Chelation in metal intoxication XXVI

The effect of pretreatment and simultaneous treatment with thiamine on therapeutic efficacy of calcium disodium edetate (CaNa₂EDTA) in lead intoxication was investigated in rats. The animals exposed to Pb as Pb (CH₃COO)₂·3 H₂O through drinking water (0.1%) for 8 wk were treated with either saline, thiamine-HCl (sc), CaNa₂EDTA (ip), or thiamine-HCl plus CaNa₂EDTA, for 3 d or thiamine-HCl for 3 d followed by thiamine, then HCl plus CaNa₂EDTA for a further 3 d. The Pb exposure caused significant accumulation of Pb in liver, kideny, and brain, inhibition in the activity of blood δ-amino-levulinic acid dehydratase (δ-ALAD), and increase in levels of urinary δ-aminolevulinic acid, homovanillic acid (HVA), vanillyl mandelic acid (VMA), brain HVA and VMA. The brain δ-ALAD and lipomide dehydrogenase remained unaffected by Pb. Thiamine significantly enhanced the urinary excretion of Pb by CaNa₂EDTA, but only marginally influenced the efficacy of CaNa₂EDTA to either mobilize tissue Pb or reverse the biochemical alterations.     

The diurnal rhythm of liver glycogen phosphorylase: correlating changes in enzyme activity and enzymic protein

The diurnal rhythm of phosphorylase activity in mouse liver extracts was correlated with the 24 h fluctuations in phosphorylase protein. This last measurement was made using rocket immunoelectrophoresis. The peak activity of phosphorylase appeared coincident (at 18:00 h) with the greatest amount of phosphorylase protein detected. Conversely, the lowest activity measured and lowest enzymic protein content both occurred at 02:00 h. Regression analysis revealed a significant positive correlation between enzyme activity and protein. Thus changes in the cellular concentration of this enzyme are implicated in the diurnal rhythm of liver glycogen.     

The rate of degradation of liver glycogen phosphorylase is specifically decreased in the C57BL/KsJ — db/db mouse

Summary We have recently demonstrated that the activity of liver glycogen phosphorylase, the rate-limiting enzyme of glycogenolysis, is elevated in genetically diabetic (db/db) mouse and that it is primarily due to the presence of increased amounts of this enzyme. In the present study, we examined the turnover of glycogen phosphorylase in vivo in order to elucidate the mechanism for this specific increase. The rate of phosphorylase synthesis was slightly decreased in the diabetic mouse compared to controls. However, the relative rates of synthesis were similar in these two groups. The rate of degradation of this enzyme was decreased 20% (p<0.05) in the diabetic mouse compared to controls. More importantly, the relative rate of degradation of phosphorylase was found to be lower in the diabetic animals. This indicates that the elevated concentration of phosphorylase in the liver of the db/db mouse is likely due to a specific decrease in its rate of degradation.     

Purification and partial characterization of glycogen synthase kinase-3 from rabbit liver

Summary Glycogen synthase kinase-3 (GSK-3) was purified from rabbit liver to homogeneity by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose, Cellulose phosphate, CM-Sephadex and Fast Protein Liquid Chromatography (FPLC) on Mono-S column. The enzyme was purified approximately 20,000 fold with an approximate 2% recovery. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. GSK-3 is a monomeric enzyme with a molecular weight of 50,000–52,000 as derived from SDS-polyacrylamide gel electrophoresis and gel filtration. The purified enzyme was indeed a GSK-3 since it phosphorylated three sites, i.e., 3a, 3b, and 3c on liver glycogen synthase. GSK-3 incorporated up to 2.6 mol Pi/mol glycogen synthase subunit with a concomitant inactivation of glycogen synthase activity.     

