Arthur D. Tinoco

Bio: Arthur D. Tinoco is an academic researcher from University of Puerto Rico, Río Piedras. The author has contributed to research in topics: Transferrin & Cytochrome c. The author has an hindex of 18, co-authored 33 publications receiving 790 citations. Previous affiliations of Arthur D. Tinoco include Harvard University & University of Puerto Rico.

Reconsideration of serum Ti(IV) transport: albumin and transferrin trafficking of Ti(IV) and its complexes.

Abstract: The trafficking of titanium(IV) by human serum transferrin (HsTf) has been implicated in the physiology of this hydrolysis-prone metal. The current work broadens to include the further interactions of Ti(IV) in serum that bear on this model. Ti2HsTf (2 equiv) binds the transferrin receptor TfR1 with Kd1 = 6.3 ± 0.4 nM and Kd2 = 410 ± 150 nM, values that are the tightest yet measured for a metal other than iron but weaker than the corresponding ones for Fe2HsTf due to both slightly slower on rates and slightly faster off rates. Comparing the affinities of metals for HsTf with the affinities of the resulting M2HsTf species for TfR1, we speculate that the formation of an M2HsTf complex of high affinity may predict a lobe-closed conformation that leads to a favorable interaction with TfR1. Human serum albumin (HSA), an important serum competitor for metal binding, can bind up to 20 equiv of Ti(IV) supplied in several forms. With some ligands, Ti(IV) may bind to the N-terminal metal binding site of albumin, fo...

Ti(IV) binds to human serum transferrin more tightly than does Fe(III).

Abstract: The binding of titanium(IV) to human serum transferrin in 50 mM Tris with 20 mM bicarbonate and 10 mM citrate at pH 7.4 was studied by UV/vis kinetics and by isothermal titration calorimetry. Ti(IV) citrate, [Ti(C6H4O7)3]8-, employed in this study was previously characterized and delivers the metal to transferrin rapidly, allowing the quantification of the intrinsic binding constants for Ti(IV) to the C- and N-sites of transferrin. The results after correcting for blood plasma conditions (pH 7.4, [HCO3-] = 27 mM) reveal that Ti(IV) binds with greater affinity (log K = 26.8 and 25.7) than Fe(III) (log K = 22.5 and 21.4) to transferrin, a finding not previously observed for other examined metal ions. The strength of metal binding to transferrin correlates with the Lewis acidity of the metal. Ti(IV) is more Lewis acidic than Fe(III) and is nearly the same size. The study also reveals that Ti(IV) binds more tightly to one site than the other, and this difference is due to both entropic and enthalpic contribut...

Calorimetric, spectroscopic, and model studies provide insight into the transport of Ti(IV) by human serum transferrin.

Abstract: Evidence suggests that transferrin can bind Ti(IV) in an unhydrolyzed form (without bound hydroxide or oxide) or in a hydrolyzed form. Ti(IV) coordination by N,N‘-di(o-hydroxybenzyl)ethylenediamine-N,N‘-diacetic acid (HBED) at different pH values models the two forms of Ti(IV)-loaded transferrin spectrally and structurally. 13C NMR and stopped-flow kinetic experiments reveal that when the metal is delivered to the protein using an unhydrolyzed source, Ti(IV) can coordinate in the typical distorted octahedral environment with a bound synergistic anion. The crystal structure of TiHBED obtained at low pH models this type of coordination. The solution structure of the complex compares favorably with the solid state from pH 3.0 to 4.0, and the complex can be reduced with E1/2 = −641 mV vs NHE. Kinetic and thermodynamic competition studies at pH 3.0 reveal that Ti(citrate)3 reacts with HBED via a dissociative mechanism and that the stability of TiHBED (log β = 34.024) is weaker than that of the Fe(III) complex....

Iron and Copper Intracellular Chelation as an Anticancer Drug Strategy.

