Implication for buried polar contacts and ion pairs in hyperthermostable enzymes

Understanding the structural basis of thermostability and thermoactivity, and their interdependence, is central to the successful future exploitation of extremophilic enzymes in biotechnology. However, the structural basis of thermostability is still not fully characterized. Ionizable residues play essential roles in proteins, modulating protein stability, folding and function. The dominant roles of the buried polar contacts and ion pairs have been reviewed by distinguishing between the inside polar contacts and the total intramolecular polar contacts, and by evaluating their contribution as molecular determinants for protein stability using various protein structures from hyperthermophiles, thermophiles and mesophilic organisms. The analysis revealed that the remarkably increased number of internal polar contacts in a monomeric structure probably play a central role in enhancing the melting temperature value up to 120 degrees C for hyperthermophilic enzymes from the genus Pyrococcus. These results provide a promising contribution for improving the thermostability of enzymes by modulating buried polar contacts and ion pairs.     

Rutaceous alkaloids as models for the design of novel antitumor drugs

The chemical diversity of alkaloids in the Rutaceae is correlated with biosynthetic pathways involving various aromatic amino acid precursors, tyrosine, tryptophan, histidine, and anthranilic acid. The interest of rutaceous polyheteroaromatic alkaloids as models for the development of anticancer agents relies on their frequent ability to interact with DNA or with systems involved in the control of its topology, repair, and replication. Fagaronine and nitidine, from Zanthoxylum, demonstrate antileukemic activity, associated with topoisomerases inhibition. Evodiamine from Euodia rutaecarpa, displays antimetastatic properties. The pyranoacridone acronycine, from Sarcomelicope, exhibits antitumor activity against a broad spectrum of solid tumors. Development of synthetic analogues based on this latter natural product template followed the isolation of the unstable acronycine epoxide, which led to a hypothesis of bioactivation of acronycine by transformation of the 1,2-double bond into the corresponding oxirane. 1,2-Diacyloxy-1,2-dihydroacronycine derivatives exhibited antitumor properties, with a broadened spectrum of activity and an increased potency. The demonstration that acronycine interacted with DNA led to develop benzo[a], [b], and [c]acronycine analogs. Benzo[a] and [b] derivatives displayed significant antitumor activities. 1,2-Dihydroxy-1,2-dihydrobenzo[b]acronycine esters and diesters were active in human orthotopic models of cancers xenografted in nude mice. The activity of these compounds was correlated with their ability to give covalent adducts with DNA, involving reaction between the N-2 amino group of guanines and the ester group at the benzylic position of the drug. Cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine, currently developed under the code S23906-1, successfully underwent phase I and is currently under phase II clinical trials.     

Model system studies of staining procedures for lysine and arginine residues

Summary Using polyacrylamide films containing poly-lysine, polyargine and DNA as test models, a variety of reportedly specific staining procedures have been examined. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin while arginine will stain with fast green, if proteins containing both amino-acids are stained with the dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acid-arginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrochloric acid in a Feulgen-like procedure which stains DNA to the same level as the classical hydrochloric acid based procedure while also staining arginine present.     

The anatomy of the greater occipital nerve: implications for the etiology of migraine headaches

An interest in pursuing new theories of the underlying etiology of migraine headaches has been sparked by previously published reports of an association between amelioration of migraine headache symptoms and corrugator resection during endoscopic brow lift. This theory has further been reinforced by recent publications documenting improvement in migraine headaches following injection of botulinum A toxin. There are thought to be four major "trigger points" along the course of several peripheral nerves that may cause migraine headaches. Among these peripheral nerves is the greater occipital nerve. For this reason, the authors have undertaken an anatomic study of this nerve to determine its usual course, potential anatomic variations, and possible points of potential entrapment or compression. The results of this anatomic study have enhanced further development of techniques designed to address these points of entrapment/compression and potentially lead to relief of migraine headaches caused by this mechanism. Twenty cadaver heads from patients with an unknown history of migraine headaches were dissected to trace the normal course of the greater occipital nerve from the semispinalis muscle penetration to the superior nuchal line. Standardized measurements were performed on 14 specimens to determine the location of the emergence of the nerve using the midline and occipital protuberance as landmarks. On the basis of this information, the location of emergence was determined to be at a point centered approximately 3 cm below the occipital protuberance and 1.5 cm lateral to the midline. This location can, in turn, be used to guide the practitioner performing chemodenervation of the semispinalis capitis muscle in an attempt to provide migraine symptom relief.     

