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The transient receptor potential, the melastatin (TRPM) subfamily, which consists of eight known members, appears to have significant importance in melanoma progression, treatment, and prognosis. As several members were originally cloned from cancerous tissue, initial studies aimed towards identifying TRPM involvement in cancer progression and tumorigenesis. For relevance in skin cancer, previous research has shown roles for several TRPM members in skin cancer progression, growth, and patient prognosis. One unique member, TRPM2, appears to have notable therapeutic potential in the treatment of melanoma. Previous and recent studies have demonstrated increased TRPM2 expression levels in melanoma, as well as important roles for TRPM2 in melanoma growth, proliferation, and survival. TRPM2 is thus an emerging target in the treatment of melanoma, where TRPM2 antagonism may offer an additional treatment option for melanoma patients in the future.

Transient receptor potential melastatin-2 (TRPM2) is an emerging chemotherapeutic target due to its involvement in poly(ADP-ribose) metabolism and the ability to induce anticancer effects after antagonism of its functions. Normally functioning as a nonspecific cation channel that is activated by free ADP-ribose, TRPM2 is involved with many cellular processes, including the induction of cell death after oxidative stress. What is becoming clear is that antagonism of TRPM2 selectively induces anticancer effects in several types of cancer. We previously demonstrated decreased growth and proliferation, increased levels of DNA damage, and the selective induction of cell death in breast cancer and melanoma cells. Due to these effects, it appears that TRPM2 has a novel role in cancer cells. Further, this novel role appears to involve nuclear function, because our studies, as well as those from other independent groups, demonstrate a nuclear localization of TRPM2 in various types of cancers. Thus, as an emerging therapeutic target, it is important to describe research techniques that can be utilized to analyze TRPM2 function, determine its effects in cancerous and noncancerous cells, and provide molecular biological methods to inhibit or downregulate its function., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein display, secretion, and export in prokaryotes are essential for utilizing microbial systems as engineered living materials, medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through just three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that modestly increased surface display from 1.4- to 3-fold as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the functional protein was displayed extracellularly, we trypsinized the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging epPCR and single-cell sorting to augment bacterial surface display rapidly that could be applied to other bacterial proteins.

The COVID-19 pandemic related measures had augmented the rise of online education. While online teaching had mitigated the negative impacts from educational institutional closures, it was unable to displace hands-on biomedical laboratory practical lessons effectively. Without practical sessions, there was concern over the imparting of laboratory skills even with video demonstrations. To investigate the effectiveness of different delivery modes in imparting laboratory skills, theoretical and practical student assessments were analyzed alongside an anonymous survey on their motivation and prior experience. The undergraduate students were exposed to (1) instructor-live demonstration; (2) video demonstration or (3) no demonstration prior to the practical test which was a plasmid extraction. Significantly higher mini-prep yields and purity were found for both instructor-live and video demonstrations compared to no demonstration. Comparison with pre-pandemic theoretical assessment performance showed no significant differences despite longer contact hours during pre-pandemic times. Prior lab experience and motivation for selecting the course did not significantly affect student mini-prep yields. In conclusion, our findings suggest that video demonstrations were as effective as instructor-live demonstrations during the pandemic without noticeably compromising the teaching and learning of biomedical laboratory skills., (© 2022 International Union of Biochemistry and Molecular Biology.)

Melanoma continues to be the most aggressive and devastating form of skin cancer for which the development of novel therapies is required. The present study aimed to determine the effects of antagonism of the transient receptor potential melastatin‑2 (TRPM2) ion channel in primary human malignant melanoma cells. TRPM2 antagonism via use of the antifungal agent, clotrimazole, led to decreases in cell proliferation, as well as dose‑dependent increases in cell death in all melanoma cell lines investigated. The targeting of TRPM2 channels was verified using TRPM2 knockdown, where treatment with TRPM2 small‑interfering RNA led to similar levels of cell death in all melanoma cell lines when compared with clotrimazole treatment. Minimal effects on proliferation and cell death were observed following antagonism or knockdown of TRPM2 in non‑cancerous human keratinocytes. Moreover, characteristics of TRPM2 were explored in these melanoma cells and the results demonstrated that TRPM2, localized to the plasma membrane as a non‑specific ion channel in non‑cancerous cells, displayed a nuclear localization in all human melanoma cell lines analyzed. Additional characterization of these melanoma cell lines confirmed that each expressed one or more established multidrug resistance genes. Results of the present study therefore indicated that antagonism of the TRPM2 channel led to antitumor effects in human melanoma cells, including those that are potentially unresponsive to current treatments due to the expression of drug resistance genes. The unique cellular localization of TRPM2 and the specificity of the antitumor effects elicited by TRPM2 antagonism suggested that TRPM2 possesses a unique role in melanoma cells. Collectively, the targeting of TRPM2 represents a potentially novel, efficacious and readily accessible treatment option for patients with melanoma.

Background: Optimizing recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialization, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterizations and investigations., Methods: Investigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analyzed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day, and common nutrient supplements., Results: Using 2 mL adherent HEK293E cell culture transfections with 25 kDa linear polyethylenimine in the most optimized parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 μg, respectively, in a cost-effective manner. With the addition of peptone, κ light chain increased by ~4-fold to 1032 μg, whereas whole antibody increased to a lesser extent by ~2.5-fold to 51 μg, with benefits potentially for antibodies limited by their light chains in production., Conclusions: Our optimized findings show promise for a more efficient and convenient antibody production method through transfection and culture optimizations that can be incorporated to scale-up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E., (© The Author(s) 2022. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Elderly people are not likely to recognize road signs due to low cognitive ability and presbyopia. In our study, three shapes of traffic symbols (circles, squares, and triangles) which are most commonly used in road driving were used to evaluate the elderly drivers' recognition. When traffic signs are randomly shown in HUD (head-up display), subjects compare them with the symbol displayed outside of the vehicle. In this test, we conducted a Go/Nogo test and determined the differences in ERP (event-related potential) data between correct and incorrect answers of EEG signals. As a result, the wrong answer rate for the elderly was 1.5 times higher than for the youths. All generation groups had a delay of 20-30 ms of P300 with incorrect answers. In order to achieve clearer differentiation, ERP data were modeled with unsupervised machine learning and supervised deep learning. The young group's correct/incorrect data were classified well using unsupervised machine learning with no pre-processing, but the elderly group's data were not. On the other hand, the elderly group's data were classified with a high accuracy of 75% using supervised deep learning with simple signal processing. Our results can be used as a basis for the implementation of a personalized safe driving system for the elderly.

