Electromechanics and Volume Dynamics in Nonexcitable Tissue Cells

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell’s lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment. Specifically, we demonstrate that a sudden cell depolarization using the whole-cell patch clamp results in a 50% increase in cell volume, whereas hyperpolarization results in a slight volume decrease. We find that cell volume can be partially controlled by changing the chloride or the sodium/potassium concentrations in the extracellular environment while maintaining a constant external osmotic pressure. Depletion of external chloride leads to a volume decrease in suspended HN31 cells. Introducing cells to a high-potassium solution causes volume increase up to 50%. Cell volume is also influenced by cortical tension: actin depolymerization leads to cell volume increase. We present an electrophysiology model of water dynamics driven by changes in membrane potential and the concentrations of permeable ions in the cells surrounding. The model quantitatively predicts that the cell volume is directly proportional to the intracellular protein content.     

Optimization of a preparative RP-HPLC method for isolation and purification of picrosides in <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis>

Preparative reversed phase high pressure liquid chromatography (prep-RP-HPLC) coupled with photodiode array (PDA) and evaporative light scattering (ELSD) detectors was employed to isolate picrosides present in Picrorhiza kurroa Royle ex Benth. A binary gradient method (water and acetonitrile) was optimized on Water Spherisorb S10 ODS2 20 mm × 250 mm Semiprep Column with a 20 mL/min flow rate at ambient temperature with linear binary gradient conditions; at 0 min 15 % acetonitrile hold for 15 min; 15 to 22 % acetonitrile in next 2 min, hold for 13 min; 22 to 15 % acetonitrile in 5 min hold for 5 min to equilibrate column for next injection. The picroside-I and picroside-II fractions were 98.6 and 99.7 % pure with 13.9 mg and 9.8 mg yield per 200 mg of crude extract, respectively from mature dry rhizomes. Structures of isolated iridoids were confirmed with UV scan, 1H-NMR and direct infusion ESI-Q-TOF-MS/MS data.     

Higher amount of steviol detected in the leaves of a non-toxic endangered medicinal herb, <Emphasis Type="Italic">Aconitum heterophyllum</Emphasis>

Aconitum heterophyllum is the only non-toxic species of Aconitum genus having immense therapeutic potential. To date, only roots of this plant species are used for the preparation of various herbal formulations while leaves are discarded due to lack of information on the presence of any therapeutically active compounds. To the best of our knowledge, this is the first report on the detection and quantification of diterpene steviol in the leaves of A. heterophyllum. Interestingly, the level of steviol is equivalent to Stevia rebaudiana leaves which is the richest source reported so far. Thus, A. heterophyllum leaves will not only provide a novel source for extraction of steviol but also benefit the harvesters to get additional economic returns on leaf biomass for this high-value non-toxic plant species.     

DNA methylation provides insight into intergenerational risk for preterm birth in African Americans

African Americans are at increased risk for spontaneous preterm birth (PTB). Though PTB is heritable, genetic studies have not identified variants that account for its intergenerational risk, prompting the hypothesis that epigenetic factors may also contribute. The objective of this study was to evaluate DNA methylation from maternal leukocytes to identify patterns specific to PTB and its intergenerational risk. DNA from peripheral leukocytes from African American women that delivered preterm (24–34 weeks; N = 16) or at term (39–41 weeks; N = 24) was assessed for DNA methylation using the HumanMethylation450 BeadChip. In maternal samples, 17,829 CpG sites associated with PTB, but no CpG site remained associated after correction for multiple comparisons. Examination of paired maternal-fetal samples identified 5,171 CpG sites in which methylation of maternal samples correlated with methylation of her respective fetus (FDR < 0.05). These correlated sites were enriched for association with PTB in maternal leukocytes. The majority of correlated CpG sites could be attributed to one or more genetic variants. They were also significantly more likely to be in genes involved in metabolic, cardiovascular, and immune pathways, suggesting a role for genetic and environmental contributions to PTB risk and chronic disease. The results of this study may provide insight into the factors underlying intergenerational risk for PTB and its consequences.     

