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Photovoltaics is amongst the most important technologies for renewable energy sources, and plays a key role in the development of a society with a smaller environmental footprint. Key parameters for solar cells are their energy conversion efficiency, their operating lifetime, and the cost of the energy obtained from a photovoltaic system compared to other sources. The optimization of these aspects involves the exploitation of new materials and development of novel solar cell concepts and designs. Both theoretical modeling and characterization of such devices require a comprehensive view including all scales from the atomic to the macroscopic and industrial scale. The different length scales of the electronic and optical degrees of freedoms specifically lead to an intrinsic need for multiscale simulation, which is accentuated in many advanced photovoltaics concepts including nanostructured regions. Therefore, multiscale modeling has found particular interest in the photovoltaics community, as a tool to advance the field beyond its current limits. In this article, we review the field of multiscale techniques applied to photovoltaics, and we discuss opportunities and remaining challenges.

The hollow protein capsids from a number of different viruses are being considered for multiple biomedical or nanotechnological applications. In order to improve the applied potential of a given viral capsid as a nanocarrier or nanocontainer, specific conditions must be found for achieving its faithful and efficient assembly in vitro. The small size, adequate physical properties and specialized biological functions of the capsids of parvoviruses such as the minute virus of mice (MVM) make them excellent choices as nanocarriers and nanocontainers. In this study we analyzed the effects of protein concentration, macromolecular crowding, temperature, pH, ionic strength, or a combination of some of those variables on the fidelity and efficiency of self-assembly of the MVM capsid in vitro. The results revealed that the in vitro reassembly of the MVM capsid is an efficient and faithful process. Under some conditions, up to ~40% of the starting virus capsids were reassembled in vitro as free, non aggregated, correctly assembled particles. These results open up the possibility of encapsidating different compounds in VP2-only capsids of MVM during its reassembly in vitro, and encourage the use of virus-like particles of MVM as nanocontainers.

Background: The coronavirus disease has led to an exhaustive exploration of the SARS-CoV-2 genome. Despite the amount of information accumulated, the prediction of short RNA motifs encoding peptides mediating protein-protein or protein-drug interactions has received limited attention., Objective: The study aims to predict short RNA motifs that are interspersed in the SARS-CoV-2 genome., Methods: A method in which 14 trinucleotide families, each characterized by being composed of triplets with identical nucleotides in all possible configurations, was used to find short peptides with biological relevance. The novelty of the approach lies in using these families to search how they are distributed across genomes of different CoV genera and then to compare the distributions of these families with each other., Results: We identified distributions of trinucleotide families in different CoV genera and also how they are related, using a selection criterion that identified short RNA motifs. The motifs were reported to be conserved in SARS-CoVs; in the remaining CoV genomes analysed, motifs contained, exclusively, different configurations of the trinucleotides A, T, G and A, C, G. Eighty-eight short RNA motifs, ranging in length from 12 to 49 nucleotides, were found: 50 motifs in the 1a polyprotein-encoding orf, 27 in the 1b polyprotein-encoding orf, 5 in the spike-encoding orf, and 6 in the nucleocapsid-encoding orf. Although some motifs (~27%) were found to be intercalated or attached to functional peptides, most of them have not yet been associated with any known functions., Conclusion: Some of the trinucleotide family distributions in different CoV genera are not random; they are present in short peptides that, in many cases, are intercalated or attached to functional sites of the proteome., Competing Interests: The authors declare no conflict of interest, financial or otherwise., (© 2022 Bentham Science Publishers.)

Damage to the retinal pigment epithelium, Bruch's membrane and/or tissues underlying macula is known to increase the risk of age-related macular degeneration (AMD). AMD is commonly categorized in two distinct types, namely, the nonexudative (dry form) and the exudative (wet form). Currently, there is no ideal treatment available for AMD. Recommended standard treatments are based on the use of vascular endothelial growth factor (VEGF), with the disadvantage of requiring repeated intravitreal injections which hinder patient's compliance to the therapy. In recent years, several synthetic and natural active compounds have been proposed as innovative therapeutic strategies against this disease. There is a growing interest in the development of formulations based on nanotechnology because of its important role in the management of posterior eye segment disorders, without the use of intravitreal injections, and furthermore, with the potential to prolong drug release and thus reduce adverse effects. In the same way, 3D bioprinting constitutes an alternative to regeneration therapies for the human retina to restore its functions. The application of 3D bioprinting may change the current and future perspectives of the treatment of patients with AMD, especially those who do not respond to conventional treatment. To monitor the progress of AMD treatment and disease, retinal images are used. In this work, we revised the recent challenges encountered in the treatment of different forms of AMD, innovative nanoformulations, 3D bioprinting, and techniques to monitor the progress.

Protein-based nanostructured materials are being developed for many biomedical and nanotechnological applications. Despite their many desirable features, protein materials are highly susceptible to disruption by mechanical stress and fatigue. This study is aimed to increase fatigue resistance and enhance self-healing of a natural protein-based supramolecular nanomaterial through permanent genetic modification. The authors envisage the conversion of a model nanosheet, formed by a regular array of noncovalently bound human immunodeficiency virus capsid protein molecules, into a supramolecular "chain mail." Rationally engineered mutations allow the formation of a regular network of disulfide bridges in the protein lattice. This network links each molecule in the lattice to each adjacent molecule through one covalent bond, analogous to the rivetting of interlinked iron rings in the chain mail of a medieval knight. The engineered protein nanosheet shows greatly increased thermostability and resistance to mechanical stress and fatigue in particular, as well as enhanced self-healing, without undesirable stiffening compared to the original material. The results provide proof of concept for a genetic design to permanently increase fatigue resistance and enhance self-healing of protein-based nanostructured materials. They also provide insights into the molecular basis for fatigue of protein materials., (© 2022 Wiley-VCH GmbH.)