Even with the ubiquity of Sanger sequencing, automated assembly software are predominantly stand-alone software packages for desktop/laptop use with very few online equivalents, thus geospatially constraining sequence analysis and assembly. With increased data output worldwide, there is also a need for automated quality checks and trimming prior to large assemblies, along with automated detection of mutations. Through web servers with expanded automation and functionalities, even smartphones/phablets can be used to perform complex analysis previously limited to desktops, especially if they can upload files from cloud storage. To facilitate such online accessible sequence assembly and analysis, we created Yet Another Quick Assembly, Analysis and Trimming Tool web server for the automated assembly of multiple .ab1 and .FASTQ sequencing reads de novo with automated trimming and scanning of the assembled sequences for single nucleotide polymorphisms and insertions or deletions without installation of software, allowing it to be accessed from anywhere with Internet access and with minimal dependency on other software and web tools., (© Association of Biomolecular Resource Facilities.)

While drug resistant mutations in HIV-1 are largely credited to its error prone HIV-1 RT, the time point in the infection cycle that these mutations can arise and if they appear spontaneously without selection pressures both remained enigmatic. Many HIV-1 RT mutational in vitro studies utilized reporter genes (LacZ) as a template to investigate these questions, thereby not accounting for the possible contribution of viral codon usage. To address this gap, we investigated HIV-1 RT mutation rates and biases on its own Gag, protease, and RT p66 genes in an in vitro selection pressure free system. We found rare clinical mutations with a general avoidance of crucial functional sites in the background mutations rates for Gag, protease, and RT p66 at 4.71 × 10 -5 , 6.03 × 10 -5 , and 7.09 × 10 -5 mutations/bp, respectively. Gag and p66 genes showed a large number of 'A to G' mutations. Comparisons with silently mutated p66 sequences showed an increase in mutation rates (1.88 × 10 -4 mutations/bp) and that 'A to G' mutations occurred in regions reminiscent of ADAR neighbor sequence preferences. Mutational free energies of the 'A to G' mutations revealed an avoidance of destabilizing effects, with the natural p66 gene codon usage providing barriers to disruptive amino acid changes. Our study demonstrates the importance of studying mutation emergence in HIV genes in a RT-PCR in vitro selection pressure free system to understand how fast drug resistance can emerge, providing transferable applications to how new viral diseases and drug resistances can emerge.

Mutations underpin the processes in life, be it beneficial or detrimental. While mutations are assumed to be random in the bereft of selection pressures, the genetic code has underlying computable probabilities in amino acid phenotypic changes. With a wide range of implications including drug resistance, understanding amino acid changes is important. In this study, we calculated the probabilities of substitutions mutations in the genetic code leading to the 20 amino acids and stop codons. Our calculations reveal an enigmatic in-built self-preserving organization of the genetic code that averts disruptive changes at the physicochemical properties level. These changes include changes to start, aromatic, negative charged amino acids and stop codons. Our findings thus reveal a statistical mechanism governing the relationship between amino acids and the universal genetic code., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

HIV protease inhibitors against the viral protease are often hampered by drug resistance mutations in protease and in the viral substrate Gag. To overcome this drug resistance and inhibit viral maturation, targeting Gag alongside protease rather than targeting protease alone may be more efficient. In order to successfully inhibit Gag, understanding of its drug resistance mutations and the elicited structural changes on protease binding needs to be investigated. While mutations on Gag have already been mapped to protease inhibitor resistance, there remain many mutations, particularly the non-cleavage mutations, that are not characterized. Through structural studies to unravel how Gag mutations contributes to protease drug resistance synergistically, it is thus possible to glean insights to design novel Gag inhibitors. In this review, we discuss the structural role of both novel and previously reported Gag mutations in PI resistance, and how new Gag inhibitors can be designed.

The elderly are more susceptible to stress than younger people. In particular, heart palpitations are one of the causes of heart failure, which can lead to serious accidents. To prevent heart palpitations, we have devised the Safe Driving Intensity (SDI) and Cardiac Reaction Time (CRT) as new methods of estimating the correlations between effects on the driver's heart and the movement of a vehicle. In SDI measurement, recommended acceleration value of vehicle for safe driving is inferred from the suggested correlation algorithm using machine learning. A higher SDI value than other people means less pressure on the heart. CRT is an estimated value of the occurring time of heart palpitations caused by stressful driving. In particular, it is proved by SDI that elderly subjects tend to overestimate their driving abilities in personal assessment questionnaires. Furthermore, we validated our SDI using other general statistical methods. When comparing the results using a t -test, we obtained reliable results for the equivalent variance. Our results can be used as a basis for evaluating elderly people's driving ability, as well as allowing for the implementation of a personalized safe driving system for the elderly.

A novel, rapid, simultaneous analysis method for five sugars (fructose, glucose, sucrose, maltose, and lactose) and eight sugar alcohols (erythritol, xylitol, sorbitol, mannitol, inositol, maltitol, lactitol, and isomalt) was developed using UPLC-ELSD, without derivatization. The analysis conditions, including the gradient conditions, modifier concentration and column length, were optimized. Thirteen sugars and sugar alcohols were separated well and the resolution of their peaks was above 1.0. Their optimum analysis condition can be analyzed within 15min. Standard curves for sugars and sugar alcohols with concentrations of 5.0-0.1% and 2.0-0.05% are presented herein, and their correlation coefficients are found to be above 0.999 and the limit of detection (LOD) was around 0.006-0.018%. This novel analysis system can be used for foodstuffs such as candy, chewing gum, jelly, chocolate, processed chocolate products, and snacks containing 0.21-46.41% of sugars and sugar alcohols., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A novel enzymatic process for cyclodextrin (CD) production was developed by utilizing sucrose as raw material instead of corn starch. Cyclodextrin glucanotransferase (CGTase) from Bacillus macerans was applied to produce the CDs from linear α-(1,4)-glucans, which were obtained by Neisseria polysaccharea amylosucrase (NpAS) treatment on sucrose. The greatest CD yield (21.1%, w/w) was achieved from a one-pot dual enzyme reaction at 40 °C for 24 h. The maximum level of CD production (15.1 mg/mL) was achieved with 0.5 M sucrose in a simultaneous mode of dual enzyme reaction, whereas the reaction with 0.1 M sucrose was the most efficient with regard to conversion yield. Consequently, dual enzyme synthesis of CDs was successfully carried out with no need of starch material. This result can be applied as a novel efficient bioconversion process that does not require the high temperature necessary for starch liquefaction by thermostable α-amylase in conventional industrial processing.

