Mitochondrial precursor proteins are imported through a hydrophilic membrane environment

We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes. F1-ATPase subunit beta (F1 beta) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates. This F1 beta translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea. By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria. These translocation intermediates were also extractable from the membranes at alkaline pH. Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants. We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites.     

Induction of sister-chromatid exchanges by restriction endonucleases

Restriction endonucleases Cfo 1, Pvu II, Sma I, Hpa II, Taq I and Hae III were tested for their ability to induce SCEs in CHO cells. The results indicate that the DNA double-strand breaks induced during S-phase by these enzymes lead to an increase in the frequencies of SCEs.     

The Effect of Microwave Radiation on Cell Sensitivity to Monohydric Alcohols in Platelet-Rich Plasma

Blood cell aggregation in platelet-rich plasma (PRP) supplemented with the aggregation inducer ristomycin was studied following exposure to low-intensity extremely high frequency (EHF) radiation. Primary alcohols were used to stimulate partial degradation of the protein–lipid bilayer in the cell membrane. Exposure to EHF radiation was shown to decrease the extent of ristomycin-induced platelet aggregation and to change the slope of the aggregation curve. A biphasic character was observed for the effect of alcohols on platelet aggregation. The first phase was characterized by a decrease in aggregation and a decrease in the slope of the aggregation curve. In the second phase, platelet aggregation and the aggregation curve slope increased with the increasing alcohol concentration. A possible mechanism is discussed for the observed effects.

     

UDP glucuronosyltransferase 1A expression levels determine the response of colorectal cancer cells to the heat shock protein 90 inhibitor ganetespib

HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC₅₀) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC₅₀s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC₅₀>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.Constant proteotoxic stress is a frequent occurrence in cancer cells and is derived from an adverse external microenvironment (hypoxia, acidosis) and internally from conformationally aberrant oncoproteins, high reactive oxygen species (ROS) levels, genomic instability, and stoichiometric imbalances in multi-protein machines. This stress condition raises the need for massive heat-shock chaperone support, especially from the heat-shock protein 90 (HSP90) system, to prevent protein aggregation and illicit interactions and promote tumor cell survival. Cancer-associated factors, such as mutant p53,^{1, 2} ErbB2,³ AKT,⁴ and macrophage migration inhibitory factor (MIF),^{5, 6} among others, represent HSP90 clients and require HSP90 for their stabilization in tumors. Hence, the multi-component HSP90 chaperone is highly upregulated and activated specifically in cancer cells as an adaptive response to malignancy.⁷HSP90 inhibitors have emerged as a highly promising class of anti-cancer compounds because of their ability to interfere with broadly active molecular networks, rather than a narrowly defined signaling pathway^{8, 9} and they enhance proteotoxic stress.¹⁰ Geldanamycin-based compounds represented the mainstay of HSP90 inhibition for the last 20 years.⁸ Clinically, however, these compounds proved to be of limited value due to their inherent liver and ocular toxicity coupled with only modest potency in vivo.¹¹ Major advances came with recently developed second-generation synthetic inhibitors such as the resorcinol containing compounds ganetespib (STA-9090)¹² and NVP-AUY922¹³ that are considerably more potent and less toxic. These compounds are currently being tested in phase II/III trials for their efficacy against various cancer types. Anaplastic lymphoma kinase (ALK)-driven NSCLC cancers showed particular clinical responsiveness to ganetespib.^{14, 15}Colorectal carcinoma (CRC) represents one of the most frequent malignancies worldwide, with a correspondingly high death toll. Moreover, the identification of predictive markers for patient stratification has proven to be difficult.¹⁶ HSP90 inhibition might be an attractive strategy for therapeutic improvement. However, this requires studies on how HSP90 inhibitors act on tumor cells in this cancer type.One approach to identify genes that determine cancer drug response was provided by highly parallel analysis of many cancer cell lines, specifically comparing their response with a large variety of small compounds with their gene expression patterns.^{17, 18} This strategy led to the identification of genes with expression patterns correlating with drug sensitivity. Notably, for many anticancer drugs this approach did not yield strong candidates, but in the case of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) a strong correlation was seen between drug sensitivity and the expression of functional NAD(P)H dehydrogenase quinone 1 (NQO1), an enzyme that can metabolize and activate 17-AAG.^{19, 20} Hence, correlating drug sensitivity and gene expression patterns in cell lines can identify mechanisms that determine drug response.Drugs are subjected to metabolic turnover, and a major route of excretion from the body consists in conjugation with a hydrophilic sugar moiety within the liver parenchyma, followed by secretion into the bile. A major group of enzymes that carry out such conjugations are the UDP glucuronosyltransferases (UGTs).^{21, 22, 23} These enzymes are the products of gene clusters that cover various substrate specificities. UGT substrates include bilirubin, amines, and phenol structures.²⁴ The existence of such mechanisms for drug conjugation in the liver raises the question if and under what circumstances they can be found directly in tumor cells, and presumably cause drug resistance when highly expressed.Here, we show that human CRC-derived cell lines fall into ganetespib-sensitive and -resistant groups. While the majority of CRC lines were sensitive, two lines were highly resistant. Importantly, resistant cancer cells show a high expression of the UGT1A gene, and high levels of UGT1A were shown to be critical for ganetespib turnover, drug inactivation, and cell resistance. Thus, UDP glucuronosyl conjugation detoxifies ganetespib not only in the liver but also in a subset of CRC cells, representing a potential predictive biomarker for ganetespib response in CRC and possibly other tumor types.     