Picornaviridae family includes several viruses of great economic and medical importance. Among all members of the family we focused our attention on the human rhinovirus, the most important etiologic agent of the common cold and on the foot-and-mouth disease virus that cause of an economically important disease in cattle. Despite the low sequence similarity of the polyprotein coding open reading frames of these highly divergent picornaviruses, they have in common structural and functional similarities including a similar genomic organization, a capsid structure composed of 60 copies of four different proteins, or 3D-structures showing similar general topology, among others. We hypothesized that such similarities could be reflected in emergent common compositional structures interspersed in their genomes which were not observed heretofore. Using a methodology categorizing nucleotide triplets by their gross-composition we have found two human rhinoviruses sharing compositional structures interspersed along their genomic RNA with three foot-and-mouth disease viruses. The shared compositional structures are in one case composed by nucleotide triplets containing all nearest-neighbours of A and G and in other case containing all nearest-neighbours of A, and C. The structures are under strong evolutionary constraints for variability, allowing the access to novel viral genomic motifs with likely biological relevance. The conserved fragments would be useful to predict critical mutation points sites important from the evolutionary point of view.

Unravelling gene structure requires the identification and understanding of the constraints that are often associated with the evolutionary history and functional domains of genes. We speculated in this manuscript with the possibility of the existence in orthologs of an emergent highly conserved gene structure that might explain their coordinated evolution during speciation events and their parental function. Here, we will address the following issues: (1) is there any conserved hypothetical structure along ortholog gene sequences? (2) If any, are such conserved structures maintained and conserved during speciation events? The data presented show evidences supporting this hypothesis. We have found that, (1) most orthologs studied share highly conserved compositional structures not observed previously. (2) While the percent identity of nucleotide sequences of orthologs correlates with the percent identity of composon sequences, the number of emergent compositional structures conserved during speciation does not correlate with the percent identity. (3) A broad range of species conserves the emergent compositional stretches. We will also discuss the concept of critical gene structure.

Icosahedral viral capsids are made of a large number of symmetrically organized protein subunits whose local movements can be essential for infection. In the capsid of the minute virus of mice, events required for infection that involve translocation of peptides through capsid pores are associated with a subtle conformational change. In vitro, this change can be reversibly induced by overcoming the energy barrier through mild heating of the capsid, but little is known about the capsid regions involved in the process. Here, we use hydrogen-deuterium exchange coupled to mass spectrometry to analyze the dynamics of the minute virus of mice capsid at increasing temperatures. Our results indicate that the transition associated with peptide translocation involves the structural rearrangement of regions distant from the capsid pores. These alterations are reflected in an increased dynamics of some secondary-structure elements in the capsid shell from which spikes protrude, and a decreased dynamics in the long intertwined loops that form the large capsid spikes. Thus, the translocation events through capsid pores involve a global conformational rearrangement of the capsid and a complex alteration of its equilibrium dynamics. This study additionally demonstrates the potential of hydrogen-deuterium exchange coupled to mass spectrometry to explore in detail temperature-dependent structural dynamics in large macromolecular protein assemblies. Most importantly, it paves the way for undertaking novel studies of the relationship between structure, dynamics, and biological function in virus particles and other large protein cages., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: The use of medicinal plants in Mexico has been documented since pre-Hispanic times. Nevertheless, the level of use of medicinal plants by health professionals in Mexico remains to be explored., Aim of the Study: To evaluate the use, acceptance and prescription of medicinal plants by health professionals in 9 of the states of Mexico., Materials and Methods: Direct and indirect interviews, regarding the use and acceptance of medicinal plants, with health professionals (n=1614), including nurses, physicians, pharmacists, and odontologists from nine states in Mexico were performed from January 2015 to July 2016. The interviews were analyzed with the factor the informant consensus (FIC)., Results: The information obtained indicated that 46% of those interviewed feel patients should not use medicinal plants as an alternative therapy. Moreover, 54% of health professionals, and 49% of the physicians have used medicinal plants as an alternative therapy for several diseases. Twenty eight percent of health professionals, and 26% of the physicians, have recommended or prescribed medicinal plants to their patients, whereas 73% of health professionals were in agreement with receiving academic information regarding the use and prescription of medicinal plants. A total of 77 plant species used for medicinal purposes, belonging to 40 botanical families were reported by the interviewed. The results of the FIC showed that the categories of diseases of the digestive system (FIC=0.901) and diseases of the respiratory system (FIC=0.898) had the greatest agreement., Conclusions: This study shows that medicinal plants are used for primary health care in Mexico by health professionals., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

The analysis of a large number of human and mouse genes codifying for a populated cluster of transmembrane proteins revealed that some of the genes significantly vary in their primary nucleotide sequence inter-species and also intra-species. In spite of that divergence and of the fact that all these genes share a common parental function we asked the question of whether at DNA level they have some kind of common compositional structure, not evident from the analysis of their primary nucleotide sequence. To reveal the existence of gene clusters not based on primary sequence relationships we have analyzed 13574 human and 14047 mouse genes by the composon-clustering methodology. The data presented show that most of the genes from each one of the samples are distributed in 18 clusters sharing the common compositional features between the particular human and mouse clusters. It was observed, in addition, that between particular human and mouse clusters having similar composon-profiles large variations in gene population were detected as an indication that a significant amount of orthologs between both species differs in compositional features. A gene cluster containing exclusively genes codifying for transmembrane proteins, an important fraction of which belongs to the Rhodopsin G-protein coupled receptor superfamily, was also detected. This indicates that even though some of them display low sequence similarity, all of them, in both species, participate with similar compositional features in terms of composons. We conclude that in this family of transmembrane proteins in general and in the Rhodopsin G-protein coupled receptor in particular, the composon-clustering reveals the existence of a type of common compositional structure underlying the primary nucleotide sequence closely correlated to function.

The polcalcin family is one of the most epidemiologically relevant families of calcium-binding allergens. Polcalcins are potent plant allergens that contain one or several EF-hand motifs and their allergenicity is primarily associated with the Ca(2+)-bound form of the protein. Conformation, stability, as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. We describe a protocol that uses three techniques (SDS-PAGE, circular dichroism spectroscopy, and ELISA) to describe the effects that calcium has on the structural changes in an allergen and its IgE binding properties.