Solution Structure of an ABC Collagen Heterotrimer Reveals a Single-register Helix Stabilized by Electrostatic Interactions

Collagen, known for its structural role in tissues and also for its participation in the regulation of homeostatic and pathological processes in mammals, is assembled from triple helices that can be either homotrimers or heterotrimers. High resolution structural information for natural collagens has been difficult to obtain because of their size and the heterogeneity of their native environment. For this reason, peptides that self-assemble into collagen-like triple helices are used to gain insight into the structure, stability, and biochemistry of this important protein family. Although many of the most common collagens in humans are heterotrimers, almost all studies of collagen helices have been on homotrimers. Here we report the first structure of a collagen heterotrimer. Our structure, obtained by solution NMR, highlights the role of electrostatic interactions as stabilizing factors within the triple helical folding motif. This addresses an issue that has been actively researched because of the predominance of charged residues in the collagen family. We also find that it is possible to selectively form a collagen heterotrimer with a well defined composition and register of the peptide chains within the helix, based on information encoded solely in the collagenous domain. Globular domains are implicated in determining the composition of several collagen types, but it is unclear what their role in controlling register may be. We show that is possible to design peptides that not only selectively choose a composition but also a specific register without the assistance of other protein constructs. This mechanism may be used in nature as well.Collagens constitute an important structural protein family. They are found in the extracellular matrix and undergo a hierarchical self-assembly into large supramolecular structures with specific morphologies carefully crafted by nature to fulfill diverse structural and functional roles in a wide variety of tissues. In total, there are 28 known isoforms of collagen in humans arranged in a variety of structures and in a wide range of tissues. The feature defining this protein family is the presence of domains with uninterrupted Xaa-Yaa-Gly sequence repeats. These domains adopt a left-handed polyproline type II conformation because of the predominance of proline in the X position and hydroxyproline (Hyp = O), a post-translationally modified amino acid with a hydroxyl group on the γ-carbon of the proline side chain, in the Y position. Three such domains associate to form tightly packed right-handed triple helices in a folding motif commonly known as the collagen triple helix.Collagens are also implicated in pivotal homeostatic events in mammals such as the production of new vascular tissue and pathological conditions such as cancer metastasis (1). These processes are notoriously governed by interactions at the molecular level between cell surface proteins and the collagen triple helix and not by the morphology of the collagen aggregates (2, 3). Thus, an understanding of the collagen molecule and its interactions with other proteins at the atomic level has been actively pursued. However, because of its complex hierarchical self-assembly, and the scale of the resulting supramolecular structures, it is difficult to obtain information at atomic resolution for collagenous proteins (4). An approach developed to overcome this limitation is the use of short model peptides that adopt a triple helical fold (5). Such peptides have been used to study the structure (6, 7), folding (8), and dynamics (9) of the triple helix. These peptides have been shown to retain the biochemical properties of the higher assemblies found in their natural counterparts, binding to cell surface proteins such as integrins (10).Most of the studies performed on collagen mimetic peptides utilize triple helices with three identical chains called homotrimers (8, 11–13). Such systems are good models for some types of collagen, like type II. However, many of the most abundant types of collagen such as type I, IV, and IX are heterotrimeric species containing two (AAB) or three (ABC) different chains. Recently we introduced a new method to prepare heterotrimeric collagen like triple helices via noncovalent interactions (14–16). These systems have been primarily characterized through CD spectroscopy, which is a good indicator for the fold and stability of the peptides but lacks the ability to give detailed structural information. There are few studies available in the literature that utilize NMR to examine collagen heterotrimers; however, none of them use the technique to examine the structures of the assemblies in detail (17–20), and none result in a complete structural determination.A system of particular interest is composed of three peptides, (Pro-Lys-Gly)₁₀, (Asp-Hyp-Gly)₁₀, and (Pro-Hyp-Gly)₁₀, which we abbreviate K, D, and O respectively. Upon mixing and annealing of the peptides, CD studies indicate that an ABC triple helix with a surprisingly high thermal stability is formed (16). We hypothesized that the high thermal stability of these systems comes from the formation of charge pairs between lysine and aspartic acid. Homotrimeric model peptides that contain the sequence KGD, which occurs both in mammalian collagen (21) and bacterial collagen-like proteins (22), apparently also use this charge pairing. However, no structural information has been available to confirm the nature of the interactions.Here we study the K·D·O system using two- and three-dimensional NMR techniques to determine the composition and fold of the components of an annealed mixture of these three peptides. Also, for the first time, we are able to study the register or relative stagger of the peptide chains in the triple helix. In a collagen triple helix, the chains assemble staggered by one amino acid, so that there is always a glycine residue in every cross-section of the helix taken perpendicular to the helical axis. This allows the peptide chains to pack tightly while avoiding steric clashes in the center of the assembly. Depending on the nature of the leading, middle, and trailing chain, a total of six different assemblies, or registers, are possible for an ABC system (Fig. 1). Given the high thermal stability of the system, which allows for the recording of high quality NMR spectra, we found that our peptides preferentially populate one register, and using samples with strategically placed isotopically labeled amino acids, we are able to determine which one. Furthermore, we are able to obtain the first structure of a collagen triple helix in solution and give direct evidence of ionic hydrogen bonds as a stabilizing factor within the triple helical folding motif.Open in a separate windowFIGURE 1.Schematic N-terminal representation of the six possible registers for the heterotrimeric triple helix. The difference in the sequence is highlighted below each representation, where the position of glycine residues is marked by colored spheres.     

