Fluorescence detection of intracellular cadmium with Leadmium Green

Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd²⁺) and lead (Pb²⁺). Leadmium Green has been used in various paradigms, such as tracking Cd²⁺ sequestration in plant cells, heavy metal export in protozoa, and Pb²⁺ absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd²⁺, Pb²⁺, and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer’s claims, Leadmium Green was sensitive to Cd²⁺ (K_D ~600 nM) and also Pb²⁺ (K_D ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca²⁺, Co²⁺, Mn²⁺ and Ni²⁺. Leadmium Green also responded to Zn²⁺ with a K_D of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd²⁺ or Zn²⁺ in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn²⁺ was much less than that produced by Cd²⁺. When tested in a model of oxidant-induced liberation of endogenous Zn²⁺, Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd²⁺, particularly in models where Cd²⁺ accumulates rapidly, and when concomitant fluctuations of intracellular Zn²⁺ are minimal.     

Magnetic Resonance Imaging of the Newborn Brain: Automatic Segmentation of Brain Images into 50 Anatomical Regions

We studied methods for the automatic segmentation of neonatal and developing brain images into 50 anatomical regions, utilizing a new set of manually segmented magnetic resonance (MR) images from 5 term-born and 15 preterm infants imaged at term corrected age called ALBERTs. Two methods were compared: individual registrations with label propagation and fusion; and template based registration with propagation of a maximum probability neonatal ALBERT (MPNA). In both cases we evaluated the performance of different neonatal atlases and MPNA, and the approaches were compared with the manual segmentations by means of the Dice overlap coefficient. Dice values, averaged across regions, were 0.81±0.02 using label propagation and fusion for the preterm population, and 0.81±0.02 using the single registration of a MPNA for the term population. Segmentations of 36 further unsegmented target images of developing brains yielded visibly high-quality results. This registration approach allows the rapid construction of automatically labeled age-specific brain atlases for neonates and the developing brain.     

Retention of cytoplasmic droplet by rat cauda epididymal spermatozoa after treatment with cytotoxic and xenobiotic agents

Spermatozoa leaving the testis contain a cytoplasmic droplet which they release during transit through the epididymis before reaching the cauda epididymidis. The cytoplasmic droplet shows P450 aromatase activity, which plays a role in synthesis of oestrogen from androgen. In the present study, 3-month-old Wistar strain male albino rats were administered with the organophosphate insecticides malathion or dichlorvos, or the phytotherapeutics andrographolide or ursolic acid. Segments of the epididymis were subjected to histopathological and ultrastructural analyses and it was found that 60-95% of the spermatozoa residing in the lumen of the cauda epididymidis retained the cytoplasmic droplet. The motility of the spermatozoa released from the cauda epididymidis was inhibited. One of the mechanisms of action of these toxicants on male reproductive function may be attributed to the retention of the cytoplasmic droplet and the resultant impairment of sperm motility.     

Cloning and expression of rabbit CCT subunits eta and beta in healing cutaneous wounds

We have previously identified the CCT subunit eta as specifically reduced in healing fetal skin wounds by differential display, and observed that this reduction is not seen with any other CCT subunit. We now report the cloning and characterization of the cDNAs for rabbit CCT-eta and its closest evolutionary homolog, CCT-beta. Quantitative examination of CCT-eta and –beta message expression in healing fetal and adult wounds at 12 h post-injury confirms that CCT-eta mRNA is decreased in fetal wound tissues, but actually elevated in adult wound tissues. CCT-beta mRNA, in contrast, remains unchanged in both fetal and adult wound tissues. CCT-eta mRNA remains persistently elevated in healing adult wounds for 28 days following injury, whereas CCT-beta mRNA remains invariant throughout. CCT-eta protein is similarly increased, whereas CCT-beta protein remains unchanged. -smooth muscle actin (-SMA), a recognized substrate of CCT known to be important in integumentary wound healing, was also measured over the course of wound healing, and both mRNA and protein levels were elevated throughout the 28 days.     

