Mechanism of extracellular ubiquitination in the mammalian epididymis.

Posttranslational modification by ubiquitination marks defective or outlived intracellular proteins for proteolytic degradation by the 26S proteasome. The ATP-dependent, covalent ligation and formation of polyubiquitin chains on substrate proteins requires the presence and activity of a set of ubiquitin activating and conjugating enzymes. While protein ubiquitination typically occurs in the cell cytosol or nucleus, defective mammalian spermatozoa become ubiquitinated on their surface during post-testicular sperm maturation in the epididymis, suggesting an active molecular mechanism for sperm quality control. Consequently, we hypothesized that the bioactive constituents of ubiquitin-proteasome pathway were secreted in the mammalian epididymal fluid (EF) and capable of ubiquitinating extrinsic substrates. Western blotting indeed detected the presence of the ubiquitin-activating enzyme E1 and presumed E1-ubiquitin thiol-ester intermediates, ubiquitin-carrier enzyme E2 and presumed E2-ubiquitin thiol-ester intermediates and the ubiquitin C-terminal hydrolase PGP 9.5/UCHL1 in the isolated bovine EF. Thiol-ester assays utilizing recombinant ubiquitin-activating and ubiquitin-conjugating enzymes, biotinylated substrates, and isolated bovine EF confirmed the activity of the ubiquitin activating and conjugating enzymes within EF. Ubiquitinated proteins were found to be enriched in the defective bull sperm fraction and appropriate proteasomal deubiquitinating and proteolytic activities were measured in the isolated EF by specific fluorescent substrates. The apocrine secretion of cytosolic proteins was visualized in transgenic mice and rats expressing the enhanced green fluorescent protein (eGFP) under the direction of ubiquitin-C promoter. Accumulation of eGFP, ubiquitin and proteasomes was detected in the apical blebs, the apocrine secretion sites of the caput epididymal epithelia of both the rat and mouse epididymal epithelium, although region-specific differences exist. Secretion of eGFP and proteasomes continued during the prolonged culture of the isolated rat epididymal epithelial cells in vitro. This study provides evidence that the activity of the ubiquitin system is not limited to the intracellular environment, contributing to a greater understanding of the sperm maturation process during epididymal passage.

Differential expression of genes encoding constitutive and inducible 20S proteasomal core subunits in the testis and epididymis of theophylline- or 1,3-dinitrobenzene-exposed rats.

Theophylline (THP) and 1,3-dinitrobenzene (DNB) are thought to induce infertility by incapacitating the nurturing Sertoli cells and causing germ cell apoptosis in the testicular seminiferous epithelium, respectively. We hypothesized that THP and DNB exposure would alter the expression of the genes within the ubiquitin-proteasome pathway (UPP), implicated in spermatogenesis and epididymal sperm quality control. Rats were fed 0 or 8000 ppm of THP and necropsied on Days 18, 30, and 42 or administered 0, 2, or 6 mg/kg DNB via oral gavage and necropsied on Day 7. Tissues were collected from the testis and the caput, corpus, and cauda regions of the epididymis for transcriptional profiling by semiquantitative RT-PCR, real-time RT-PCR, and histopathology. Target UPP genes included those encoding for constitutive the 20S proteasomal core subunits Psmb1 (beta1), Psmb2 (beta2), and Psmb5 (beta5); the inducible 20S core subunits Psmb9 (LMP2), Psmb8 (LMP7), and Psmb10 (LMP10); and Ube1 (ubiquitin-activating enzyme E1), Ube2d3 (ubiquitin-conjugating enzyme E2), and Uchl1 (ubiquitin C-terminal hydrolase PGP9.5). Spermatozoa were collected from the cauda epididymis for analysis by light microscopy and flow cytometric evaluation of sperm surface ubiquitin. These data show that reprotoxic exposure alters the tissue-specific expression of UPP genes in the testis and epididymis, which may contribute to the aberrant spermatogenesis and epididymal processing of both normal and defective spermatozoa. Transcriptional profiling and flow cytometric analysis of the UPP thus captures the prodromal effects of reproductive toxicity not captured by conventional histology and functional cytology. Complementing seminal analysis with these measures may be useful in screening drug-induced toxicity or environmental infertility.

Investigating cytoskeletal alterations as a potential marker of retinal and lens drug-related toxicity.

Actin filaments play a critical role in the normal physiology of lenticular and retinal cells in the eye. Disruption of the actin cytoskeleton has been associated with retinal pathology and lens cataract formation. Ocular toxicity is an infrequent observation in drug safety studies, yet its impact to the drug development process is significant. Recognizing compounds through screening with a potential ocular safety liability is one way to prioritize development candidates while reducing development attrition. Lens epithelial cells from human, dog, and rat origins and retinal pigmented epithelium cells from human, monkey, and rat origins were cultured and investigated with immunocytochemical techniques. Cells were treated using noncytotoxic doses of the compound, fixed, stained for actin with rhodamine phalloidin, and counterstained for nuclei with TOTO-3, followed by confocal imaging. Tamoxifen and several experimental compounds known to be in vivo lens and retinal toxicants caused a reduction in F-actin fluorescence at noncytotoxic concentrations in all cells tested as observed by confocal microscopy. Developing an assay that predicts ocular toxicity helps the development process by prioritizing compounds for further investigation. Drug-induced cytoskeletal alterations may be useful as a potential safety-screening marker of retinal and lens toxicity. The knowledge of actin molecular biology and the application of other mechanistic screens to toxicology are discussed. Reducing this work to a high-throughput platform will enable chemists to select compounds with a reduced risk of ocular toxicity.

