Development and forensic validation of human genomic DNA quantification kit.

DNA quantification is an essential step for successful multiplex short tandem repeat (STR) polymerase chain reactions (PCR), which are used for confirming identities using human genomic DNA. The new DNA quantification kit, named the National Forensic Service Quantification (NFSQ) kit, simultaneously provides total human DNA concentration, human male DNA concentration, and a DNA degradation index (DI) using multiplex TaqMan fluorescent probes. The NFSQ was validated according to developmental validation guidelines from the SWGDAM and MIQE. NFSQ detected up to 0.00128 ng/µL and could detect male DNA up to a 1:8000 ratio of male to female DNA. In PCR inhibitor tests, NFSQ could measure DNA at a concentration of 200 ng/µL of humic acid and 600 µM of hematin. The NFSQ kit showed a DI value trend similar to other qPCR kits. In the reproducibility study, the coefficient of variation of the NFSQ kit was within 10%. The quantitative results of the casework samples obtained using the NFSQ kit were consistent with the STR interpretation results. The NFSQ kit can be useful in the human identification process, as it has detection capabilities similar to those of other comparable quantification kits.

Triglyceride-induced macrophage cell death is triggered by caspase-1.

Triglyceride (TG) induces macrophage cell death which contributes to the development of atherosclerosis. We confirmed that exogenous TG accumulates in human THP-1 macrophages and causes cell death. TG treated THP-1 macrophages exhibited no change in tumor necrosis factor (TNF)-α, interleukin (IL)-18, macrophage inflammatory protein (MIP)-1α, and IL-1R1 receptor mRNA expression. However, there was a marked decrease in IL-1ß mRNA expression but an increase in IL-1ß protein secretion. Decreased expression of IL-1ß mRNA and increased secretion of IL-1ß protein was not the direct cause of cell death. Until now, TG was assumed to induce necrotic cell death in macrophages. Since caspase-1 is known to be involved in activation and secretion of IL-1ß protein and pyroptotic cell death, next we determined whether caspase-1 is associated with TG-induced macrophage cell death. We found an increase in caspase-1 activity in TG-treated THP-1 macrophages and inhibition of caspase-1 activity using a specific inhibitor partially rescued cell death. These results suggest activation of the pyroptotic pathway by TG. This is the first report implicating the activation of caspase-1 and the triggering of the pyroptosis pathway in TG-induced macrophage cell death.

REBA HPV-ID® for efficient genotyping of human papillomavirus in clinical samples from Korean patients.

This study was conducted to evaluate the overall performance of a reverse blot hybridization-based assay, REBA HPV-ID® (Molecules and Diagnostics, Wonju, Korea) for genotyping human papillomaviruses (HPV). HPV Genotyping on 356 specimens examined cytologically was performed using the REBA HPV-ID®, and its results were compared with those obtained using the MyHPV DNA Chip® (Mygene, Seoul, Korea), DNA chip-based HPV genotyping assay. The results from this study showed that the positivity rate of the REBA HPV-ID® for abnormal cytological samples was higher (80.9%) than that of the MyHPV DNA chip (69.8%). In addition, the REBA HPV-ID® positivity rate with normal cytological samples was higher (64.4%) than that obtained using DNA chips (34.4%). Subsequently, sequence analysis was performed with specimens that generated conflicting test results. Sequence analysis confirmed that the specimens which were positive by REBA HPV-ID® did indeed contain HPV sequences. The results of this study suggest that the REBA HPV-ID® is a sensitive test for genotyping HPV of clinical specimens.

Neuroprotective effects of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride against oxidative stress.

Oxidative stress, glutamate excitotoxicity, and inflammation are the important pathological mechanisms in neurodegenerative diseases. Recently, we reported that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects rat glial cells against glutamate-induced excitotoxicity. In this study, we report the effects of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride on primary cultured cortical astrocytes after exposure to hydrogen peroxide (H2O2). Pretreatment of cells with 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride prior to H2O2 exposure attenuated the H2O2-induced reductions in cell survival and superoxide dismutase, catalase, glutathione, and glutathione peroxidase activities. It also reduced H2O2-induced increases in reactive oxygen species levels, malondialdehyde content, and production of nitric oxide. These effects were all concentration-dependent. Our results suggest that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects against oxidative stress.