Abstract: A very promising direction in the development of anticancer drugs is inhibiting the molecular pathways that keep cancer cells alive and able to metastasize. Copper and iron are two essential metals that play significant roles in the rapid proliferation of cancer cells and several chelators have been studied to suppress the bioavailability of these metals in the cells. This review discusses the major contributions that Cu and Fe play in the progression and spreading of cancer and evaluates select Cu and Fe chelators that demonstrate great promise as anticancer drugs. Efforts to improve the cellular delivery, efficacy, and tumor responsiveness of these chelators are also presented including a transmetallation strategy for dual targeting of Cu and Fe. To elucidate the effectiveness and specificity of Cu and Fe chelators for treating cancer, analytical tools are described for measuring Cu and Fe levels and for tracking the metals in cells, tissue, and the body.

Expanding the dipeptidyl peptidase 4-regulated peptidome via an optimized peptidomics platform.

Abstract: In recent years, the biological sciences have seen a surge in the development of methods, including high-throughput global methods, for the quantitative measurement of biomolecule levels (i.e., RNA, proteins, metabolites) from cells and tissues. Just as important as quantitation of biomolecules has been the creation of approaches that uncover the regulatory and signaling connections between biomolecules. Our specific interest is in understanding peptide metabolism in a physiological setting, and this has led us to develop a multidisciplinary approach that integrates genetics, analytical chemistry, synthetic chemistry, biochemistry, and chemical biology to identify the substrates of peptidases in vivo. To accomplish this we utilize a liquid chromatography-mass spectrometry (LC-MS)-based peptidomics platform to measure changes in the peptidome as a function of peptidase activity. Previous analysis of mice lacking the enzyme dipeptidyl peptidase 4 (DPP4(-/-) mice), a biomedically relevant peptidase, using this approach identified a handful of novel endogenous DPP4 substrates. Here, we utilize these substrates and tissues from DPP4(-/-) mice to improve the coverage of the peptidomics platform by optimizing the key steps in the workflow, and in doing so, discover over 70 renal DPP4 substrates (up from 7 at the beginning of our optimization), a 10-fold improvement in our coverage. The sequences of these DPP4 peptide substrates support a broad role for DPP4 in proline-containing peptide catabolism and strengthen a biochemical model that interlinks aminopeptidase and DPP4 activities. Moreover, the improved peptidome coverage also led to the detection of greater numbers of known bioactive peptides (e.g., peptide hormones) during the analysis of gut samples, suggesting additional uses for this optimized workflow. Together these results strengthen our ability to identify endogenous peptide substrates through improved peptidome coverage and demonstrate a broader potential of this peptidomics platform.

Methods in Enzymology.

Abstract: I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Peptidomic discovery of short open reading frame–encoded peptides in human cells

Abstract: The amount of the transcriptome that is translated into polypeptides is of fundamental importance. We developed a peptidomic strategy to detect short ORF (sORF)-encoded polypeptides (SEPs) in human cells. We identified 90 SEPs, 86 of which are novel, the largest number of human SEPs ever reported. SEP abundances range from 10-1000 molecules per cell, identical to known proteins. SEPs arise from sORFs in non-coding RNAs as well as multi-cistronic mRNAs, and many SEPs initiate with non-AUG start codons, indicating that non-canonical translation may be more widespread in mammals than previously thought. In addition, coding sORFs are present in a small fraction (8/1866) of long intergenic non-coding RNAs (lincRNAs). Together, these results provide the strongest evidence to date that the human proteome is more complex than previously appreciated.