This report deals with the study of compositional properties of human gene sequences evaluating similarities and differences among functionally distinct sectors of the gene independently of the reading frame. To retrieve the compositional information of DNA, we present a neighbor base dependent coding system in which the alphabet of 64 letters (DNA triplets) is compressed to an alphabet of 14 letters here termed triplet composons. The triplets containing the same set of distinct bases in whatever order and number form a triplet composon. The reading of the DNA sequence is performed starting at any letter of the initial triplet and then moving, triplet-to-triplet, until the end of the sequence. The readings were made in an overlapping way along the length of the sequences. The analysis of the compositional content in terms of the composon usage frequencies of the gene sequences shows that: (i) the compositional content of the sequences is far from that of random sequences, even in the case of non-protein coding sequences; (ii) coding sequences can be classified as components of compositional clusters; and (iii) intron sequences in a cluster have the same composon usage frequencies, even as their base composition differs notably from that of their home coding sequences. A comparison of the composon usage frequencies between human and mouse homologous genes indicated that two clusters found in humans do not have their counterpart in mouse whereas the others clusters are stable in both species with respect to their composon usage frequencies in both coding and noncoding sequences.

UV-B absorbing compounds (UACs) in present-day and fossil pollen, spores, cuticles, seed coats and wood have been evaluated as a proxy for past UV. This proxy may not only provide information on variation of stratospheric ozone and solar UV in the period preceding and during the Antarctic ozone hole (1974-present day), but also on the development and variation of the stratospheric ozone layer and solar surface UV during the evolution of life on Earth. Sporopollenin and cutin are highly resistant biopolymers, preserving well in the geological record and contain the phenolic acids p-coumaric (pCA) and ferulic acid (FA). pCA and FA represent a good perspective for a plant-based proxy for past surface UV radiation since they are induced by solar UV-B via the phenylpropanoid pathway (PPP). UV-B absorption by these monomers in the wall of pollen and spores and in cuticles may prevent damage to the cellular metabolism. Increased pCA and FA in pollen of Vicia faba exposed to enhanced UV-B was found in greenhouse experiments. Further correlative evidence comes from UV-absorbing compounds in spores from 1960-2000 comparing exposure of land plants (Lycopodium species) to solar UV before and during ozone depletion and comparing plants from Antarctica (severe ozone depletion), Arctic, and other latitudes with less or negligible ozone depletion. Wood-derived compounds guaiacyl (G), syringyl (S), and p-hydroxyphenyl (P) are produced via the PPP. The proportions of P, G, and S in the lignin differ between various plant groups (e.g. dicotyledons/monocotyledons, gymnosperms/angiosperms). It is hypothesized that this lignin composition and derived physiological and physical properties of lignin (such as tree-ring wood density) has potential as a proxy for palaeo-UV climate. However validation by exposure of trees to enhanced UV is lacking. pCA and FA also form part of cutin polymers and are found in extant and fossil Ginkgo leaf cuticles as shown by thermally-assisted hydrolysis and methylation (THM)-pyrolysis-GC-MS. Potentially, the time scale for reconstruction of ozone column thickness and UV-B based on the UAC UV proxy may be decadal, centennial, millennial and possibly billenial. For further development of the UACs and pCA and FA-based UV proxy, it is necessary to obtain the UV dose-response (content of UACs, pCA and FA in sporopollenin and cutin) relationships for validation, based on outdoor UV radiation manipulations experiments with plants, and comparative analysis of stored plants (herbaria) or fossil material of the same or related plant species.

Leishmaniasis comprises a spectrum of parasitic illnesses caused by several species of the protozoan kinetoplastid parasite, Leishmania spp. The disease affects 12 million people around the world with an annual death rate of approximately 80,000 people. Several drugs are available for treating leishmaniasis. For example, pentavalent antimonial compounds, such as sodium stibogluconate and meglumine antimonite are the drugs used in first-line chemotherapy. As second-line drugs, amphotericin B and pentamidine are used. However, current treatments against leishmaniasis are usually unsatisfactory due to some limitations including the route of administration of the drugs, their unaffordable cost and toxicity. Efforts have been made to develop new leishmanicidal drugs and to find new strategies of drug design. Hence, it is interesting to point out that the effectiveness of certain molecules as both anticancer drugs and antiprotozoal agents suggested that this class of compounds and their derivatives might be useful as antileishmanial agents. This review summarizes the anticancer compounds that have been investigated against leishmaniasis. Some of such agents include: compounds with in vitro antileishmanial activities, molecules tested in clinical trials and registered patents. We finally discuss challenges in chemotherapy and future prospects in the treatment of leishmaniasis.

Background: Controversies about laparoscopic appendectomy (LA) focus mainly on the high intraabdominal infection rate. In 2005, Serour et al described a distinct complication specific to LA, termed "postlaparoscopic appendectomy complication" (PLAC). This complication is an intraabdominal infection, without abscess formation, which develops after laparoscopic appendectomy for non-complicated appendicitis (simple, phlegmonous, or normal appendix) and is observed in patients discharged after an uneventful postoperative period. We reviewed our case series to establish our intraabdominal infection rate in appendectomy and to identify cases similar to this newly described complication., Material and Method: We retrospectively reviewed 651 clinical records of appendectomy performed by the laparoscopic (LA) or open approach (OA) over an 11-year period in our hospital. The criteria for a diagnosis of PLAC were as follows: a) clinical criteria: uneventful appendectomy (OA or LA), asymptomatic status on hospital discharge, and onset of right lower quadrant pain, fever, and elevated white blood cell count after discharge; b) pathologic criteria: non-complicated appendicitis (gangrenous or perforated appendicitis were excluded), and c) ultrasound scan showing characteristic features., Results: A total of 432 LA and 219 OA were reviewed. The conversion rate was 11.1%. The main complications (intention-to-treat analysis) were wound infection (6.3% in LA versus 7.8% in OA) and intraabdominal infection (4.2% in LA versus 2.3% in OA). Four out of 18 cases of intraabdominal infection after LA fulfilled PLAC criteria, representing 1% of all LA and 22% of intraabdominal infections after LA., Conclusions: LA seems to be associated with an increased risk of intraabdominal infection. Our results suggest that a distinct form of intraabdominal infection specific to laparoscopic appendectomy may exist.

