–15 mM) or nickel (0–500 µM) to compare their responses to those ions. Increased external calcium generally led to increased callus biomass, however, the increase was greater for untransformed callus. Further, increased external calcium led to increased callus calcium concentrations. Transformed callus was less nickel tolerant than untransformed callus: under increasing nickel concentrations callus relative growth rate was significantly less for transformed callus. Transformed callus also contained significantly less nickel than untransformed callus when exposed to the highest external nickel concentration (200 µM). We suggest that transformation with CAX1 decreased cytosolic calcium and resulted in decreased nickel tolerance. This in turn suggests that, at low cytosolic calcium concentrations, other nickel tolerance mechanisms (e.g., complexation and vacuolar sequestration) are insufficient for nickel tolerance. We propose that high cytosolic calcium is an important mechanism that results in nickel tolerance by nickel hyperaccumulator plants.     

Copper Affects the Cotyledonary Carbohydrate Status During the Germination of Bean Seed

Seeds of bean (Phaseolus vulgaris L.) were germinated by soaking in distilled water or copper chloride solution. The relationships among copper excess treatment, germination rate, dry weight, sugar contents, and carbohydrase activities in cotyledon were investigated. Heavy metal stress provoked a diminution in germination rate and biomass mobilization, as compared with the control. A drastic disorder in soluble sugars export, especially glucose and fructose liberation, was also imposed after exposure to excess copper. This restricted the starch and sucrose breakdown in reserve tissue, as evidenced by the inhibition in the activities of α-amylase and invertase isoenzymes (soluble acid, soluble neutral, cell wall-bound acid).     

The <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds

Background  

The Arabidopsis response regulator 22 (ARR22) is one of two members of a recently defined novel group of two-component system (TCS) elements. TCSs are stimulus perception and response modules of prokaryotic origin, which signal by a His-to-Asp phosphorelay mechanism. In plants, TCS regulators are involved in hormone response pathways, such as those for cytokinin and ethylene. While the functions of the other TCS elements in Arabidopsis, such as histidine kinases (AHKs), histidine-containing phosphotransfer proteins (AHPs) and A-type and B-type ARRs are becoming evident, the role of ARR22 is poorly understood.     

MRI Features in a Canine Model of Ischemic Stroke: Correlation between Lesion Volume and Neurobehavioral Status during the Subacute Stage

The purpose of this study was to evaluate the diagnostic value of magnetic resonance imaging (MRI) and assess the correlation between the volume of the ischemic lesion and neurobehavioral status during the subacute stage of ischemic stroke. Ischemic stroke was induced in 6 healthy laboratory beagles through permanent occlusion of the middle cerebral artery (MCAO). T2-weighted and fluid-attenuated inversion recovery (FLAIR) imaging, diffusion-weighted imaging (DWI), measurement of the apparent diffusion coefficient (ADC) ratio, and neurobehavioral evaluation were performed 3 times serially by using a 1.5-T MR system: before and 3 and 10 d after MCAO. Ischemic lesions demonstrated T2 hyperintensity, FLAIR hyperintensity, and DWI hyperintensity. The ADC ratio was decreased initially but then was increased at 10 d after MCAO. Ischemic lesion volumes on T2-weighted and FLAIR imaging were not significantly different from those on DWI. The lesion volume and neurobehavioral score showed strong correlation. Our results suggest that conventional MRI may be a reliable diagnostic tool during the subacute stage of canine ischemic stroke.Abbreviations: ADC, apparent diffusion coefficient; DWI, diffusion-weighted imaging; FLAIR, fluid-attenuated inversion recovery; MCAO, middle cerebral artery occlusion; MRI, magnetic resonance imaging; PWI, perfusion-weighted imagingIn human medicine, stroke is a leading cause of adult mortality and neurologic disability worldwide.¹ Strokes previously were thought to be uncommon in small animals, but the true prevalence is unknown.⁴ These events are now recognized more frequently in dogs because of increased use of magnetic resonance imaging (MRI).^5,14,17Because the infusion of thrombolytic agents, such as urokinase or tissue plasminogen activator, within 3 to 6 h of the onset symptoms is effective in restoring blood flow and improving stroke outcome in humans,¹⁹ the detection of early ischemic changes is now thought to be necessary to improve patient outcome. Computed tomography and conventional MRI are not sufficiently sensitive to predict the presence and extent of ischemic damage during the acute stage after a stroke.^12,20 Therefore several MRI sequences, such as fluid-attenuated inversion recovery (FLAIR), diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), and MR angiography, have been developed for early diagnosis and subsequent follow-up of ischemic stroke.³ High-field magnetic strengths (at least 1.5 T) are necessary to perform these sequences.In contrast to the situation in humans, ischemic stroke in many dogs is diagnosed during the subacute stage—24 h to 6 wk after the vascular insult—due to the time lag between the onset of clinical signs to referral and to the lack of standard diagnostic protocols for ischemic stroke in dogs. In most reports of strokes in dogs, the median interval between the onset of neurologic dysfunction and performance of an MRI was more than 2 d.^5,14,17 Whereas DWI has marked sensitivity to very early ischemic changes in the brain, T2-weighted and FLAIR images gradually become more hyperintense later (that is, during the first 24 h after the insult).³ Therefore, hyperintensity on T2-weighted and FLAIR images is believed to be representative of mature lesions.¹⁵ In light of these findings, we hypothesized that conventional MR sequences, such as T2-weighted and FLAIR imaging as well as DWI would be used for the diagnosis of the subacute stage of ischemic stroke in dogs.The purpose of this study was to evaluate the diagnostic value of MRI and assess the correlation between the volume of ischemic lesions and neurobehavioral status during the subacute stage of ischemic stroke in dogs. We therefore investigated the lesion volume of T2-weighted and FLAIR images compared with that on DWI images. Furthermore, we assessed the relationship between the apparent diffusion coefficient (ADC) of the ischemic lesions and the neurobehavioral status of the dogs.     