In Situ Imaging of Metals in Cells and Tissues

Abstract: Approximately a third of the human proteome contains metal cations, either in form of cofactors with catalytic functions, or as structural support elements.1,2 To guarantee a proper maintenance of this metal ion pool, both at the cellular and whole organism levels, nature has evolved a highly sophisticated machinery comprised of a complex interplay between DNA, proteins, and biomolecules.3 Over the past decades, a steadily growing number of diseases have been identified, which are characterized by metal imbalance in cells and tissues. Among the most prominent examples rank Alzheimer’s disease and Parkinson’s disease, two neurodegenerative disorders that involve abnormal accumulation of transition metals in brain tissue.4 While some progress has been made at understanding the molecular basis of these disorders, many important questions remain unanswered. For example, little is known about the cellular structures that are involved in transiently storing metal ions prior to their incorporation into metalloproteins or the fate of metal ions upon protein degradation. An important first step towards unraveling the regulatory mechanisms involved in trace metal transport, storage, and distribution represents the identification and quantification of the metals, ideally in context of their native physiological environment in tissues, cells, or even at the level of individual organelles and subcellular compartments. Since the inception of the first histochemical methods for the microscopic demonstration of transition metals in tissues more than 140 years ago,5 many highly sensitive microanalytical techniques and instruments have been developed for the in situ analysis of trace metals. The aim of this review is to provide an overview of the most recent achievements in trace metal imaging while at the same time also offering a historical perspective of this rapidly evolving research field. Although this survey has been structured according to the various analytical techniques, particular emphasis is given to the biological background for a better understanding of the context and importance of each discussed study. An overview of the most important microanalytical techniques currently available for the in situ detection of trace metals in cells and tissues is compiled in Table 1. Depending on the task, each technique may offer specific advantages and, of course, also disadvantages. Currently, synchrotron- and focused ion-beam microprobes presumably offer the best combination of sensitivity and spatial resolution; however, the ionizing high-energy excitation beam is not compatible with studying live organisms. Conversely, techniques that have been specifically developed for physiological imaging in clinical medicine, notably magnetic resonance imaging and positron emission tomography, inherently offer only a low spatial resolution and are merely suitable for obtaining information at the organ or tissue level. Although fluorescence microscopy based methods provide very high sensitivity down to the single molecule level while being at the same time compatible with live cell and tissue studies, scattering and limited penetration depth renders these techniques unsuitable for imaging opaque specimens. There are also important differences regarding the type of quantitative information that can be gained by each of these analytical techniques. For example, the histochemical detection with chromogenic and fluorogenic dyes relies on a competitive exchange of the metal ion within its native environment, most likely coordinated to endogenous ligands. Depending on the exchange kinetics and thermodynamic affinity of the histochemical indicator, only a fraction of the total metal ion contents in a cell or tissue can be probed. Nevertheless, this kinetically labile pool is particularly of interest in context of understanding the uptake, distribution, and regulation of trace elements at the cellular level, and in this regard, these methods offer unique opportunities to dynamically image metal ion fluxes in live cells with high sensitivity and spatial resolution. At the same time, organelles and proteins of interest can be readily labeled with genetically encoded green fluorescent protein tags,12 thus providing direct insights into dynamic processes within a larger cellular and biochemical context. In contrast, similar correlative information is difficult to gain with the fully quantitative micro beam methods, which require xenobiotic elemental tags for identifying subcellular structures. Autoradiographic tracer experiments offer much improved resolution over PET; however, the technique is only applicable to fixed or frozen tissues and cells. Furthermore, tracer studies cannot provide direct information regarding the endogenous metal composition of cells or tissues, and are therefore primarily limited to metal uptake, distribution, and release studies. Finally, mass spectrometric analyses are surface-based methods that destroy the sample while measuring its elemental composition. Clearly, only the combination of several analytical techniques and specific biochemical studies may lead to a fully comprehensive analysis of a biological system. Table 1 Spatially resolved microanalytical techniques for in situ imaging of trace metals in biology.6–11 2. Histochemical Techniques Histology is the branch of biology dealing with the study of microscopic anatomy of cells and tissues of plants and animals. Histological studies are typically carried out on thin sections of tissue or with cultured cells. To visualize and identify particular structures, a broad spectrum of histological stains and indicators are available. Among the most widely used dyes are hematoxylin and eosin, which stain nuclei blue and the cytoplasm pink, respectively.13 The history of detecting biological trace metal by histological methods dates back more than 140 years. Although these techniques have been today mostly replaced by the much more sensitive modern analytical methods described in this review article, histochemical approaches for visualizing metals mark the very beginning in the exploration of the inorganic physiology of transition metals. Given this special place in history, we deemed it necessary to briefly review some of the early achievements in this field. 2.1. Chromogenic Detection with Chelators and Ligands Ever since the inception of Perls Prussian blue method for staining of non-heme iron, numerous indicators have been developed for the in situ visualization of trace metals in biological tissues and cells.13 Due to their limited sensitivity; however, most of these techniques were only suitable for the diagnosis of pathological conditions, typically associated with excess metal accumulations, thus preventing their application for routine staining of normal tissue. Furthermore, because the dyes are engaged in a competitive exchange equilibrium with endogenous ligands, histological stains are not suitable for the analytical determination of the total metal contents in tissues and thus limited to the visualization of the histologically reactive fraction of loosely bound labile metal ions. 2.1.1. Histochemistry of Iron The histochemical demonstration of labile iron reported by Perls in 1867 is among the earliest accounts describing the in situ visualization of a trace metal in biological tissues.5 The method was originally described by Grohe, who observed the formation of a blue coloration when he treated cadaver tissues with potassium ferrocyanide in acidic solution.14 Due to its low cost and simplicity, the technique is still used today for the histological visualization of non-heme iron. Some variations focused on optimizing the concentrations and proportions of the reagents,15–17 among which Lison’s protocol17 appears to be most popular today. An intensification of Perls’ staining can be obtained by exploiting the use of ferric ferrocyanide in catalyzing the oxidation of diaminobenzidine (DAB) to polymeric benzidine black by hydrogen peroxide.18 An alternative method employs the reaction of ferricyanide with Fe(II) resulting in Turnbull blue.19 Since almost all of the Fe in tissues is in the ferric form, the staining procedure requires the in situ conversion of Fe(III) to Fe(II) with ammonium sulfide.15 Due to often incomplete reduction, the method never gained much attention. More recently, an application of Turnbull blue, named the ‘perfusion Turnbull method’ has been developed, where in vivo perfusion of acidic ferricyanide is followed by DAB intensification.20 The direct in vivo perfusion avoids artifacts associated with tissue fixation, including the loss of loosely bound iron and oxidation of Fe(II) to Fe(III). Similarly, Perls method was modified by employing in vivo perfusion with acidic ferrocyanide. Both methods are capable of identifying organs and tissues containing histochemically reactive iron over a broad pH range, including the low endosomal pH.21,22 The history of iron histochemistry would be incomplete without mentioning Quincke’s method, which employed ammonium sulfide for the precipitation of tissue iron as its sulfide.23 A detailed account on the various techniques, including a comprehensive historical overview of non-heme iron chemistry, has been recently published.24