Solvent free synthesis,anti-inflammatory and anticancer activity evaluation of tricyclic and tetracyclic benzimidazole derivatives

Heterocyclic benzimidazole derivatives 3a–h, 5a–c and 7a–d have been synthesized by condensation of succinic acid (1) homophthalic acid (4) and 2,3-pyrazinedicarboxlic acid (6) with various substituted diamines under microwave irradiation in good yields. Structures assigned to 3a–h, 5a–c and 7a–d are fully supported by spectral data. All these compounds were screened for anti-inflammatory and anticancer activities. At a dose of 50 mg/kg po compounds 3b (39.4%) and 3c (39.2%) exhibited anti-inflammatory activity, comparable to standard ibuprofen which showed 39% activity at 50 mg/kg po and compound 7c exhibit good anticancer activity against ovary (IGR-OV-1), breast (MCF-7) and CNS(SF-295) human cancer cell lines.     

SLC9B1 methylation predicts fetal intolerance of labor

Fetal intolerance of labor is a common indication for delivery by Caesarean section. Diagnosis is based on the presence of category III fetal heart rate tracing, which is an abnormal heart tracing associated with increased likelihood of fetal hypoxia and metabolic acidemia. This study analyzed data from 177 unique women who, during their prenatal visits (7-15 weeks and/or 24–32 weeks) to Atlanta area prenatal care clinics, consented to provide blood samples for DNA methylation (HumanMethylation450 BeadChip) and gene expression (Human HT-12 v4 Expression BeadChip) analyses. We focused on 57 women aged 18–36 (mean 25.4), who had DNA methylation data available from their second prenatal visit. DNA methylation patterns at CpG sites across the genome were interrogated for associations with fetal intolerance of labor. Four CpG sites (P value <8.9 × 10^?9, FDR <0.05) in gene SLC9B1, a Na⁺/H⁺ exchanger, were associated with fetal intolerance of labor. DNA methylation and gene expression were negatively associated when examined longitudinally during pregnancy using a linear mixed-effects model. Positive predictive values of methylation of these four sites ranged from 0.80 to 0.89, while negative predictive values ranged from 0.91 to 0.92. The four CpG sites were also associated with fetal intolerance of labor in an independent cohort (the Johns Hopkins Prospective PPD cohort). Therefore, fetal intolerance of labor could be accurately predicted from maternal blood samples obtained between 24–32 weeks gestation. Fetal intolerance of labor may be accurately predicted from maternal blood samples obtained between 24–32 weeks gestation by assessing DNA methylation patterns of SLC9B1. The identification of pregnant women at elevated risk for fetal intolerance of labor may allow for the development of targeted treatments or management plans.