Prokaryotic DNA replication is compartmentalized at the cellular membrane. The Bacillus subtilis phage varphi29-encoded membrane protein p16.7 is one of the few proteins known to be involved in the organization of prokaryotic membrane-associated DNA replication. The functional DNA binding domain of p16.7 is constituted by its C-terminal half, p16.7C, which forms high affinity dimers in solution and which can form higher order oligomers. Recently, the solution and crystal structures of p16.7C and the crystal structure of the p16.7C-DNA complex have been solved. Here, we have studied the p16.7C dimerization process and the structural and functional roles of p16.7 residues Trp-116 and Asn-120 and its last nine C-terminal amino acids, which form an extended tail. The results obtained show that transition of folded dimers into unfolded monomers occurs without stable intermediates and that both Trp-116 and the C-terminal tail are important for dimerization and functionality of p16.7C. Residue Trp-116 is involved in formation of a novel aromatic cage dimerization motif, which we call "Pro cage." Finally, whereas residue Asn-120 plays a minor role in p16.7C dimerization, we show that it is critical for both oligomerization and DNA binding, providing further evidence that DNA binding and oligomerization of p16.7C are coupled processes.

Aims and Background: The purpose of the study was to test the immunological and clinical effects of infusions of dendritic cells pulsed with autologous tumor lysate in patients with advanced cancer., Patients and Methods: Peripheral blood mononuclear cells from 15 patients with metastatic cancer (melanoma in 10, lung cancer in 2, renal cell carcinoma in 1, sarcoma in 1, breast cancer in 1) were harvested by leukapheresis after mobilization with GM-CSF (5 microg/kg/day s.c. for 4 days). Mononuclear cells were separated and cultured in GM-CSF (1000 U/ml) and interleukin-4 (1000 U/ml) for 7 days. Phenotype was assessed by 2-color flow cytometry and immunocytochemistry. On day 6, dendritic cells were pulsed with 1 g of fresh autologous tumor lysate for 24 h and infused intravenously. Interleukin-2 (6 million IU), interferon a (4 million IU) and GM-CSF (400 microg) were injected s.c. daily for 10 days beginning on the day of dendritic cell infusion. Treatment was repeated every 21 days for 3 courses., Results: The morphology, immunocytochemistry and phenotype of cultured cells was consistent with dendritic cells: intense positivity for HLA-DR and CD86, with negativity for markers of other lineages, including CD3, CD4, CD8 and CD14. More than 5 x 10(7) dendritic cells were injected in all patients. Nine patients developed >5 mm delayed type cutaneous hypersensitivity reactions to tumor lysate+/-GM-CSF after the first immunization (larger than GM-CSF in all cases). Median delayed type cutaneous hypersensitivity to lysate +/- GM-CSF was 3 cm after the third immunization. One melanoma patient with skin, liver, lung and bone metastases had a partial response lasting 8 months (followed by progression in the brain). Seven patients had stable disease for >3 months, and 7 had progression., Conclusions: Infusion of tumor lysate-pulsed dendritic cells induces a strong cell-mediated antitumor immune reaction in patients with advanced cancer and has some clinical activity.

Since the discovery by Rosenberg and collaborators of the antitumor activity of cisplatin 35 years ago, three platinum antitumor drugs (cisplatin, carboplatin and oxaliplatin) have enjoyed a huge clinical and commercial hit. Ever since the initial discovery of the anticancer activity of cisplatin, major efforts have been devoted to elucidate the biochemical mechanisms of antitumor activity of cisplatin in order to be able to rationally design novel platinum based drugs with superior pharmacological profiles. In this report we attempt to provide a current picture of the known facts pertaining to the mechanism of action of the drug, including those involved in drug uptake, DNA damage signals transduction, and cell death through apoptosis or necrosis. A deep knowledge of the biochemical mechanisms, which are triggered in the tumor cell in response to cisplatin injury not only may lead to the design of more efficient platinum antitumor drugs but also may provide new therapeutic strategies based on the biochemical modulation of cisplatin activity.

Cisplatin is one of the most widely used antitumor drugs. However, as all the anticancer drugs currently used in clinic, cisplatin shows the phenomenon of drug resistance (intrinsic or acquired) against a wide variety of tumors. Poly (ADP-ribose) polymerase-1 is an enzyme involved in DNA repair and apoptotic cell death, which may be inhibited to increase cisplatin chemosensitivity of tumor cells so that cisplatin resistance may be circumvented. In the present study we report that PARP-1 inhibitor 3-aminobenzamide (3-AB) increases the cytotoxic activity of the platinum compounds cisplatin, trans-[PtCl(2)(4-picoline)(piperazine)] and transplatin against CH1cisR cisplatin-resistant ovarian tumor cells. In fact, a concentration of 3-AB of 1 mM not only increases the cytotoxic activity of these platinum complexes but also switches the mode of cell death from necrosis to apoptosis. Altogether, these data suggest that pharmacological modulation of PARP-1 by inhibitors may be a suitable strategy to fight against tumor resistance to platinum drugs.

Poly(ADP-ribose) polymerases (PARPs) are defined as a family of cell signaling enzymes present in eukaryotes, which are involved in poly(ADP-ribosylation) of DNA-binding proteins. The best studied of these enzymes (PARP-1) is involved in the cellular response to DNA damage so that in the event of irreparable DNA damage overactivation of PARP-1 leads to necrotic cell death. Inhibitors of PARP-1 activity in combination with DNA-binding antitumor drugs may constitute a suitable strategy in cancer chemotherapy. When DNA is moderately damaged, PARP-1 participates in the DNA repair process and the cell survives. However, in the case of extensive DNA damage PARP-1 overactivation induces a decrease of NAD+ and ATP levels leading to cell dysfunction or even to necrotic cell death. So, due to PARP-1 involvement in cell death, pharmacological inhibition of PARP-1 activity by PARP-1 inhibitors may constitute a suitable target to enhance the activity of antitumor drugs through inhibition of necrosis and activation of apoptosis. PARP-1 inhibitors such as 3-aminobenzamide, 1,5-dihydroxyisoquinolinone and the recently patented tryciclic benzimidazoles have shown potent inhibitory effects of PARP-1 activity in tumor cells. The present review gives an update of the state-of-the-art of inhibition of PARP-1 activity as adjuvant therapy in cancer treatment.