Stimulated endocytosis in penetratin uptake: effect of arginine and lysine

Cell-penetrating peptides can deliver macromolecular cargo into cells and show promise as vectors for intracellular drug delivery. Internalization occurs predominantly via endocytosis, but the exact uptake mechanisms are not fully understood. We show quantitatively how penetratin, a 16-residue cationic peptide, stimulates fluid-phase endocytosis and triggers its own uptake into Chinese hamster ovarian cells, using a 70 kDa dextran to indicate macropinocytosis. The total cellular endocytotic rate is significantly less affected and we therefore propose up-regulation of macropinocytosis to occur at the expense of other types of endocytosis. By comparing penetratin to its analogs PenArg and PenLys, enriched in arginines and lysines, respectively, we show how these side-chains contribute to uptake efficiency. The degree of peptide and dextran uptake follows similar patterns regarding peptide concentration and arginine/lysine content (PenArg > penetratin > PenLys), indicating that a high content of arginines is beneficial but not necessary for stimulating endocytosis.     

Identification of the characteristics that underlie botulinum toxin potency: implications for designing novel drugs

Botulinum toxin is a uniquely potent substance whose natural site of action is the peripheral cholinergic nerve ending. A substantial amount of information on the cellular, subcellular and molecular aspects of toxin action has been accumulated, and as a result a sound understanding of the basis for toxin potency has been developed. The principal characteristics of the toxin molecule that account for its potency are its ability: a) to be absorbed from the gut with minimal degradation; b) to bind to receptors that maximize the prospects of a pathophysiologic outcome; c) to act by a multiplicative (viz., enzymatic) mechanism; and d) to modify a substrate that is essential for neuronal function. Interestingly, the same properties that account for potency can also be exploited to utilize the toxin as a research tool and as a therapeutic agent. Several specific examples of ways to use the toxin advantageously are presented, including: a) development of oral medications and vaccines; b) analysis of subcellular mechanisms that govern transcytosis; c) identification of cell surface markers characteristic of cholinergic nerve endings; and d) analysis of specific aspects of exocytosis, such as spontaneous quantal release and synchronous quantal release. In all likelihood, further studies on the mechanism of botulinum toxin action will reveal yet further opportunities for utilizing it as a research tool or therapeutic agent.     

The dual biosynthetic capability of N-acetylornithine aminotransferase in arginine and lysine biosynthesis

The genes encoding the seven enzymes needed to synthesize L-lysine from aspartate semialdehyde and pyruvate have been identified in a number of bacterial genera, with the single exception of the dapC gene encoding the PLP-dependent N-succinyl-L, L-diaminopimelate:alpha-ketoglutarate aminotransferase (DapATase). Purification of E. coli DapATase allowed the determination of both the amino-terminal 26 amino acids and a tryptic peptide fragment. Sequence analysis identified both of these sequences as being identical to corresponding sequences from the PLP-dependent E. coli argD-encoded N-acetylornithine aminotransferase (NAcOATase). This enzyme performs a similar reaction to that of DapATase, catalyzing the N-acetylornithine-dependent transamination of alpha-ketoglutarate. PCR cloning of the argD gene from genomic E. coli DNA, expression, and purification yielded homogeneous E. coli NAcOATase. This enzyme exhibits both NAcOATase and DapATase activity, with similar specificity constants for N-acetylornithine and N-succinyl-L,L-DAP, suggesting that it can function in both lysine and arginine biosynthesis. This finding may explain why numerous investigations have failed to identify genetically the bacterial dapC locus, and suggests that this enzyme may be an attractive target for antibacterial inhibitor design due to the essential roles of these two pathways in bacteria.     

Model system studies of staining procedures for lysine and arginine residues.

Using polyacrylamide films containg poly-lysine, polyarginine and DNA as test models, a variety of reportedly specific staining procedures have been examine. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin arginine will stain with fast green, if proteins containing both amino-acids are stained with dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acidarginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrocholoric acid in a Feulgen-like procedure which stains DNA to the same level as the classiclal hydrochloric acid based procedure while also staining arginine present.     

The maximal and submaximal vertical jump: implications for strength and conditioning

The vertical jump is a widely used activity to develop explosive strength, particularly in plyometric and maximal power training programs. It is a multijoint action that requires substantial muscular effort from primarily the ankle, knee, and hip joints. It is not known if submaximal performances of a vertical jump have a proportional or differential training effect on the major lower-limb muscles compared to maximal jump performance. Therefore, the purpose of this study was to investigate the contribution that each of the major lower-limb joints makes to vertical jump performance as jump height increases and to comment on the previously mentioned uncertainty. Adult males (N = 20) were asked to perform a series of submaximal (LOW and HIGH) and maximal (MAX) vertical jumps while using an arm swing. Force, motion, and electromyographical data were recorded during each performance and used to compute a range of kinematic and kinetic data, including ankle, knee, and hip joint torques, powers, and work done. It was found that the contribution to jump height made by the ankle and knee joints remains largely unchanged as jump height increases (work done at the ankle: LOW =1.80, HIGH = 1.97, MAX = 2.06 J.kg(-1), F = 3.596, p = 0.034; knee: LOW = 1.62, HIGH = 1.77, MAX = 1.94 J.kg(-1), F = 1.492, p = 0.234) and that superior performance in the vertical jump is achieved by a greater effort of the hip extensor muscles (work done at the hip: LOW = 1.03, HIGH = 1.84, MAX = 3.24 J.kg(-1), F = 110.143, p < 0.001). It was concluded that the role of submaximal and maximal jumps can be differentiated in terms of their effect on ankle, knee, and hip joint muscles and may be of some importance to training regimens in which these muscles need to be differentially trained.     