Chelation in metal intoxication. XXVIII: Effect of thiochelators on mercury (II) toxicity: pre- and post treatment

The effect of treatment with alpha-mercapto-beta-(2-furyl)acrylic acid (MFA), N-(N-mercaptopropionyl) glycine (MPG) and N-acetylcysteine (NAC) compared to spironolactone (SPL), a steroid, before and after 203 mercury (II) exposure, on the disposition of Hg and induction of tissue metallothionein (MT), was investigated in rats. The pretreatment with SPL, MFA and MPG enhanced faecal elimination of Hg and reduced its accumulation in liver particularly, the "heat stable fraction" resulting in lowered hepatic MT induction. Neither the renal uptake of Hg nor induction of tissue MT was affected by pre-treatment with the chelating agents; SPL and MFA causing re-distribution of Hg among the renal sub-cellular fractions. The post-Hg exposure treatment with MFA enhanced the faecal and MPG the urinary excretion of Hg. However, both the chelating agents increased the hepatic burden of Hg as reflected in the subcellular fractions and increased MT contents indicating mobilization of Hg from other tissue binding sites. The post-treatment with MPG however, depleted renal Hg as reflected by the sub-cellular distribution, without affecting renal MT levels. The results show that MFA and MPG are more promising preventive than therapeutic agents in Hg intoxication acting as metal chelators.     

Purification and properties of a heat-stable protein inhibitor of phosphoprotein phosphatase from rabbit liver.

A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95 degrees followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The purified inhibitor showed a single band when examined by gel electrophoresis S20, w and Stokes radius values were 1.45 and 25.5, respectively. Using these two values, the molecular weight and frictional ratio was calculated to be 15,500 and 3.40, respectively. The molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was found to be 14,200. The inhibition of phosphoprotein phosphatase was linear up to 40% inhibition with respect to inhibitor was constant with time of incubation for at least 30 min. The optimum pH for the inhibition was between 6.8 and 7.6. A kinetic analysis of the effect of the inhibitor on the dephosphorylation of [32P]phosphorylase a by rabbit liver phosphoprotein phosphatase indicated a noncompetitive inhibition with respect to phosphorylase a. Purified liver inhibitor inhibited the phosphoprotein phosphatase activity in all rat tissues examined. Utilizing purified rabbit liver phosphoprotein phosphatase, the presence of inhibitor activity was also demonstrated in all rat tissues tested.     

Moderate Zinc Supplementation During Prolonged Steroid Therapy Exacerbates Bone Loss in Rats

The present study was conducted to understand the influence of zinc on bone mineral metabolism in prednisolone-treated rats. Disturbance in bone mineral metabolism was induced in rats by subjecting them to prednisolone treatment for a period of 8 weeks. Female rats aged 6–8 weeks weighing 150 to 200 g were divided into four treatment groups, viz., normal control, prednisolone-treated (40 mg/kg body weight orally, thrice a week), zinc-treated (227 mg/L in drinking water, daily), and combined prednisolone?+?zinc-treated groups. Parameters such as changes in mineral levels in the bone and serum, bone mineral density (BMD), bone mineral content (BMC), and bone 99m-technetium-labeled methylene diphosphonate (^99mTc-MDP) uptake were studied in various treatment groups. Prednisolone treatment caused an appreciable decrease in calcium levels both in the bone and serum and also in bone dry weight, BMC, and BMD in rats. Prednisolone-treated rats when supplemented with zinc showed further reduction in calcium levels, bone dry weight, BMD, and BMC. The study therefore revealed that moderate intake of zinc as a nutritional supplement during steroid therapy could enhance calcium deficiency in the body and accelerate bone loss.     

pH modulates the TGF‐β ligands binding to the receptors: a computational analysis