We have determined the cytotoxic properties of pentamidine isethionate (2) towards the promastigotes of the protozoan parasite Leishmania infantum. The leishmanicidal activity of 2 was 60 times higher after 72 h of incubation than that of cisplatin (4). The pentamidine salt 2 induced a higher amount of programmed cell death (PCD) than cisplatin, which is associated with inhibition of DNA synthesis and cell-cycle arrest in the G2/M phase. Circular dichroism (CD) data indicate that binding of 2 to calf-thymus DNA (CT-DNA) induces conformational changes in the DNA double helix, consistent with a B-->A transition. Moreover, the interaction of 2 with ubiquitin led to a 6% increase in the beta-sheet content of the protein as observed by CD spectroscopy. Fluorescence-spectroscopy studies agreed with the CD data, showing that the pentamidine portion of 2 induces a significant decrease in the fluorescence of the Ub residues Phe4 and Phe45 located on the beta-cluster of the molecule, but not of Tyr59 on the alpha-cluster. These data indicate that pentamidine specifically modifies the beta-cluster, i.e., the 'basic face' of ubiquitin. Our results suggest that the biochemical mechanism of action of pentamidine may be a consequence of its dual binding to DNA and proteins.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes, which show differences in structure, cellular location and functions. However, all these enzymes possess poly(ADP-ribosyl)ation activity. Overactivation of PARP enzymes has been implicated in the pathogenesis of several diseases, including stroke, myocardial infarction, diabetes, shock, neurodegenerative disorder and allergy. The best studied of these enzymes (PARP-1) is involved in the cellular response to DNA damage so that in the event of irreparable DNA damage overactivation of PARP-1 leads to necrotic cell death. Inhibitors of PARP-1 activity in combination with DNA-binding antitumor drugs may constitute a suitable strategy in cancer chemotherapy. In addition, PARP inhibitors may be also useful to restore cellular functions in several pathophysiological states and diseases. This review gives an update of the state-of-the-art concerning PARP enzymes and their exploitation as pharmacological targets in several illnesses.

We have evaluated the cytotoxic properties against the protozoan Leishmania infantum of four water soluble cationic trans-Pt(II)Cl(2) compounds containing as inert groups NH3 and piperazine (1), 4-picoline and piperazine (2), n-butylamine and piperazine (3), and NH3 and 4-piperidino-piperidine (4). The leishmanicidal activity of compounds 3 and 4 against promastigotes of the parasite Leishmania infantum was 2.5- and 1.6-times higher than that of the cytotoxic drug cis-diamminedichloroplatinum(II), respectively. Interestingly, compounds 3 and 4 produce in Leishmania infantum promastigotes a higher amount of programmed cell death than cisplatin, which is associated with cell cycle arrest in G2/M. In contrast to cis-diamminedichloroplatinum(II), binding of compounds 3 and 4 to calf thymus DNA induces conformational changes more similar to those of trans-diamminedichloroplatinum(II) that may be attributed to denaturation of the double helix. Similarly to cis-diamminedichloroplatinum(II) and trans-diamminedichloroplatinum(II), the interaction of compounds 3 and 4 with ubiquitin results in an increase of the alpha-helix content of the protein as observed by circular dichroism spectroscopy. However, fluorescence studies indicate that compounds 3 and 4 produce a decrease in the fluorescence of the tyrosine 59 residue of ubiquitin higher than both cis-diamminedichloroplatinum(II) and trans-diamminedichloroplatinum(II). Altogether, our results suggest that the biochemical mechanism of cytotoxic activity of compounds 3 and 4 against Leishmania infantum must be different from that of cis-diamminedichloroplatinum(II). To the best of our knowledge, compounds 3 and 4 are the first reported trans-platinum complexes that show antiparasitic activity.

Paired helical filaments (PHFs) isolated from patients with Alzheimer's disease (AD) mainly consist of the microtubule-associated protein tau in a hyperphosphorylated form. It has been found that PHFs are the first example of pathological protein aggregation associated with formation of alpha-helices [Biochemistry (2002) 41, 7150-5]. In an effort to investigate the interplay between phosphorylation and the putative role of short regions of alpha-helix in the polymerization of tau, we have focused on the region of tau encompassing residues 317 to 335. This region is able to form protein fibrils in vitro and has two serines that are often found phosphorylated in PHFs. Using trifluoroethanol as an indicator of the alpha-helix, we find that the stability of the alpha-helix conformation is enhanced by phosphorylation. Circular dichroism data show that the phosphorylated peptide in water presents a content in alpha-helix similar to the unphosphorylated peptide at 40% of trifluoroethanol. Phosphorylation also stimulates the effect of juglone in promoting the in vitro polymerization. Furthermore, Fourier transformed infrared spectroscopy of samples of phosphorylated peptide polymerized with juglone renders a spectrum with maxima at approximately 1665 and approximately 1675 cm(-1), which are suggestive of a mixture of turns and alpha-helix conformations. Our results provide a direct mechanistic connection between phosphorylation and polymerization in tau. The connection between phosphorylation and polymerization appears to involve formation of alpha-helix structure.

Programmed cell death (PCD) is a biochemical process that plays an essential role in the development of multicellular organisms. However, accumulating evidence indicates that PCD is also present in single-celled eukaryotes. Thus, trypanosomatids might be endowed with a PCD mechanism that is derived from ancestral death machinery. PCD in trypanosomatids could be a process without a defined function, inherited through eukaryotic cell evolution, which might be triggered in response to diverse stimuli and stress conditions. However, recent observations suggest that PCD might be used by trypanosomatids to maximize their biological fitness. Therefore, PCD could represent a potential pharmacological target for protozoan control.