The primordial metabolism: an ancestral interconnection between leucine,arginine, and lysine biosynthesis

Background

It is generally assumed that primordial cells had small genomes with simple genes coding for enzymes able to react with a wide range of chemically related substrates, interconnecting different metabolic routes. New genes coding for enzymes with a narrowed substrate specificity arose by paralogous duplication(s) of ancestral ones and evolutionary divergence. In this way new metabolic pathways were built up by primordial cells. Useful hints to disclose the origin and evolution of ancestral metabolic routes and their interconnections can be obtained by comparing sequences of enzymes involved in the same or different metabolic routes. From this viewpoint, the lysine, arginine, and leucine biosynthetic routes represent very interesting study-models. Some of the lys, arg and leu genes are paralogs; this led to the suggestion that their ancestor genes might interconnect the three pathways. The aim of this work was to trace the evolutionary pathway leading to the appearance of the extant biosynthetic routes and to try to disclose the interrelationships existing between them and other pathways in the early stages of cellular evolution.

Results

The comparative analysis of the genes involved in the biosynthesis of lysine, leucine, and arginine, their phylogenetic distribution and analysis revealed that the extant metabolic "grids" and their interrelationships might be the outcome of a cascade of duplication of ancestral genes that, according to the patchwork hypothesis, coded for unspecific enzymes able to react with a wide range of substrates. These genes belonged to a single common pathway in which the three biosynthetic routes were highly interconnected between them and also to methionine, threonine, and cell wall biosynthesis. A possible evolutionary model leading to the extant metabolic scenarios was also depicted.

Conclusion

The whole body of data obtained in this work suggests that primordial cells synthesized leucine, lysine, and arginine through a single common metabolic pathway, whose genes underwent a set of duplication events, most of which can have predated the appearance of the last common universal ancestor of the three cell domains (Archaea, Bacteria, and Eucaryotes). The model proposes a relative timing for the appearance of the three routes and also suggests a possible evolutionary pathway for the assembly of bacterial cell-wall.

     

The survival benefit of short-chain organic acids and the inducible arginine and lysine decarboxylase genes for Escherichia coli

D.E. GUILFOYLE AND I.N. HIRSHFIELD. 1996. The short-chain organic acids (SCOAs), acetic and propionic acids, are used widely as food preservatives. The production of these two acids plus butyric acid in the colon by anaerobes serves as a mechanism for controlling the numbers of enterobacteria (which can be pathogens) in this organ. It has been found in this study that the acid tolerance of cells initially grown at near neutral pH (6.5) to a lethal pH of 3.5 is enhanced by their exposure to 0.1% propionate or butyrate. The data also indicate that the inducible arginine and lysine decarboxylases are important for the survival of Escherichia coli exposed to a combination of mildly acidic pH (5.5) and 0.5% butyrate. This study suggests that the presence of SCOAs could trigger an adaptive survival response which may be important in the survival of food-borne pathogens.     

Immune-based mechanisms of cytotoxic chemotherapy: implications for the design of novel and rationale-based combined treatments against cancer

Conventional anticancer chemotherapy has been historically thought to act through direct killing of tumor cells. This concept stems from the fact that cytotoxic drugs interfere with DNA synthesis and replication. Accumulating evidence, however, indicates that the antitumor activities of chemotherapy also rely on several off-target effects, especially directed to the host immune system, that cooperate for successful tumor eradication. Chemotherapeutic agents stimulate both the innate and adaptive arms of the immune system through several modalities: (i) by promoting specific rearrangements on dying tumor cells, which render them visible to the immune system; (ii) by influencing the homeostasis of the hematopoietic compartment through transient lymphodepletion followed by rebound replenishment of immune cell pools; (iii) by subverting tumor-induced immunosuppressive mechanisms and (iv) by exerting direct or indirect stimulatory effects on immune effectors. Among the indirect ways of immune cell stimulation, some cytotoxic drugs have been shown to induce an immunogenic type of cell death in tumor cells, resulting in the emission of specific signals that trigger phagocytosis of cell debris and promote the maturation of dendritic cells, ultimately resulting in the induction of potent antitumor responses. Here, we provide an extensive overview of the multiple immune-based mechanisms exploited by the most commonly employed cytotoxic drugs, with the final aim of identifying prerequisites for optimal combination with immunotherapy strategies for the development of more effective treatments against cancer.