Combination therapy enhances the inhibition of tumor growth with the fully human anti-type 1 insulin-like growth factor receptor monoclonal antibody CP-751,871.

PURPOSE: The insulin-like growth factor (IGF) signaling pathway is implicated in cellular mitogenesis, angiogenesis, tumor cell survival, and tumorigenesis. Inhibition of this pathway results in decreased cell growth, inhibition of tumor formation in animal models, and increased apoptosis in cells treated with cytotoxic chemotherapy. We generated and characterized a human monoclonal antibody that targeted the IGF receptor. EXPERIMENTAL DESIGN: By use of XenoMouse technology, we generated CP-751,871, a fully human IgG2 antibody with high affinity (K(d) = 1.5 nmol/L) for human IGF-1R and evaluated its biological, pharmacologic, and antitumor properties. RESULTS: This antibody blocks binding of IGF-1 to its receptor (IC(50) 1.8 nmol/L), IGF-1-induced receptor autophosphorylation (IC(50) 0.42 nmol/L) and induced the down-regulation of IGF-1R in vitro and in tumor xenografts. The extent of IGF-1R down-regulation in vivo was proportional to CP-751,871 concentrations in the serum of tumor-bearing mice. Pharmacokinetic profiles in cynomolgus monkeys indicated a close to linear increase of exposure following i.v. dosing of antibody in the range of 3 to 100 mg/kg. CP-751,871 showed significant antitumor activity both as a single agent and in combination with Adriamycin, 5-fluorouracil, or tamoxifen in multiple tumor models. A biomarker assay was developed to establish the relationship between circulating antibody concentrations and down-regulation of IGF-1R in peripheral blood cells. The concentration of CP-751,871 required to down-regulate 50% of IGF-1R on peripheral blood cells was 0.3 nmol/L. CONCLUSION: These data suggest that inhibition of the IGF cascade by use of this monoclonal antibody may be of clinical benefit in the treatment of human cancers.

Pentamidine reduces hERG expression to prolong the QT interval.

Pentamidine, an antiprotozoal agent, has been traditionally known to cause QT prolongation and arrhythmias; however, its ionic mechanism has not been illustrated. In a stable HEK-293 cell line, we observed a concentration-dependent inhibition of the hERG current with an IC50 of 252 microM. In freshly isolated guinea-pig ventricular myocytes, pentamidine showed no effect on the L-type calcium current at concentrations up to 300 microM, with a slight prolongation of the action potential duration at this concentration. Since the effective concentrations of pentamidine on the hERG channel and APD were much higher than clinically relevant exposures (approximately 1 microM free or lower), we speculated that this drug might not prolong the QT interval through direct inhibition of I(Kr) channel. We therefore incubated hERG-HEK cells in 1 and 10 microM pentamidine-containing media (supplemented with 10% serum) for 48 h, and examined the hERG current densities in the vehicle control and pentamidine-treated cells. In all, 36 and 85% reductions of the current densities were caused by 1- and 10-microM pentamidine treatment (P<0.001 vs control), respectively. A similar level of reduction of the hERG polypeptides and a reduced intensity of the hERG protein on the surface membrane in treated cells were observed by Western blot analysis and laser-scanning confocal microscopy, respectively. Taken together, our data imply that chronic administration of pentamidine at clinically relevant exposure reduces the membrane expression of the hERG channel, which may most likely be the major mechanism of QT prolongation and torsade de pointes reported in man.

Image compression in morphometry studies requiring 21 CFR Part 11 compliance: procedure is key with TIFFs and various JPEG compression strengths.

This study aims to compare the integrity and reproducibility of measurements created from uncompressed and compressed digital images in order to implement compliance with 21 CFR Part 11 for image analysis studies executed using 21 CFR Part 58 compliant capture systems. Images of a 400-mesh electron microscope grid and H&E stained rat liver tissue were captured on an upright microscope with digital camera using commercially available analysis software. Digital images were stored as either uncompressed TIFFs or in one of five different levels of JPEG compression. The grid images were analyzed with automatic detection of bright objects while the liver images were segmented using color cube-based morphometry techniques, respectively, using commercially-available image analysis software. When comparing the feature-extracted measurements from the TIFF uncompressed to the JPEG compressed images, the data suggest that JPEG compression does not alter the accuracy or reliability to reproduce individual data point measurements in all but the highest compression levels. There is, however, discordance if the initial measure was obtained with a TIFF format and subsequently saved as one of the JPEG levels, suggesting that the use of compression must precede feature extraction. It is a common practice in software packages to work with TIFF uncompressed images. However, this study suggests that the use of JPEG compression as part of the analysis work flow was an acceptable practice for these images and features. Investigators applying image file compression to other organ images will need to validate the utility of image compression in their work flow. A procedure to digitally acquire and JPEG compress images prior to image analysis has the potential to reduce file archiving demands without compromising reproducibility of data.