Carboplatin is a low-molecular-weight anticancer drug that acts by binding to the nuclear DNA of cells. Thus, efficient delivery of the platinum drugs to the nucleus of the cancer cells may enhance the cytotoxicity of the drug. Efficient drug delivery to the nucleus of cancer cells requires three levels of localization: targeting to the cancerous tissue, accumulation in the cancer cells, and intracellular localization in the nucleus. Nuclear localization signals (NLS) are short positively charged basic peptides that actively transport large proteins across the nuclear membrane. We have prepared conjugates in which the NLS is tethered to poly(ethyleneglycol)carboplatin conjugate (NLS-PEG-Pt) and compared their pharmacological properties to those of their untargeted analogues that do not possess the NLS (PEG-Pt). NLS-PEG-Pt conjugates are rapidly internalized into cancer cells and accumulate in the nucleus. Despite their rapid nuclear localization, they form less Pt-DNA adducts than the untargeted analogues, PEG-Pt, and are also less cytotoxic. These results support the hypothesis that carboplatin (unlike cisplatin) may require cytosolic activation prior to its binding to nuclear DNA.

The quantization of small amounts of chemical denaturants as urea or guanidine hydrochloride in protein solutions after dialysis is a difficult task in the molecular biology laboratory practice. Refractometric methods are useful to quantify a denaturant in the molar range but this methodology is not helpful when the denaturant is present in small amounts. The method herein described is a new comparative method that requires, a priori, the quantification of the stock solutions of urea (8 M) and guanidine hydrochloride (6 M) by refractometry to prepare by sequential dilution the standards used for comparison in the spectropolarimeter. The method is based on the observation that the wavelengths, at which the absorbance of polarized light increases in the far-UV region, as observed by spectropolarimetry, is related to the concentration of the chemical denaturant present in the protein solution. In the quantitation method herein reported, the urea and guanidine hydrochloride detection limits range from 1.2 x 10(-4) to 6 x 10(-6) M depending on the protein dialysis buffer used for a standard cell path length of 1 cm. The sensibility of this method results to be comprised in a range 4-5 orders of magnitude higher than that measured by refractometry. The determinations in both the sample and the control preparations are virtually completed within approximately 10 min.

The stability of the substrate-binding region of human inducible Hsp70 was studied by a combination of spectroscopic and calorimetric methods. Thermal denaturation of the protein involves four accessible states: the native state, two largely populated intermediates, and the denatured state, with transition temperatures of 52.8, 56.2 and 71.2 degrees C, respectively, at pH 6.5. The intermediate spectroscopic properties resemble those of molten globules but they still retain substantial enthalpy and heat capacity of unfolding. Moreover, the similar heat capacities of the first intermediate and the native state suggests that the hydrophobic core of the intermediate would be highly native-like and that its formation would involve an increased disorder in localized portions of the structure rather than formation of a globally disordered state. The structure of the C-terminal of Hsp70 is destabilized as the pH separates from neutrality. The intermediates become populated under heat shock conditions at acidic and basic pHs. Denaturation by guanidine chloride also indicated that the protein undergoes a sequential unfolding process. The free energy change associated to the loss of secondary structure at 20 degrees C (pH 6.5) is 3.1 kcal.mol(-1) at high salt conditions. These values agree with the free energy changes estimated from differential scanning calorimetry for the transition between the second intermediate and the final denatured state.

The new potential antitumour soluble drug K[Ru(eddp)Cl(2)].3H(2)O, (eddp=ethylenediamine-N,N'-di-3-propionate) has been isolated and characterized. The analysis of the interaction of this complex with pBR322 plasmid DNA by circular dichroism spectroscopy shows that the ruthenium complex initially induces alteration of both CD positive and negative features resembling those previously observed for monofunctional platinum complexes. Further addition of drug at r(i) higher than 0.50 suggests appreciable conformational alterations of typical secondary structure of B-type DNA, implying loss of DNA helicity and unwinding of the double helix. The results reported herein about the binding of K[Ru(eddp)Cl(2)] to the named plasmid performed by electrophoresis indicate that the Ru(III) center preferentially forms initial monofunctional adducts with this plasmid. In addition, the DNA binding data suggest that the plasmid is cleaved by K[Ru(eddp)Cl(2)] in the presence of physiological concentrations of ascorbate. These results support the hypothesis that reactive Ru(II) species may be formed from Ru(III) upon incubation with a reductant agent such as ascorbate. The testing of the cytotoxic activity of this complex against several human cancer cell lines evidenced that K[Ru(eddp)Cl(2)] complex had a remarkable and selective antiproliferative effect against the cervix carcinoma HeLa and colon adenocarcinoma HT-29, behaving in these two cases as an antineoplastic drug.

The antitumor and cellular pharmacological properties of the trans-Pt(IV) complex, trans-[PtCl(2)(OH)(2)(dimethylamine)(isopropylamine)] (compound 2) has been evaluated in comparison with its corresponding trans-Pt(II) counterpart, trans-[PtCl(2)(dimethylamine)(isopropylamine)] (compound 1). The results reported here indicate that compound 2 markedly circumvents cisplatin resistance in 41McisR and CH1cisR ovarian tumor cell lines endowed with different mechanisms of resistance (decreased platinum accumulation and enhanced DNA repair/tolerance, respectively). However, compound 1 is able to circumvent cisplatin resistance only in CH1cisR cells. Interestingly, at equitoxic concentrations, compounds 1 and 2 induce a higher amount of apoptotic cells than cisplatin in CH1cisR cells. Moreover, the number of apoptotic cells induced by compounds 1 and 2 correlates with their ability to form DNA interstrand cross-links in CH1cisR cells. Although compounds 1 and 2 showed remarkable cytotoxic activity, only compound 2 was able to inhibit the growth of CH1 human ovarian carcinoma xenografts in mice. Binding studies with serum albumin indicate that compound 1 possesses a much higher reactivity against albumin than compound 2. Moreover, the level of binding of compound 1 to plasma proteins during the period 15 min to 1 h after administration to mice (15 mg/kg, i.p.) is 2.5-fold higher than that of compound 2. Therefore, the lack of in vivo antitumor activity shown by compound 1 might be related to its extracellular inactivation before reaching the tumor site because of its high rate of binding to plasma proteins.