Pharmacology, Physiology, and Mechanisms of Action of Dipeptidyl Peptidase-4 Inhibitors

Abstract: Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane molecule and a soluble circulating protein. Both membrane-associated and soluble DPP4 exert catalytic activity, cleaving proteins containing a position 2 alanine or proline. DPP4-mediated enzymatic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the molecular mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biology of DPP4 with a focus on: 1) identification of pharmacological vs physiological DPP4 substrates; and 2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chemical reduction of DPP4 activity. We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4 inhibitors in both preclinical and clinical studies. Finally, we highlight experimental pitfalls and technical challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.

High surface area crystalline titanium dioxide: potential and limits in electrochemical energy storage and catalysis.

Abstract: Titanium dioxide is one of the most intensely studied oxides due to its interesting electrochemical and photocatalytic properties and it is widely applied, for example in photocatalysis, electrochemical energy storage, in white pigments, as support in catalysis, etc. Common synthesis methods of titanium dioxide typically require a high temperature step to crystallize the amorphous material into one of the polymorphs of titania, e.g. anatase, brookite and rutile, thus resulting in larger particles and mostly non-porous materials. Only recently, low temperature solution-based protocols gave access to crystalline titania with higher degree of control over the formed polymorph and its intra- or interparticle porosity. The present work critically reviews the formation of crystalline nanoscale titania particles via solution-based approaches without thermal treatment, with special focus on the resulting polymorphs, crystal morphology, surface area, and particle dimensions. Special emphasis is given to sol–gel processes via glycolated precursor molecules as well as the miniemulsion technique. The functional properties of these materials and the differences to chemically identical, non-porous materials are illustrated using heterogeneous catalysis and electrochemical energy storage (battery materials) as example.