In spite of showing high sequence similarity and forming structurally similar ternary complex in vitro, the in vivo role of TGF‐β1 and TGF‐β3 ligands suggests against their functional redundancy and necessitates the importance for the study of the specificity of these ligands. A comparative computational analysis of binary and ternary complexes of these two ligands shows that anchor residues of ligand and receptor at TGF‐β:TβR2 interface are similar in both complexes. However, the potential anchor residues of TGF‐β at TGF‐β:TβR1 interface are different, Tyr50 and Lys51 in TGF‐β3 complex and Lys60 and Tyr6 in TGF‐β1 complex. Pro55 and Asp57 of TβRI may act as anchor residues in complexes of both ligands along with Ile54 for TGF‐β3 complex and Val61 for TGF‐β1 complex. Arg58 of TβR1 acts as a potential hot residue for TGF‐β3 ternary complex but not for TGF‐β1 ternary complex formation whereas Pro55 and Phe60 may act as hot residues for both complexes. The Delphi analysis of the pH dependence of the binding energy indicates that pH has a remarkable effect on the binding energy of TβR2 to the open form of TGF‐β3. Lowering of pH from 7 to 4 favors binding of the open form of TGF‐β3 to TβR2. Now, apart from the residues at pH 7, residues Arg25, Lys31 and Arg94 of TGF‐β3 and Asp118 and Glu119 of TβR2 also contribute significantly to the binding energy. Contrary to the binding energy of TβR2 to TGF‐β3/TGF‐β1, TβR1 shows appreciable pH dependence for its binding in ternary complex of TGF‐β3/TGF‐β1. In TGF‐β3 ternary complex, the TβR1 electrostatic interaction energy disfavors complex formation at pH 7 while it is favored at pH 4. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative biology: where modern biology meets physical sciences