The ileal brake mechanism which induces a potentially beneficial slower gastric emptying rate and increased satiety is triggered by macronutrients including glucose from glycemic carbohydrates. For optimization of this diet-induced health benefit, there is the need for a way to determine the location of glucose deposition in the small intestine. Labeled 2-deoxyglucose (2-DG) can be used to trace the location of glucose absorption due to its accumulative property in the small intestine enterocytes. However, because pure glucose, or 2-DG, is directly absorbed in the proximal small intestine, we designed 2-DG containing maltooligosaccharides (2-DG-MOs) that can be used with a mild α-glucosidase inhibitor to attain an analytical method for determining location-specific delivery of glucose and its physiological effect., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We previously demonstrated a unique protective role for the transient receptor potential, melastatin-2 (TRPM2) cation channel in breast cancer cells. In the present study, we investigated the chemotherapeutic effects elicited by inhibiting this protective role in metastatic breast adenocarcinoma cells. TRPM2 inhibition led to dose-dependent increases in MDA-MB-231 breast adenocarcinoma cell death after treatment with doxorubicin or the DNA-methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine. Similar results were observed after RNAi silencing of TRPM2 in these cells after doxorubicin treatment. However, TRPM2 RNAi silencing also led to increased MCF-7 breast adenocarcinoma cell death after tamoxifen treatment, yet not in non-cancerous human mammary epithelial cells. These results thus revealed that TRPM2 inhibition selectively increased cytotoxicity in a triple-negative and an estrogen receptor-positive breast cancer cell line, with minimal deleterious effects in non-cancerous breast cells. Analysis of DNA damage revealed enhanced DNA damage levels in MCF-7 cells treated with doxorubicin due to TRPM2 inhibition. Analysis of cell death demonstrated that inhibition of apoptosis, caspase-independent cell death or autophagy failed to significantly reduce cell death induced by TRPM2 inhibition and chemotherapy. These results indicate that TRPM2 inhibition activates alternative pathways of cell death in breast cancer cells. Taken together, our results provide significant evidence that TRPM2 inhibition is a potential strategy to induce triple-negative and estrogen receptor-positive breast adenocarcinoma cell death via alternative cell death pathways. This is expected to provide a basis for inhibiting TRPM2 for the improved treatment of breast cancer, which potentially includes treating breast tumors that are resistant to chemotherapy due to their evasion of apoptosis.

Transient receptor potential, melastatin-2 (TRPM2) is a plasma membrane cation channel with important roles in sensory functions and promoting cell death. However, we demonstrated here that TRPM2 was present in the nuclei of MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, and its pharmacologic inhibition or RNAi silencing caused decreased cell proliferation. Neither an effect on proliferation nor a localization of TRPM2 in the nucleus was observed in noncancerous HMEC and MCF-10A human mammary epithelial cells. Investigation of possible effects of TRPM2 function in the nucleus demonstrated that pharmacologic inhibition or RNAi silencing of TRPM2 in MCF-7 and MDA-MB-231 human breast adenocarcinoma cells caused up to 4-fold increases in DNA damage levels, as compared to noncancerous breast cells after equivalent treatments. These results indicate that TRPM2 has a novel nuclear function in human breast adenocarcinoma cells that facilitates the integrity of genomic DNA, a finding that is distinct from its previously reported role as a plasma membrane cation channel in noncancerous cells. In summary, we report here a novel effect promoted by TRPM2, where it functions to minimize DNA damage and thus may have a role in the protection of genomic DNA in breast cancer cells. Our study therefore provides compelling evidence that TRPM2 has a unique role in breast adenocarcinoma cells. Accordingly, these studies suggest that TRPM2 is a potential therapeutic target, where its pharmacologic inhibition may provide an innovative strategy to selectively increase DNA damage levels in breast cancer cells.

Background: The incidence and mortality of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) is higher in African Americans (AA) than other racial/ethnic groups in the U.S., but the reasons for this disparity are unknown. There is an urgent need for the discovery of novel molecular signatures for HCV disease progression to understand the underlying biological basis for this cancer rate disparity to improve the clinical outcome., Methods: We performed differential proteomics with isobaric labeling tags for relative and absolute quantitation (iTRAQ) and MS/MS analysis to identify proteins differentially expressed in cirrhotic (CIR) and HCC as compared to normal tissues of Caucasian American (CA) patients. The raw data were analyzed using the ProteinPilot v3.0. Searches were performed against all known sequences populating the Swiss-Prot, Refseq, and TrEMBL databases. Quality control analyses were accomplished using pairwise correlation plots, boxplots, principal component analysis, and unsupervised hierarchical clustering. Supervised analysis was carried out to identify differentially expressed proteins. Candidates were validated in independent cohorts of CA and AA tissues by qRT-PCR or Western blotting., Results: A total of 238 unique proteins were identified. Of those, around 15% were differentially expressed between normal, CIR & HCC groups. Target validation demonstrates racially distinct alteration in the expression of certain proteins. For example, the mRNA expression levels of transferrin (TF) were 2 and18-fold higher in CIR and HCC in AA as compared to CA. Similarly; the expression of Apolipoprotein A1 (APOA1) was 7-fold higher in HCC of AA. This increase was mirrored in the protein expression levels. Interestingly, the level of hepatocyte nuclear factor4a (HNF4a) protein was down regulated in AA, whereas repression of transcription is seen more in CA compared to AA. These data suggest that racial disparities in HCC could be a consequence of differential dysregulation of HNF4a transcriptional activity., Conclusion: This study identifies novel molecular signatures in HCV-induced HCC using iTRAQ-based tissue proteomics. The proteins identified will further enhance a molecular explanation to the biochemical mechanism(s) that may play a role in HCC racial disparities.