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs.     

Design and synthesis of diarylamines and diarylethers as cytotoxic antitumor agents

Based on a shared structural core of diarylamine in several known anticancer drugs as well as a new cytotoxic hit 6-chloro-2-(4-cyanophenyl)amino-3-nitropyridine (7), 30 diarylamines and diarylethers were designed, synthesized, and evaluated for cytotoxic activity against A549, KB, KB-vin, and DU145 human tumor cell lines (HTCL). Four new leads 11e, 12, 13a, and 13b were discovered with GI(50) values ranging from 0.33 to 3.45μM. Preliminary SAR results revealed that a diarylamine or diarylether could serve as an active structural core, meta-chloro and ortho-nitro groups on the A-ring (either pyridine or phenyl ring) were necessary and crucial for cytotoxic activity, and the para-substituents on the other phenyl ring (B-ring) were related to inhibitory selectivity for different tumor cells. In an investigation of potential biological targets of the new leads, high thoughput kinase screening discovered that new leads 11e, 12 and 13b especially inhibit Mer tyrosine kinase, a proto-oncogene associated with munerous tumor types, with IC(50) values of 2.2-3.0μM. Therefore, these findings provide a good starting point to optimize a new class of compounds as potential anticancer agents, particularly targeting Mer tyrosine kinase.     

Asoprisnil (J867): a selective progesterone receptor modulator for gynecological therapy

Asoprisnil is a novel selective steroid receptor modulator that shows unique pharmacodynamic effects in animal models and humans. Asoprisnil, its major metabolite J912, and structurally related compounds represent a new class of progesterone receptor (PR) ligands that exhibit partial agonist and antagonist activities in vivo. Asoprisnil demonstrates a high degree of receptor and tissue selectivity, with high-binding affinity for PR, moderate affinity for glucocorticoid receptor (GR), low affinity for androgen receptor (AR), and no binding affinity for estrogen or mineralocorticoid receptors. In the rabbit endometrium, both asoprisnil and J912 induce partial agonist and antagonist effects. Asoprisnil induces mucification of the guinea pig vagina and has pronounced anti-uterotrophic effects in normal and ovariectomized guinea pigs. Unlike antiprogestins, asoprisnil shows only marginal labor-inducing activity during mid-pregnancy and is completely ineffective in inducing preterm parturition in the guinea pig. Asoprisnil exhibits only marginal antiglucocorticoid activity in transactivation in vitro assays and animal models. In male rats, asoprisnil showed weak androgenic and anti-androgenic properties. In toxicological studies in female cynomolgus monkeys, asoprisnil treatment abolished menstrual cyclicity and endometrial atrophy. Early clinical studies of asoprisnil in normal volunteers demonstrated a dose-dependent suppression of menstruation irrespective of the effects on ovulation, with no change in basal estrogen concentrations and no antiglucocorticoid effects. Unlike progestins, asoprisnil does not induce breakthrough bleeding. With favorable safety and tolerability profiles thus far, asoprisnil appears promising as a novel treatment of gynecological disorders, such as uterine fibroids and endometriosis.     

Population structure of North Atlantic and North Pacific sei whales (<Emphasis Type="Italic">Balaenoptera borealis</Emphasis>) inferred from mitochondrial control region DNA sequences and microsatellite genotypes

Currently, three stocks of sei whales (Balaenoptera borealis) are defined in the North Atlantic; the Nova Scotian, Iceland-Denmark Strait and Eastern North Atlantic stocks, which are mainly based upon historical catch and sighting data. We analyzed mitochondrial control region DNA (mtDNA) sequences and genotypes from 7 to 11 microsatellite loci in 87 samples from three sites in the North Atlantic; Iceland, the Gulf of Maine and the Azores, and compared against the North Pacific using 489 previously published samples. No statistically significant deviations from homogeneity were detected among the North Atlantic samples at mtDNA or microsatellite loci. The genealogy estimated from the mtDNA sequences revealed a clear division of the haplotypes into a North Atlantic and a North Pacific clade, with the exception of one haplotype detected in a single sample from the Azores, which was included in the North Pacific clade. Significant genetic divergence between the North Atlantic and North Pacific Oceans was detected (mtDNA Φ_ST?=?0.72, microsatellite Weir and Cockerham’s ? = 0.20; p?<?0.001). The coalescent-based estimate of the population divergence time between the North Atlantic and North Pacific populations from the sequence variation among the mtDNA sequences was at 163,000 years ago. However, the inference was limited by an absence of samples from the Southern Hemisphere and uncertainty regarding mutation rates and generation times. The estimates of inter-oceanic migration rates were low (Nm at 0.007 into the North Pacific and at 0.248 in the opposite direction). Although estimates of genetic divergence among the current North Atlantic stocks were low and consistent with the extensive range of movement observed in satellite tagged sei whales, the high uncertainty of the genetic divergence estimates precludes rejection of multiple stocks in the North Atlantic.