Quantitative methods and approaches have been playing an increasingly important role in cell biology in recent years. They involve making accurate measurements to test a predefined hypothesis in order to compare experimental data with predictions generated by theoretical models, an approach that has benefited physicists for decades. Building quantitative models in experimental biology not only has led to discoveries of counterintuitive phenomena but has also opened up novel research directions. To make the biological sciences more quantitative, we believe a two-pronged approach needs to be taken. First, graduate training needs to be revamped to ensure biology students are adequately trained in physical and mathematical sciences and vice versa. Second, students of both the biological and the physical sciences need to be provided adequate opportunities for hands-on engagement with the methods and approaches necessary to be able to work at the intersection of the biological and physical sciences. We present the annual Physiology Course organized at the Marine Biological Laboratory (Woods Hole, MA) as a case study for a hands-on training program that gives young scientists the opportunity not only to acquire the tools of quantitative biology but also to develop the necessary thought processes that will enable them to bridge the gap between these disciplines.What does a mathematician looking at bacterial division under a microscope have in common with a biologist programming a stochastic simulation of microtubule growth? For one, both can be found at the Physiology Course at the Marine Biological Laboratory (MBL) in Woods Hole, MA, which brings together graduate students and young postdocs who have a passion for quantitative biology. Students enter the course with a wide array of scientific backgrounds, including chemistry, molecular biology, mathematics, and theoretical physics. Although at first hesitant to step outside their comfort zones, students leave the course confident and courageous in their abilities to work across traditional academic boundaries. Having experienced this transformation ourselves as participants in the 2014 Physiology Course, we wanted to share some of our insights and how they have influenced our perspectives on the present challenges and exciting future of quantitative cell biology.“When you cannot express [what you are speaking about] in numbers, your knowledge is of a meager and unsatisfactory kind; it may be the beginning of knowledge, but you have scarcely, in your thoughts, advanced to the stage of science.” The need for quantification in the life sciences could not have been better worded than it is in this quote from Lord Kelvin. One of the key take-home messages from the course has been the crucial need for advancement of quantitative cell biology, which uses accurate measurements to refine a hypothesis, with the aim of comparing experimental data with predictions generated by theoretical models. We strongly believe that quantitative approaches not only aid in better addressing existing biological questions but also enable the formulation of new ones.The present time is particularly ripe for implementing quantitative approaches in cell biology, due to the wealth of data available and the depth of control we now have over many experimental systems. In the past 20 years, we have sequenced the human genome, broken the diffraction limit in microscopy, and begun to explore the possibilities of the micron-scaled experiments with microfluidics. With these tools in hand, the means to obtain quantitative data are not limited to a select few model systems; this level of experimental detail allows us to craft theoretical models that not only fit the data but have real predictive power. We can then return to our respective experimental systems with new hypotheses and interrogate them anew, reaping the benefits of an approach that has benefited physicists for decades.Building quantitative models in biology has been a powerful approach that has often revealed counterintuitive phenomena and insights while at the same time leading to novel research directions. This is of particular importance today, as experiments are becoming increasingly expensive and are rapidly accumulating vast amounts of data. It is now possible to perform “virtual” preliminary experiments in silico using quantitative models and pre-existing data and only then move to “real” laboratory experiments to test the developed hypotheses. Researchers trained this way can perform more focused experiments instead of adopting the traditional exploratory mode in the lab, saving both time and resources. However, we recognize that a majority of biology graduates have not been rigorously trained in the mathematical and physical sciences. Similarly, many physics graduates often remember their introductory biology classes simply for the rote memorization of protein names and signaling pathways, leading to the wrong assumption that biology is all about remembering three-letter abbreviations such as WNT, MYC, and so on. This can often create a misleading picture of biology.These challenges could be overcome by finding a common language between biologists, physicists, and mathematicians. A simple example of this is the word “model.” The same word can mean very different things to scientists depending upon their training: to a physicist it refers to quantitative visualization of a process via certain well-defined mathematical parameters; a biologist, on the other hand, might use the word to refer to a schematic depiction (also called a cartoon) of a biochemical reaction. We aim to reduce this gap between biological and physical sciences and bring these two communities together.One way to train young scientists in such an approach is to provide opportunities for hands-on engagement with the methods and thought processes necessary to partake in both fields. The MBL Physiology Course is an excellent case study for a training program that gives young scientists the building blocks and community necessary for success in bridging quantitative/physical sciences and biology. The course starts with a weeklong boot camp designed to bring students of different backgrounds up to speed on basic tools in quantitative biology. Students purify proteins, program in MATLAB, and build microscopes. The most important skill that biologists acquire is not simply learning how to write lines of MATLAB code, but rather phrasing biological phenomena in mathematical terms through equations and simulations. Building confocal microscopes and optical tweezers on a bare optical table creates trust in the tools we depend on to acquire quantitative data. Physicists, on the other hand, learn to purify motor proteins like