The genome-protecting role of poly(ADP-ribose) (PAR) has identified PAR polymerase-1 (PARP-1) and PAR glycohydrolase (PARG), two enzymes responsible for the synthesis and hydrolysis of PAR, as chemotherapeutic targets. Each has been previously individually evaluated in chemotherapy, but the effects of combination PARP-1 and PARG inhibition in cancer cells are not known. Here we determined the effects of the inhibition of PARP-1 and the absence or RNAi knockdown of PARG on PAR synthesis, cell death after chemotherapy and long-term viability. Using three experimental/clinical PARP-1 inhibitors in PARG-null cells, we show decreased levels of PAR and increased short‑term and long‑term viability with each inhibitor, with the exception of DPQ. Treatment with the experimental chemotherapeutic agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), led to increased cell death in PARG-null cells, but decreased cell death when pretreated with each PARP-1 inhibitor. Similar results were observed in MNNG-treated HeLa cells, where RNAi knockdown of PARG or pretreatment with ABT-888 led to increased HeLa cell death, whereas combination PARG RNAi knockdown + ABT-888 failed to produce increased cell death. The results demonstrate the ability of the PARP-1 inhibitors to decrease PAR levels, maintain viability and decrease PAR-mediated cell death after chemotherapeutic treatment in the absence of PARG. Further, the results demonstrate that the combination of PARP-1 and PARG inhibition in chemotherapy does not produce increased HeLa cell death. Thus, the results indicate that inhibiting both PARP-1 and PARG, which both are chemotherapeutic targets that increase cancer cell death, does not lead to synergistic cell death in HeLa cells. Therefore, strategies that target PAR metabolism for the improved treatment of cancer may be required to target PARP-1 and PARG individually in order to optimize cancer cell death.

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme that catalyzes the hydrolysis of poly(ADP-ribose) (PAR), an essential biopolymer that is synthesized by poly(ADP-ribose) polymerases (PARPs) in the cell. By regulating the hydrolytic arm of poly(ADP-ribosyl)ation, PARG participates in a number of biological processes, including the repair of DNA damage, chromatin dynamics, transcriptional regulation, and cell death. Collectively, the research investigating the roles of PARG in the cell has identified the importance of PARG and its value as a therapeutic target. However, the biological role of PARG remains less understood than the role of PAR synthesis by the PARPs. Further complicating the study of PARG is the existence of multiple PARG isoforms in the cell, the lack of optimal PARG inhibitors, and the lack of viable PARG-null animals. This review will present our current knowledge of PARG, with a focus on its roles in DNA-damage repair and cell death., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Cell death induced by poly(ADP-ribose) (PAR) and mediated by apoptosis-inducing factor (AIF) is well-characterized in models of ischemic tissue injury, but their roles in cancer cell death after chemotherapy are less understood., Methods: Here we investigated the roles of PAR and AIF by RNA interference (RNAi) in MDA-MB-231 and MCF-7 breast adenocarcinoma cells after chemotherapy. Differences in effects were statistically tested by analysis-of-variance and unpaired student's t-test., Results: Silencing of AIF by RNAi led to decreased MDA-MB-231 and MCF-7 breast cancer cell death after chemotherapy, which demonstrates a critical role for AIF. RNAi silencing of PAR glycohydrolase (PARG), the primary enzyme that catalyzes the hydrolysis of PAR, led to increased PAR levels but decreased cell death. Further investigation into the possible role of PAR in apoptosis revealed decreased caspase-3/7/8/9 activity in PARG-null cells. Interestingly, the pharmacologic inhibition of caspase activity in PARG-silenced breast cancer cells led to increased cell death after chemotherapy, which indicates that an alternative cell death pathway is activated due to elevated PAR levels and caspase inhibition. AIF silencing in these cells led to profound protection from chemotherapy, which demonstrates that the increased cell death after PARG silencing and caspase inhibition was mediated by AIF., Conclusions: The results show a role for AIF in breast cancer cell death after chemotherapy, the ability of PAR to regulate caspase activity, and the ability of AIF to substitute as a primary mediator of breast cancer cell death in the absence of caspases. Thus, the induction of cell death by PAR/AIF may represent a novel strategy to optimize the eradication of breast tumors by activating an alternative cell death pathway.

DNA-alkylating agents in combination with poly (ADP-ribose) (PAR) synthesis inhibitors are a promising treatment for cancer. In search of other efficacious alternatives, we hypothesized that the absence of poly(ADP-ribose) glycohydrolase (PARG), which leads to the inhibition of PAR hydrolysis, would lead to increased DNA alkylation after treatment with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). At a sublethal dose, MNNG shows synergistic cytotoxicity in PARG-null embryonic trophoblast stem (TS) cells. The PAR modifications of histone H1 and histone H2B are much more pronounced in PARG null-TS cells exposed to MNNG, suggesting their relevance in the efficacy of this combination therapy. Because the PAR modification of these chromatin binding proteins leads to chromatin remodeling, a possible mechanism for the observed synergistic effects involves the subsequent decondensation of chromatin, which may cause the genomic DNA to be more accessible to MNNG alkylation. Further analysis demonstrated chromatin decondensation in PARG null-TS cells as visualized by electron microscopy. In addition, treatment with MNNG led to an increase in O6- methylguanine levels in PARG null-TS cells compared to wild-type, which demonstrates increased DNA alkylation in the absence of PARG. Taken together, we provide compelling evidence that the absence of PARG leads to chromatin decondensation, which in turn leads to increased amounts of DNA alkylation and cell death induced by low doses of MNNG. Therefore, combination therapy of PARG inhibition and a DNA- alkylating agent is a potential treatment to induce the death of cancer cells.

We previously demonstrated that the absence of poly(ADP-ribose) glycohydrolase (PARG) led to increased cell death following DNA-damaging treatments. Here, we investigated cell death pathways following UV treatment. Decreased amounts of PARG-null embryonic trophoblast stem (TS) cells were observed following doses of 10-100 J/m2 as compared to wild-type cells. In wild-type cells, caspase-cleaved poly(ADP-ribose) polymerase-1 (PARP-1) and activated caspase-3 were detected 12-24 h after UV treatment. Surprisingly, both were detected at decreased levels only after 24 h in PARG-null TS cells, indicating a decreased level and delayed presence of caspase-mediated events. Further, a time- and dose-dependent accumulation of poly(ADP-ribose) (PAR) levels after UV was observed in PARG-null TS cells and not in wild-type cells. Determination of the levels of nicotinamide adenine dinucleotide (NAD+), the substrate for PAR synthesis and a coenzyme in cellular redox reactions, demonstrated a UV dose-dependent decrease in the level of NAD+ in wild-type cells, while NAD+ levels in PARG-null TS cells remained at higher levels. This indicates no depletion of NAD+ in PARG-null TS cells following increased levels of PAR. Lastly, cell death mediated by apoptosis-inducing factor (AIF) was analyzed because of its dependence on increased PAR levels. The results demonstrate nuclear AIF translocation only in PARG-null TS cells, which demonstrates the presence of AIF-mediated cell death. Herein, we provide compelling evidence that the absence of PARG leads to decreased caspase-3 activity and the specific activation of AIF-mediated cell death. Therefore, the absence of PARG may provide a strategy for specifically inducing an alternative apoptotic pathway.