Although cisplatin, cis-diamminedichloroplatinum(II), has been successfully used in the chemotherapy of cancer for more than 25 years, its biochemical mechanism of action is still unclear. The current accepted paradigm about cisplatin mechanism of action is that the drug induces its cytotoxic properties through binding to nuclear DNA and subsequent interference with normal transcription, and/or DNA replication mechanisms. If cisplatin-DNA adducts are not efficiently processed by cell machinery, cytotoxic processes eventually end up in cell death. However, before cisplatin enters the cell it may bind to phospholipids and phosphatidylserine in the cell membrane. In addition, in the cytoplasm many potential platinum-binding sites are also available, including RNA and sulfur-containing biomolecules. Moreover, there is much evidence suggesting that the cytotoxic effects induced by binding of cisplatin to non-DNA targets (especially proteins) may contribute to its biochemical mechanism of action. On the other hand, it has been found that several factors such as the dose of drug as well as the metabolic condition of the cell subjected to cisplatin aggression, may determine that cancer cells die through apoptosis or necrosis. In fact, it has recently been reported that both mechanisms of cell demise work in concert so that within a population of tumour cells there is a continuum of possible modes of cell death.

The limitations of cisplatin as an anticancer drug have stimulated the search for other antitumor-active platinum complexes with improved pharmacological properties. The two main goals in the search for new platinum anti-cancer agents are the reduction of the dose-limiting toxicities of cisplatin and the circumvention of cisplatin resistance. However, it should be pointed out that this has proven to be a difficult task. In fact, less than 1% of the thousand of platinum complexes tested for pre-clinical antitumor activity have entered clinical trials in the past 30 years. Nonetheless, right now, several new platinum complexes are in clinical trials, a proof of the continued belief that platinum complexes may still fulfil the needs for novel antitumor drugs. This review will focus on the three main innovative approaches found in the platinum anticancer-field, namely, (1) compounds with decreased reactivity against nucleophiles, (2) compounds with carrier ligands, and (3) compounds which bind differently to DNA as compared to cisplatin. In the latter class, special attention is paid to dinuclear and polinuclear platinum complexes.

We have investigated the cytotoxic activity, the induction of apoptosis, and the interstrand cross-linking efficiency in the A2780cisR ovarian tumor cell line, after replacement of the two NH3 nonleaving groups in trans-[PtCl2(NH3)2] (trans-DDP) by dimethylamine and isopropylamine. The data show that trans-[PtCl2(NH(CH)2)(NHCH(CH3)2)] is able to circumvent resistance to cis-[PtCl2(NH3)2] (cis-DDP, cisplatin) in A2780cisR cells. In fact, trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] shows a cytotoxic potency higher than that of cis-DDP and trans-DDP, with the mean IC50 values being 11, 58, and 300 microM, respectively. In addition, at equitoxic doses (concentrations of the platinum drugs equal to their IC50 values) and after 24 hours of drug treatment, the level of induction of apoptosis by trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is twice that produced by cis-DDP. Under the same experimental conditions, trans-DDP does not induce significant levels of apoptosis in A2780cisR cells. After 24 hours of incubation of A2780cisR cells at concentrations equal to the IC0o value of the platinum drugs, the level of DNA interstrand cross-links (ICLs) induced by trans-[PtCI2(NH(CH)2)(NHCH(CH3)] is two and three times higher, respectively, than those induced by cis-DDP and trans-DDP. We also found that trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] formed DNA ICLs between guanine and complementary cytosine. We propose that, in A2780cisR cells, the induction of apoptosis by trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is related to its greater ability (relative to cis-DDP and trans-DDP) to form DNA ICLs.

The analysis of the kinetic data of the B-Z conformational changes induced by salt in sized double-stranded poly[d(G-C)].poly[d(G-C)] and poly[d(G-m5C)].poly[d(G-m5C)] polymers indicated that there exists a salt threshold which reveals some largely, as yet, unrecognised characteristics of the transition. It was observed that there is a direct correlation between the length of the polymer and the rate of the B-Z transition when the salt concentration in the polymer solution is lower than the salt threshold. The correlation is inverse when the salt concentration is higher than the salt threshold. Thus, the molecular mechanism of the B- to Z-DNA transition varies depending on whether the salt concentration is higher or lower than the threshold. In this context, we have found that the contrasting results reported in the literature describing the rate of the B-Z transition are not contradictory but complementary. The finding of a salt threshold leads to the establishment of a relationship between the cooperativity index of the B-Z transition and the polymer chain length. That relationship is dependent on the chemical structure of the polymer but is temperature independent.