kinesins and dyneins from native sources (squid), in the process coming face-to-face with the natural context of the biological questions that they are addressing.This interdisciplinary approach helps students from diverse backgrounds develop a common language. After the boot camp, students work together on three 2-week-long research projects under the guidance of leading scientists. Projects range from studying the spatial organization of the human oral microbiome and observing the development of the Caenorhabditis elegans embryo all the way to performing computational simulations of cytoskeletal polymers. By working together in a highly informal and stimulating environment, physicists learn to appreciate biological problems and biologists begin to see biological phenomena in a new light as a result of the novel physical tools and methodologies they learn from their peers. As an example, course participants Rikki Garner and Daniel Feliciano successfully collaborated to study how competition between two highly processive microtubule motors that work in opposition controls microtubule length. While Rikki (mentored by Jané Kondev) tackled the question theoretically using a random walk model, Daniel, under the mentorship of Joe Howard, carried out the experimental measurements via an in vitro assay to test Rikki''s predictions. Other examples of quantitative and biological expertise coming together to address biological questions include studying the displacement and transport of proteins at the interface between cells and synthetic supported lipid bilayers (Figure 1), observing and quantifying the cytoplasmic streaming as well as the filter-feeding flow vortices in the giant single-celled organism Stentor coeruleus (Figure 2), and imaging the spatial organization of complex oral microbial communities (Figure 3).Open in a separate windowFIGURE 1:Proteins are organized based on size at the membrane interface. The membrane interface between a cell and a supported lipid bilayer (SLB) was formed by the interaction of synthetic adhesion molecules, one protein (bound to the membrane via a His-tag) and another protein that interacts and binds with the membrane-bound protein (expressed in the S2 cells). Note that the long noninteracting protein (21 nm, magenta colored) but not the shorter noninteracting protein (8 nm, magenta colored), which is bigger than the synthetic interacting dimer (16 nm, red–green colored) is excluded from the cell–SLB interface. Both of these noninteracting proteins are bound to the SLB and do not interact with the cell. Scale bar: 10 μm. (Prepared by L.Z. and Nan Hyung Hong under the guidance of Matt Bakalar, Eva Schmid, and Dan Fletcher.)Open in a separate windowFIGURE 2:Stentor coeruleus is a giant single-celled organism that feeds by creating flow vortices in water and directing prey into its oral opening using this flow. (A) Maximum intensity projection of time-lapse images showing flow fields in the feeding flow generated by S. coeruleus. The flow is generated by the coordinated ciliary beating of the mouth cilia. (B) Flow velocity and flow directions were quantified by the particle image velocimetry method. The circularity of flow has also been indicated—the blue cloud around the oral cilia indicates clockwise flow, and red indicates anticlockwise flow. Scale bar: 100 μm. (Prepared by S.S. under the guidance of Mark Slabodnick, Tatyana Makushok, and Wallace Marshall in collaboration with Jack Costello, Providence College.)Open in a separate windowFIGURE 3:Spatial organization of complex microbial communities in an oral plaque sample taken from a volunteer as seen by combinatorial labeling and spectral imaging–fluorescent in situ hybridization (CLASI-FISH). Microbes seen here are Corynebacterium (pink), Neisseriaceae (blue), Fusobacterium (green), Pasteurellaceae (yellow), Streptococcus (cyan), and Actinomyces (red). (Prepared by Bryan Weinstein, Lishibanya Mohapatra, and Matti Gralka under the guidance of Blair Rossetti, Jessica Mark Welch, and Gary Borisy.)An invaluable aspect of the course is the informal nature of the interaction. There are a wide variety of morning seminar speakers, and in the question-and-answer sessions following the talks, the speakers discuss not only science but also the successes and failures they experienced while moving across the boundaries of biological and physical sciences. The interactive and collaborative nature of the course encourages students to not just learn from one another but to actually teach one another. “Chalk talks” and interactions happen spontaneously and are the strongest indication of the richness of the intellectual exchange among members of the community. Prime examples in the 2014 course are the chalk talks on Python (Bryan Weinstein), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) cloning (Dan Dickinson), and the basics of microfluidics (Sindy Tang).Although we have benefited immensely from this interdisciplinary course, we understand that it might not be feasible for all graduate students to participate in such courses. Nevertheless, we believe that the scientific community should work together to replicate elsewhere, at least partially, the strengths of this course to allow students to benefit from this approach. Students should be encouraged to host and attend seminars from speakers with diverse backgrounds, which will expose them to research areas different from their own. Biology students should also be exposed to mathematical and statistical instruction early in their research careers, preferably at the undergraduate level, to enable them to build strong foundations. Students should also be encouraged to participate in short-term, low-pressure interdisciplinary collaborations to broaden their understanding and initiate interactions with other fields. Short summer/winter schools—for example, the physical biology of the cell courses at Cold Spring Harbor and the International Centre for Theoretical Physics (Italy)–International Centre for Theoretical Sciences (India) Winter School on Quantitative Systems Biology, 2013, in Bangalore, India—can serve as the perfect stage for this.In conclusion, we expect that quantitative approaches will be indispensable for better addressing biological questions in the future. Our experience is that combining traditional experimental cell biology with quantitative thinking leads to hitherto unknown scientifically rich domains, and we ourselves have found this exploratory journey to be both achievable and rewarding. Although bridging the gap may appear to be difficult at times, it is extremely satisfying when accomplished, and doing it within a highly motivated and supportive community is what makes the connection possible and extremely useful.