Studies suggest that inhibiting the hydrolysis of poly(ADP-ribose) (PAR) by targeting the enzyme PAR glycohydrolase (PARG) is a potential chemotherapeutic strategy to induce cell death. However, the lack of structural data for PARG has hindered the discovery of specific PARG inhibitors and thus hampered the search for cellular effects dependent on the hydrolysis of PAR. We previously generated a murine PARG null cell model to identify the intracellular processes mediated by PARG. Using this system, the only mammalian system to date that completely lacks PARG activity, we have shown that the absence of PARG leads to massive amounts of cell death due to increased levels of PAR. Further, we have shown that PARG null-TS cells exhibit profound hypersensitivity to low doses of DNA-damaging agents. This hypersensitivity most likely results from the high levels of DNA damage that occur after treatment of these cells with nonlethal doses of DNA-damaging agents.

We consider two interacting semiflexible charged chains of length L(c) under shape fluctuations, where the interplay of electric and mechanical properties is found to yield rigidity-sensitive charge modulation and interdistance-dependent persistence length ℓ(p). The resulting conformation is characterized by equilibrium force between the chains and their fractal dimensions. It turns out that ℓ(p) and L(c) emerge as critical factors to determine the force nature as well as chain shapes. We show that conformational fluctuations cause the repulsion of nonsteric origin, and its competition with charge fluctuation effects yields the interchain force modulated by the length scales and counterion valence. As a result, it is predicted that flexible short chains can be more strongly repulsive than rigid long chains, although they carry smaller amount of net charges.

Poly(ADP-ribose) (PAR) is a therapeutic target primarily identified through inhibiting its synthesis by PAR polymerase-1 (PARP-1). However, inhibiting its hydrolysis by PAR glycohydrolase (PARG) has therapeutic potential in cancer. Unknown is the effect of elevated PAR levels on cellular processes and if this effect can enhance the therapeutic value of PARG. Here, we demonstrate in PARG null embryonic trophoblast stem (TS) cells that the absence of PAR hydrolysis led to PAR-modified histones H1, H2A, and H2B. To determine if this led to the differential vulnerability of DNA to modification, TS cells were treated with DNA-modifying agents. The results demonstrate increased DNA laddering by micrococcal nuclease and an increased amount of DNA intercalation by acridine orange in PARG null-TS cells. This increased access to PARG null-TS cell DNA was further verified by the detection of increased DNA damage following treatment with UV radiation and a minimal dose of the DNA-alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Further, this DNA damage was predominantly unrepaired 12 h after treatment in PARG null-TS cells. Finally, TS cells were treated with DNA-modifying chemotherapeutic agents. The results demonstrate up to 4-fold increases in cell death in PARG null-TS cells after treatment with epirubicin or sub-IC(50) doses of cisplatin and cyclophosphamide. Taken together, we provide compelling evidence that increased DNA access induced by the absence of PARG enhances the efficacy of DNA-modifying agents. Thus, this study demonstrates that greater DNA accessibility, increased DNA damage, and increased cell death all contribute to the PARG null cell phenotype in response to genotoxic stress.

Background/aims: Treatment of patients with hepatocellular carcinoma (HCC) depends on the tumour extent and underlying liver function. Antiviral therapy with nucleoside/nucleotide analogues has been shown to be effective in improving the liver function of chronic hepatitis B (CHB) patients. We assessed whether lamivudine could induce biochemical and virological improvements in patients with hepatitis B virus-related HCC., Patients/methods: Of 148 CHB patients treated with 100 mg/day lamivudine for at least 6 months, 80 had HCC (CHB/HCC group) and 68 did not (CHB group). Biochemical and virological parameters were serially monitored., Results: Compared with the CHB group, the CHB/HCC group was older, had higher male predominance, bilirubin levels and liver cirrhosis rate, and lower albumin and hepatitis B virus (HBV) DNA levels and hepatitis B e antigen (HBeAg) positivity (P<0.05 each). The two groups showed similar cumulative rates of alanine aminotransferase normalization, HBV DNA seroconversion, HBeAg loss and viral breakthrough during 12 months of lamivudine treatment. After 12 months, the CHB/HCC group showed, relative to baseline, increased albumin levels (3.51+/-0.5 vs. 3.72+/-0.5 mg/ml) and decreased ascites scores (1.63+/-0.7 vs. 1.45+/-0.6) and Child-Pugh scores (6.92+/-1.9 vs. 6.02+/-1.38) (P<0.05 each)., Conclusion: Lamivudine had comparable antiviral effects both in patients with CHB and CHB/HCC, and improved underlying liver function in the latter group. Treatment of HBV may increase the chance of curative treatments in patients with HBV-related HCC.

Amputation neuroma or traumatic neuroma is a tumor-like secondary hyperplasia that may develop after an accidental or surgical trauma. Amputation neuroma of the bile duct has occasionally been reported which occurred in the cystic duct stump late after the cholecystectomy. However, even if the amputation neuroma is suspected in a patient with late-onset jaundice after cholecystectomy, the differential diagnosis from a malignancy is difficult preoperatively. We experienced a case of the amputation neuroma of common bile duct (CBD) developed in a 70-year-old man who presented with a polypoid mass in CBD. He had undergone cholecystectomy 25 years ago and choledochojejunostomy 12 years ago, respectively. We have performed pylorus-preserving pancreatico-duodenectomy (PPPD) under the impression of CBD cancer. He had not been diagnosed of amputation neuroma until having undergone PPPD. We report a case of CBD neuroma mimicking CBD cancer, which was confirmed after PPPD.