INVESTIGATIONS ON RESISTANCE TO DISEASE AMONG SPECIES OF THE GENUS AVENA

Fifty-three samples representing species of the genus Arena were tested for resistance to infection by Ustilago avenae and U. kolleri following artificial inoculation. Among the diploid and tetraploid species tested, eleven out of thirty-seven samples of Arena strigosa subsp. strigosa showed complete resistance to all the cultures with which they were inoculated. Avena strigosa subsp. barbata (three samples), and Avena strigosa subsp. abyssinica (one sample), also proved to be resistant to all the available races.
Variation in morphological and physiological characteristics within species and samples (varieties?) of the lower chromosome groups of Avena were observed and its consequences in breeding and race identification discussed.
Race identification was carried out on the eight Ustilago cultures and the existence of at least six races established. The tester varieties used in the present study proved inadequate for the complete separation of the smut races.     

Silk fibroin hydrogels for potential applications in photodynamic therapy

In this study, we prepared translucid hydrogels with different concentrations of silk fibroin, extracted from raw silk fibers, and used them as a matrix to incorporate the photosensitizer 5-(4-aminophenyl)-10,15,20-tris-(4-sulphonatophenyl) porphyrin trisodium for application in photodynamic therapy (PDT). The hydrogels obtained were characterized by rheology, spectrophotometry, and scattering techniques to elucidate the factors involved in the formation of the hydrogel, and to characterize the behavior of silk fibroin (SF) after incorporating of the porphyrin to the matrix. The rheology results demonstrated that the SF hydrogels had a shear thinning behavior. In addition, we were able to verify that the structure of the material was able to be recovered over time after shear deformation. The encapsulation of porphyrins in hydrogels leads to the formation of self-assembled peptide nanostructures that prevent porphyrin aggregation, thereby greatly increasing the generation of singlet oxygen. Also, our findings suggest that porphyrin can diffuse out of the hydrogel and permeate the outer skin layers. This evidence suggests that SF hydrogels could be used as porphyrin encapsulation and as a drug carrier for the sustained release of photosensitizers for PDT.     

Role of Ca2+ in induction and secretion of dengue virus-induced cytokines

The present study was undertaken to investigate the role of calcium ions (Ca₂₊) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca₂₊ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.