It is generally accepted that DNA damage and subsequent induction of apoptosis may be the primary cytotoxic mechanism of cisplatin and other DNA-binding antitumor drugs (Fisher,1994). Because the final step of apoptosis is characterized by morphological changes in the nucleus, the death signals of the execution phase must be transmitted from the cytoplasm to the nucleus. Thus, the recognition and processing of cisplatin-induced DNA damage through"classic" apoptosis, requires that a nuclear signal, generated at the initiation phase, be transmitted to the cytoplasm to be processed through the effector and execution phases. At the end of the execution phase, the apoptotic signal must come back to the nucleus to produce internucleosomal DNA degradation. Therefore, the induction of apoptosis from detection and subsequent processing of cisplatin-induced DNA damage seems to be a long and complex process of cell death. However, because cisplatin is a nonspecific drug and reacts not only with DNA but also with proteins,we cannot rule out the possibility that in some cases of cisplatin-induced apoptosis, an easier process of initiation, such as damage to cytoplasmic proteins, may take place (Pérez, 1998). Thus, damage to proteins is worth considering as a factor contributing to cisplatin-induced apoptosis. Moreover, it is possible that cisplatin damage to proteins could induce apoptosis at the execution phase level. In fact, initiation of apoptosis at the execution phase (activation of caspases) has been previously reported for the cell killing produced by cytotoxic T lymphocytes (Golstein et al., 1991). Although apoptosis and necrosis are conceptually distinct forms of cell death with very different morphological and biochemical characteristics, these two types of demise may occur simultaneously in tissues or cell cultures exposed to the same insult (Eguchi et al., 1997, Zhan et al., 1999). In fact, both types of cell death have been found in the same population of cisplatin-treated cells (Pestell et al., 2000). Moreover, it has been hypothesized that in a tissue or cell population,apoptosis and necrosis might be two extremes of a continuum of possible types of cell demise. Individual cell death would be decided by factors such as the availability of energy and the metabolic condition of the cell (Leist et al., 1997). Thus, some cells might die as a result of an unfinished apoptotic program. In fact, in L1210 leukemic cells, cisplatin-induced cell death seems to be the result of a defective apoptotic program that lacks some morphological and biochemical characteristics attributed to apoptosis (Segal-Bendirdjian and Jacquemin-Sablon, 1995). In addition, at high doses, cisplatin could damage molecules involved in cellular energy supply (i.e., ATP) and also proteins directly or indirectly involved in the apoptotic process (i.e., p53, Bax, Bcl-2, and caspases), leading to necrotic cell death. In fact, in cisplatin-resistant keratinocytes transformed by H-ras oncogene, a high dose of cisplatin (312 microM) induces characteristic features of necrotic cell death(Pérez et al., 1999). Thus, depending on the level of cellular damage induced by cisplatin, necrosis could take place either directly or as a consequence of an unfinished apoptotic program. In summary, a growing body of evidence suggests that cisplatin-induced cell death does not always come from "classic"apoptosis. Depending on both cisplatin dose and cellular status, cisplatin may also induced cell death by a defective apoptotic program or even by necrosis. Elucidation of the conditions under which the apoptotic program induced by cisplatin as well as other antitumor drugs is totally or partially executed may have important implications for the outcome of cancer chemotherapy.

Total-reflection X-ray fluorescence has been used to study whether the Leishmania infantum kinetoplastid membrane protein-11 is a Ca2+-binding protein. The 108 amino acid helix-loop-helix protein has the loop region located between residues 45 and 57, having similarity to the EF-hand motifs. In particular, the sequence alignment of the putative motif revealed the existence of 67% similarity and 33% identity with the EF-hand of the plasmodia-specific 40-kDa protein from Physarum polycephalum. To address the type of conformational changes induced by Ca2+ binding, circular dichroism and fluorescence spectroscopy were used. The data showed that Ca2+ induces changes in both the secondary and tertiary structure of the protein in a temperature- and pH-dependent way. It also induces the precipitation of the protein at pH 7.5, in contrast with what occurs at pH 5.0, and the precipitation process can be reverted by addition of EGTA. At acidic pH values the complex EGTA-Ca2+ causes drastic structural changes, forcing the protein to adopt a structure close to that of a random coil. Because, at acidic pH values, protein:Ca2+:EGTA ternary complexes may be formed, the drastic change may be attributed to the presence of a high density of EGTA negative charges in the neighborhood of the alpha-helices.

Trans-[PtCl(2)(N,N-dimethylamine)(isopropylamine)] is a novel trans-platinum compound that shows cytotoxic activity in several cisplatin resistant cell lines. The aim of this paper was to analyse, by means of molecular cell biology techniques and total reflection X-ray fluorescence (TXRF), the cytotoxic activity, the induction of apoptosis, the cellular uptake and the DNA binding of trans-[PtCl(2)(N,N-dimethylamine)(isopropylamine)] in the cisplatin resistant cell line A2780cisR. The results show that this drug is more cytotoxic and induces a higher amount of apoptotic cells than cisplatin in A2780cisR cells. However, the intracellular accumulation and extent of binding to DNA of trans-[PtCl(2)(N,N-dimethylamine)( isopropylamine)] is lower than that of cis-DDP. Moreover, trans-[PtCl(2)(N,N-dimethylamine)(isopropylaminae)] is partially inactivated by intracellular levels of glulathione. The result suggest that circumvention of ciplatin resistance by trans-[PtCl(2)(N,N-dimethylamine)(isopropylamine)] in A2780cisR cells might be related with the ability of this drug to induce apoptosis.

The discovery in the 1990s of several trans-Pt complexes with in vitro and in vivo activity against tumor cells sensitive and/or resistant to cisplatin has forced the re-evaluation of the structure-activity relationships for platinum antitumor drugs. Because the determinant factors of cytotoxic activity of trans-platinum complexes do not follow the same patterns as those found for cisplatin and its analogues, the differences in cellular and biochemical pharmacology between trans-platinum antitumor complexes and cisplatin might be systematically exploited to design novel trans-platinum complexes with a clinical profile complementary to that of cisplatin and related analogues. Therefore, there may exist a novel molecular rationale for new platinum antitumor drugs development in the twenty-first century.

We analyzed both the cytotoxicity and the type of cell death produced by the novel binuclear Pt(III) compound Pt-Spym ([Pt(2)(2-mercaptopyrimidine)(4)Cl(2)]) in kidney human fibroblasts and in human tumor cell lines (HeLa, CH1, CH1cisR and HL-60). The data showed that Pt-Spym displayed higher cytotoxicity against these tumor cells than cisplatin. In contrast, Pt-Spym had low toxicity against normal human fibroblasts. Interestingly, Pt-Spym circumvented cisplatin resistance in CH1cisR cells. We also observed that Pt-Spym induced the characteristic changes attributed to apoptosis in cells with normal levels of p53 protein (CH1 and CH1cisR) and with low levels of p53 protein (HeLa), but not in cells lacking p53 (HL-60). Interestingly, Western blot data indicated that apoptosis induction by Pt-Spym in HeLa, CH1, and CH1cisR cells was not associated with drastic changes in p53 levels. However, cis-DDP strongly decreased p53 levels in CH1 and CH1cisR cells and abolish p53 protein in HeLa cells. Altogether, these results suggest that induction of apoptosis by Pt-Spym requires the presence of p53 protein.