Purpose: To investigate whether three-dimensional conformal radiotherapy (3D-CRT) influences hepatitis B virus (HBV) reactivation and chronic hepatitis B (CHB) exacerbation in patients with HBV-related hepatocellular carcinoma (HCC)., Methods and Materials: Of the 48 HCC patients with HBV who underwent 3D-CRT to the liver, 16 underwent lamivudine therapy before and during 3D-CRT (Group 1) and 32 did not receive antiviral therapy before 3D-CRT (Group 2). To analyze spontaneous HBV reactivation, we included a control group of 43 HCC patients who did not receive any specific treatment for HCC or CHB., Results: The cumulative rate of radiation-induced liver disease for Groups 1 and 2 was 12.5% (2 of 16) and 21.8% (7 of 32), respectively (p > 0.05). The cumulative rate of HBV reactivation was significantly greater in Group 2 (21.8%, 7 of 32) than in Group 1 (0%, 0/16) or the control group (2.3%, 1 of 43; p < 0.05 each). The cumulative rate of CHB exacerbation, however, did not differ significantly between Groups 2 (12.5%, 4 of 32) and 1 (0%, 0 of 16) or the control group (2.3%, 1 of 43; p > 0.05 each). The CHB exacerbations in the 4 Group 2 patients had radiation-induced liver disease features but were differentiated by serum HBV DNA changes. Two of these patients required antiviral therapy and effectively recovered with lamivudine therapy., Conclusions: In patients with HBV-related HCC undergoing 3D-CRT, HBV reactivation and consequent CHB exacerbation should be considered in the differential diagnosis of radiation-induced liver disease, and antiviral therapy might be considered for the prevention of liver function deterioration after RT.

Poly(ADP-ribose) polymerases (PARPs) are members of a family of enzymes that utilize nicotinamide adenine dinucleotide (NAD(+)) as substrate to form large ADP-ribose polymers (PAR) in the nucleus. PAR has a very short half-life due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). PARP-1 mediates acute neuronal cell death induced by a variety of insults including cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism, and CNS trauma. While PARP-1 is localized to the nucleus, PARG resides in both the nucleus and cytoplasm. Surprisingly, there appears to be only one gene encoding PARG activity, which has been characterized in vitro to generate different splice variants, in contrast to the growing family of PARPs. Little is known regarding the spatial and functional relationships of PARG and PARP-1. Here we evaluate PARG expression in the brain and its cellular and subcellular distribution in relation to PARP-1. Anti-PARG (alpha-PARG) antibodies raised in rabbits using a purified 30 kDa C-terminal fragment of murine PARG recognize a single band at 111 kDa in the brain. Western blot analysis also shows that PARG and PARP-1 are evenly distributed throughout the brain. Immunohistochemical studies using alpha-PARG antibodies reveal punctate cytosolic staining, whereas anti-PARP-1 (alpha-PARP-1) antibodies demonstrate nuclear staining. PARG is enriched in the mitochondrial fraction together with manganese superoxide dismutase (MnSOD) and cytochrome C (Cyt C) following whole brain subcellular fractionation and Western blot analysis. Confocal microscopy confirms the co-localization of PARG and Cyt C. Finally, PARG translocation to the nucleus is triggered by NMDA-induced PARP-1 activation. Therefore, the subcellular segregation of PARG in the mitochondria and PARP-1 in the nucleus suggests that PARG translocation is necessary for their functional interaction. This translocation is PARP-1 dependent, further demonstrating a functional interaction of PARP-1 and PARG in the brain.

Extrahepatic metastasis in patients with hepatocellular carcinoma (HCC) occurs frequently. The most common site of metastasis is the lung, followed by regional lymph nodes and bones. However, gastrointestinal metastasis of HCC is a rare condition and solitary polypoid metastatic lesion on stomach without any evidence of direct invasion from primary mass is very rare. These metastatic lesions are usually asymptomatic, and most are discovered at postmortem examination or are found incidentally during laparotomy. The choice of treatment for gastrointestinal metastatic lesion of HCC includes surgery, transarterial chemoembolization, and local injection but the treatment is often difficult and unsuccessful. We report a case of 69 years old man who presented disappearance of a polypoid metastatic lesion of HCC on the gastric fundus by transarterial chemoembolization.

Excessive activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) plays a prominent role in various of models of cellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer, a product of PARP-1 activity, as a previously uncharacterized cell death signal. PAR polymer is directly toxic to neurons, and degradation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG) or phosphodiesterase 1 prevents PAR polymer-induced cell death. PARP-1-dependent, NMDA excitotoxicity of cortical neurons is reduced by neutralizing antibodies to PAR and by overexpression of PARG. Neuronal cultures with reduced levels of PARG are more sensitive to NMDA excitotoxicity than WT cultures. Transgenic mice overexpressing PARG have significantly reduced infarct volumes after focal ischemia. Conversely, mice with reduced levels of PARG have significantly increased infarct volumes after focal ischemia compared with WT littermate controls. These results reveal PAR polymer as a signaling molecule that induces cell death and suggests that interference with PAR polymer signaling may offer innovative therapeutic approaches for the treatment of cellular injury.

Poly(ADP-ribose) polymerase-2 (PARP-2) is a member of the PARP enzyme family, and, similarly to PARP-1, catalyzes the formation of ADP-ribose polymers in response to DNA damage. While PARP-1 overactivation contributes to ischemic cell death, no information is available regarding the role of PARP-2. In this study, we evaluated the impact of PARP-2 deletion on histopathological outcome from two different experimental models of cerebral ischemia. Male PARP-2-/- mice and wild-type (WT) littermates were subjected to either 2 h of middle cerebral artery occlusion (MCAO) followed by 22 h reperfusion, or underwent 10 mins of KCl-induced cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR) and 3-day survival. After MCAO, infarct volume was reduced in PARP-2-/- mice (38%+/-12% of contralateral hemisphere) compared with WT (64%+/-16%). After CA/CPR, PARP-2 deletion significantly increased neuronal cell loss in the hippocampal CA1 field (65%+/-36% ischemic neurons) when compared with WT mice (31%+/-33%), with no effect in either striatum or cortex. We conclude that PARP-2 is a novel executioner of cell death pathways in focal cerebral ischemia, but might be a necessary survival factor after global ischemia to mitigate hippocampal delayed cell death.