Insulin-secretagogue, antihyperlipidemic and other protective effects of gallic acid isolated from Terminalia bellerica Roxb. in streptozotocin-induced diabetic rats

Diabetes mellitus causes derangement of carbohydrate, protein and lipid metabolism which eventually leads to a number of secondary complications. Terminalia bellerica is widely used in Indian medicine to treat various diseases including diabetes. The present study was carried out to isolate and identify the putative antidiabetic compound from the fruit rind of T. bellerica and assess its chemico-biological interaction in experimental diabetic rat models. Bioassay guided fractionation was followed to isolate the active compound, structure was elucidated using (1)H and (13)C NMR, IR, UV and mass spectrometry and the compound was identified as gallic acid (GA). GA isolated from T. bellerica and synthetic GA was administered to streptozotocin (STZ)-induced diabetic male Wistar rats at different doses for 28 days. Plasma glucose level was significantly (p<0.05) reduced in a dose-dependent manner when compared to the control.Histopathological examination of the pancreatic sections showed regeneration of β-cells of islets of GA-treated rats when compared to untreated diabetic rats. In addition, oral administration of GA (20mg/kg bw) significantly decreased serum total cholesterol, triglyceride, LDL-cholesterol, urea, uric acid, creatinine and at the same time markedly increased plasma insulin, C-peptide and glucose tolerance level. Also GA restored the total protein, albumin and body weight of diabetic rats to near normal. Thus our findings indicate that gallic acid present in fruit rind of T. bellerica is the active principle responsible for the regeneration of β-cells and normalizing all the biochemical parameters related to the patho-biochemistry of diabetes mellitus and hence it could be used as a potent antidiabetic agent.     

Galantamine reverses scopolamine-induced behavioral alterations in Dugesia tigrina

In planaria (Dugesia tigrina), scopolamine, a nonselective muscarinic receptor antagonist, induced distinct behaviors of attenuated motility and C-like hyperactivity. Planarian locomotor velocity (pLMV) displayed a dose-dependent negative correlation with scopolamine concentrations from 0.001 to 1.0 mM, and a further increase in scopolamine concentration to 2.25 mM did not further decrease pLMV. Planarian hyperactivity counts was dose-dependently increased following pretreatment with scopolamine concentrations from 0.001 to 0.5 mM and then decreased for scopolamine concentrations ≥1 mM. Planarian learning and memory investigated using classical Pavlovian conditioning experiments demonstrated that scopolamine (1 mM) negatively influenced associative learning indicated by a significant decrease in % positive behaviors from 86 % (control) to 14 % (1 mM scopolamine) and similarly altered memory retention, which is indicated by a decrease in % positive behaviors from 69 % (control) to 27 % (1 mM scopolamine). Galantamine demonstrated a complex behavior in planarian motility experiments since co-application of low concentrations of galantamine (0.001 and 0.01 mM) protected planaria against 1 mM scopolamine-induced motility impairments; however, pLMV was significantly decreased when planaria were tested in the presence of 0.1 mM galantamine alone. Effects of co-treatment of scopolamine and galantamine on memory retention in planaria via classical Pavlovian conditioning experiments showed that galantamine (0.01 mM) partially reversed scopolamine (1 mM)-induced memory deficits in planaria as the % positive behaviors increased from 27 to 63 %. The results demonstrate, for the first time in planaria, scopolamine’s effects in causing learning and memory impairments and galantamine’s ability in reversing scopolamine-induced memory impairments.     

Whole Plastome Sequences from Five Ginger Species Facilitate Marker Development and Define Limits to Barcode Methodology

Plants from the Zingiberaceae family are a key source of spices and herbal medicines. Species identification within this group is critical in the search for known and possibly novel bioactive compounds. To facilitate precise characterization of this group, we have sequenced chloroplast genomes from species representing five major groups within Zingiberaceae. Generally, the structure of these genomes is similar to the basal angiosperm excepting an expansion of 3 kb associated with the inverted repeat A region. Portions of this expansion appear to be shared across the entire Zingiberales order, which includes gingers and bananas. We used whole plastome alignment information to develop DNA barcodes that would maximize the ability to differentiate species within the Zingiberaceae. Our computation pipeline identified regions of high variability that were flanked by highly conserved regions used for primer design. This approach yielded hitherto unexploited regions of variability. These theoretically optimal barcodes were tested on a range of species throughout the family and were found to amplify and differentiate genera and, in some cases, species. Still, though these barcodes were specifically optimized for the Zingiberaceae, our data support the emerging consensus that whole plastome sequences are needed for robust species identification and phylogenetics within this family.     

Response of Cassava canopy to mid-day pseudo sunrise induced by solar eclipse

Variations in CO₂ concentration over a cassava canopy were measured during a solar eclipse at Thiruvananthapuram, India. The analysis presented attempts to differentiate between the eclipse effect and the possible effect of thick clouds, taking CO₂ as a proxy for photosynthesis. CO₂ and water vapor were measured at a rate of 10 Hz, and radiation at 1 Hz, together with other meteorological parameters. A rapid reduction in CO₂ observed post-peak eclipse, due apparently to intense photosynthesis, appears similar to what happens at daybreak/post-sunrise. The increase in CO₂ (4 ppm) during peak eclipse, with radiation levels falling below the photosynthesis cut-off for cassava, indicates domination of respiration due to the light-limiting conditions.