Poly(ADP-ribosyl)ation plays an important role in modulating the cellular response to stress. The extent of poly(ADP-ribosyl)ation, chiefly via the activation of the poly(ADP-ribose) polymerase-1 (PARP-1), correlates with the severity of genotoxic stress and this determines the cellular response. Under mild and moderate stress, it plays important roles in DNA processing and it participates in the proinflammatory/cellular defense via transcriptional regulation. However, severe stress following acute neuronal injury causes the overactivation of PARP-1, which results in unregulated poly(ADP-ribose) (PAR) synthesis and widespread neuronal cell death. Previously, this PARP-1-dependent cell death mechanism was manifest solely through necrosis, but apoptotic mechanisms are also evident. Poly(ADP-ribosyl)ation directly induces the nuclear translocation of apoptosis-inducing factor, which results in caspase-independent cell death significant in many neurodegenerative conditions. Further, the hydrolysis of PAR by poly(ADP-ribose) glycohydrolase (PARG) has a protective role, since the accumulation of PAR leads to cell death by apoptosis. Thus, PAR signaling, regulated by PARP-1 and PARG, mediates cell death. Accordingly, modulation of PAR synthesis or degradation through the targeting of PARP-1 or PARG holds particular promise in the treatment of conditions such as cancer, stroke, and Parkinson's disease.

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of alpha(1''-->2') or alpha(1'''-->2'') O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG 'signature sequence' [vDFA-X3-GGg-X6-8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610-795 in bovine PARG.

Poly(ADP-ribosyl)ation is required by multicellular eukaryotes to ensure genomic integrity under conditions of mild to moderate genotoxic stress. However, severe stress following acute neuronal injury causes overactivation of poly(ADP-ribose) polymerase-1, which results in unregulated poly(ADP-ribose) (PAR) synthesis and widespread neuronal cell death. Once thought to be a necrotic cell death resulting from energy failure, PARP-1 activation is now known to induce the nuclear translocation of apoptosis-inducing factor, which results in caspase-independent cell death. Conversely, poly(ADP-ribose) glycohydrolase, once thought to contribute to neuronal injury, now appears to have a protective role as demonstrated by recent studies utilizing gene disruption technology. Thus, the emerging mechanism dictating the fate of neurons appears to involve the regulation of PAR levels in neurons. Therefore, therapies targeting poly(ADP-ribosyl)ation in the treatment of neurodegenerative conditions such as stroke and Parkinson's disease are required to inhibit PAR synthesis and/or facilitate its degradation.

Unlike poly(ADP-ribose) polymerase-1 (PARP-1), poly(ADP-ribose) glycohydrolase (PARG) has long been a difficult protein to study. However, the complete absence of PARG activity was recently characterized in mice via disruption of the murine PARG gene. As expected, PARG is critical for the maintenance of steady-state poly(ADP-ribose) levels. But surprisingly, the disruption of PARG led to embryonic lethality and increased susceptibility to mild cell stress. Therefore, the protective role of PARG and its involvement in development indicate that these roads to viability go through PARG.

The metabolism of poly(ADP-ribose) (PAR) is critical for genomic stability in multicellular eukaryotes. Here, we show that the failure to degrade PAR by means of disruption of the murine poly(ADP-ribose) glycohydrolase (PARG) gene unexpectedly causes early embryonic lethality and enhanced sensitivity to genotoxic stress. This lethality results from the failure to hydrolyze PAR, because PARG null embryonic day (E) 3.5 blastocysts accumulate PAR and concurrently undergo apoptosis. Moreover, embryonic trophoblast stem cell lines established from early PARG null embryos are viable only when cultured in medium containing the poly(ADP-ribose) polymerase inhibitor benzamide. Cells lacking PARG also show reduced growth, accumulation of PAR, and increased sensitivity to cytotoxicity induced by N-methyl-N'-nitro-N-nitrosoguanidine and menadione after benzamide withdrawal. These results provide compelling evidence that the failure to degrade PAR has deleterious consequences. Further, they define a role for PARG in embryonic development and a protective role in the response to genotoxic stress.

The profound neuroprotection observed in poly(ADP-ribose) polymerase-1 (PARP-1) null mice to ischemic and excitotoxic injury positions PARP-1 as a major mediator of neuronal cell death. We report here that apoptosis-inducing factor (AIF) mediates PARP-1-dependent glutamate excitotoxicity in a caspase-independent manner after translocation from the mitochondria to the nucleus. In primary murine cortical cultures, neurotoxic NMDA exposure triggers AIF translocation, mitochondrial membrane depolarization, and phosphatidyl serine exposure on the cell surface, which precedes cytochrome c release and caspase activation. NMDA neurotoxicity is not affected by broad-spectrum caspase inhibitors, but it is prevented by Bcl-2 overexpression and a neutralizing antibody to AIF. These results link PARP-1 activation with AIF translocation in NMDA-triggered excitotoxic neuronal death and provide a paradigm in which AIF can substitute for caspase executioners.

Polyadenosine diphosphoribose glycohydrolase (PARG) catalyzes the intracellular hydrolysis of adenosine diphosphoribose polymers. Because structure-activity data are lacking for PARG, the specific inhibitor adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) was utilized to determine the effects of structure on inhibitor potency using PARG isolated from bovine thymus (bPARG) and recombinant bovine PARG catalytic fragment (rPARG-CF). Both enzymes were strongly inhibited by submicromolar levels of ADP-HPD, but ADP and the phosphorylated pyrrolidine displayed no activity. Utilizing ADP-HPD analogues containing 2-, N(6), or 8-adenosyl substituents or guanine instead of adenine, the importance of adenine ring recognition as well as a correlation between loss of PARG inhibition and the length and bulkiness of 8-adenosyl substituents was shown. Utilization of ADP-HPD analogues lacking one or both pyrrolidine cis-hydroxyls demonstrated their importance for inhibitor binding. Last, the similarity between naturally occurring bPARG and heterologously expressed rPARG-CF was demonstrated. Therefore, readily available rPARG-CF is suitable for use in future studies to determine the structural aspects of PARG.

Polymers of ADP-ribose involved in the maintenance of genomic integrity are converted to free ADP-ribose by the action of poly(ADP-ribose) glycohydrolase (PARG). As an approach to mapping functions of PARG onto the amino acid sequence of the protein, we report here experiments that identify an amino acid residue involved in the binding of potent PARG inhibitors. A photoreactive inhibitor, [alpha-(32)P]-8-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol (8-N(3)-ADP-HPD), was used to photolabel a recombinant bovine PARG catalytic fragment (rPARG-CF). N-Terminal sequencing of tryptic and subtilitic peptides of photoderivatized rPARG-CF identified tyrosine 796 (Y796), a residue conserved in PARG across a wide range of organisms, as a site of photoderivatization. Site-directed mutants where this tyrosine residue was replaced with an alanine residue (Y796A) had a nearly 8-fold decrease in catalytic efficiency (k(cat)/K(M)), while replacement with a tryptophan residue (Y796W) had little effect on catalytic efficiency. Surface plasmon resonance spectroscopy using the PARG inhibitor 8-(aminohexyl)amino-ADP-HPD demonstrated that the binding constant of the inhibitor for Y796A was 21-fold lower (K(D) = 170 nM) than that of wild-type PARG (K(D) = 8.2 nM), while Y796W displayed a binding affinity similar to that of the wild-type enzyme. Our results indicate that Y796 is involved in inhibitor binding to PARG via a ring stacking interaction and identify a highly conserved region of the protein that putatively contains other residues involved in catalytic activity and/or substrate recognition.

Primary adenosquamous carcinoma of the liver is a very rare type of cholangiocarcinoma and is defined as a cancer containing both squamous and adenomatous components in the same lesion. Recently, we experienced a primary adenosquamous carcinoma of the liver presented as liver abscess. A 63-year-old man was presented with a 4-day history of fever and chill. The radiologic study showed a 4 cm-sized, central hypoattenuated mass with peripheral rim enhancement in the left lobe of the liver. Ultrasonography-guided aspiration and biopsy suggested an adenocarcinoma with abscess in the liver. At laparotomy, the tumor occupied the left lobe of the liver and invaded the right diaphragm. An extended left lobectomy and a partial excision of the involved diaphragm were done. Grossly, the tumor was 6 x 5 x 5 cm in size and had an eccentric necrosis. Microscopically, the tumor was composed of adenocarcinoma and squamous cell carcinoma with a transitional area.

Poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), the only enzyme known to synthesize ADP-ribose polymers from NAD+, is activated in response to DNA strand breaks and functions in the maintenance of genomic integrity. Mice homozygous for a disrupted gene encoding PARP are viable but have severe sensitivity to gamma-radiation and alkylating agents. We demonstrate here that both 3T3 and primary embryo cells derived from PARP-/- mice synthesized ADP-ribose polymers following treatment with the DNA-damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine, despite the fact that no PARP protein was detected in these cells. ADP-ribose polymers isolated from PARP-/- cells were indistinguishable from that of PARP+/+ cells by several criteria. First, they bound to a boronate resin selective for ADP-ribose polymers. Second, treatment of polymers with snake venom phosphodiesterase and alkaline phosphatase yielded ribosyladenosine, a nucleoside diagnostic for the unique ribosyl-ribosyl linkages of ADP-ribose polymers. Third, they were digested by treatment with recombinant poly(ADP-ribose) glycohydrolase, an enzyme highly specific for ADP-ribose polymers. Collectively, these data demonstrate that ADP-ribose polymers are formed in PARP-/- cells in a DNA damage-dependent manner. Because the PARP gene has been disrupted, these results suggest the presence of a previously unreported activity capable of synthesizing ADP-ribose polymers in PARP-/- cells.

Two isomeric azidoadenosyl analogues of adenosine diphosphate (hydroxymethyl)pyrrolidinediol [ADP-HPD; Slama, J. T., et al. (1995) J. Med. Chem. 38, 389-393] were synthesized as photoaffinity labels for poly(ADP-ribose) glycohydrolase. 8-Azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol (8-N3-ADP-HPD) inhibited the enzyme activity by 50% at ca. 1 microM, a concentration 80-fold lower than that where the isomeric 2-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol did. [alpha-32P]-8-N3-ADP-HPD was therefore synthesized and used to photoderivatize poly(ADP-ribose) glycohydrolase. Irradiation of recombinant poly(ADP-ribose) glycohydrolase and low concentrations of [alpha-32P]-8-N3-ADP-HPD with short-wave UV light resulted in the covalent incorporation of the photoprobe into the protein, as demonstrated by gel electrophoresis followed by autoradiography or acid precipitation of the protein followed by scintillation counting. No photoincorporation occurred in the absence of UV light. The photoincorporation saturated at low concentrations of the photoprobe and photoprotection was observed in the presence of low concentrations of ADP-HPD, an indication of the specificity of the photoinsertion reaction. These results demonstrate that [alpha-32P]-8-N3-ADP-HPD can be used to specifically covalently photoderivatize the enzyme to characterize the polypetides that constitute the ADP-HPD binding site of poly(ADP-ribose) glycohydrolase. The photoincorporation reaction was further used to determine the ability of ADP-ribose polymers of varying size to compete with [alpha-32P]-8-N3-ADP-HPD for binding to the enzyme. Photoincorporation of [alpha-32P]-8-N3-ADP-HPD was inhibited by 80% in the presence of low concentrations of short, unbranched ADP-ribose oligomers (5-15 ADP-ribose units in length). No similar photoprotection was afforded by the addition of a high-molecular weight highly branched polymer. These results indicate that the photolabel shares a binding site with the short, linear polymer, but not with the long, highly branched polymer.

Elastic and collagen fibers confer recoil and tensile strength on the pulmonary vasculature, airways, alveolar walls, and pleura. These durable extracellular matrix components are primarily synthesized during lung development and growth, and are expressed at very low levels in healthy adult lung. However, reinitiation of elastin and collagen synthesis in diseases of adult lung, such as idiopathic pulmonary fibrosis, often leads to excessive or aberrant deposition of elastin and collagen which contribute to the pathophysiology of these diseases. We used an experimental model of postpneumonectomy lung growth to determine whether normal patterns of synthesis and deposition of these critical structural components can occur in the adult lung. Male Sprague-Dawley rats (250-300 grams) were subjected to left pneumonectomy and right lobectomy. The remaining lung tissue was harvested for analysis after 3, 7, or 14 days. Compensatory growth of the remaining right lung progressed throughout the time course. Total desmosine and hydroxyproline content increased in the postpneumonectomy lung, reflecting increased elastin and collagen accumulation, but both were normal in content per weight of lung tissue. Northern analysis demonstrated induction of tropoelastin and type I procollagen mRNA expression in lungs of pneumonectomy rats. In situ hybridization localized tropoelastin and type I procollagen mRNA expression to anatomical sites similar to those seen during lung development. These data indicate that the adult lung can reinitiate elastin and collagen production and deposit these extracellular matrix components in a normal pattern.