Suppression of glutamate-induced excitotoxicity by 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride in rat glial cultures.

We have screened new drugs with a view to developing effective drugs against glutamate-induced excitotoxicity. In the present work, we show effects of a new drug, 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride against glutamate-induced excitotoxicity in primary rat glial cultures. Pretreatment of glial cells with 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride for 2 h significantly protected glial cells against glutamate-induced excitotoxicity in a time- and dose-dependent manner with an optimum concentration of 100 microM. The drug significantly reduced production of proinflammatory cytokines, tumor necrosis factor-alpha, and interlukin-1beta in glutamate-induced excitotoxicity. The drug also prevented glutamate-induced intracellular Ca2+ influx and reduced the subsequent overproduction of nitric oxide and reactive oxygen species. Furthermore, the drug preserved the mitochondrial potential and inhibited the overproduction of cytochrome c. In addition, the drug effectively attenuated the protein level changes of beta-catenin and glycogen synthase kinase-3beta. These results suggest that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride effectively protected primary cultures of rat glial cells against glutamate-induced excitotoxicity.

Correction: ΔNp63 to TAp63 expression ratio as a potential molecular marker for cervical cancer prognosis.

[This corrects the article DOI: 10.1371/journal.pone.0214867.].

ΔNp63 to TAp63 expression ratio as a potential molecular marker for cervical cancer prognosis.

p63 is a transcription factor p53 family. Two major isoforms of p63, TAp63 with transactivation (TA) domain and ΔNp63 with truncated TA domain, have been reported to play opposing roles either in tumor suppression or oncogenic function. Little is known about the association of these two isoforms of p63 in the carcinogenesis of cervical cancer. In this study, the mRNA expression levels of TAp63 and ΔNp63 in 40 normal, 30 low-grade squamous intraepithelial lesions (LSIL), 38 high-grade squamous intraepithelial lesions (HSIL), and 52 cervical cancer formalin-fixed paraffin-embedded tissues were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). We analyzed the association between the ΔNp63 and ΔN/TAp63 mRNA expression ratio and clinicopathological parameters and compared disease-specific survival of each ΔNp63 mRNA expression and ΔN/TAp63 mRNA expression ratio. The ΔN/TAp63 mRNA expression ratio in cervical cancer showed higher sensitivity than the mRNA expression levels of ΔNp63 (52.0% vs 44.2%). The level of ΔN/TAp63 mRNA expression ratio in precancerous LSIL and HSIL was higher than in normal tissues (P = 0.01 and P = 0.003) and lower than in cervical cancer tissues (P = 0.03 and P = 0.02). Besides, the positive ΔN/TAp63 mRNA expression ratio was associated with bulky tumor size and high expression of Ki-67, the proliferation marker, in cervical cancer (P = 0.04 and P = 0.02). The cervical cancer patients with the positive ΔN/TAp63 mRNA expression ratio showed worse survival compared to those who with the negative expression ratio of ΔN/TAp63 (HR = 5.7, 95% CI: 1.6-19.9). In conclusion, the balance of TAp63 and ΔNp63 is closely related to the carcinogenesis of cervical cancer. The ΔN/TAp63 mRNA expression ratio could be useful as a diagnostic and prognostic marker of cervical cancer.

Protective effect of benzothiazole derivative KHG21834 on amyloid beta-induced neurotoxicity in PC12 cells and cortical and mesencephalic neurons.

We have investigated the effect of KHG21834, a benzothiazole derivative, on the amyloid beta protein (Abeta)-induced cell death in rat pheochromocytoma (PC12) cells and rat cortical and mesencephalic neuron-glia cultures. KHG21834 attenuated the Abeta(25-35)-induced apoptotic death in PC12 cells determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL). In the cortical neuron-glia cultures, KHG21834 reduced the Abeta(25-35)-induced apoptosis determined by TUNEL staining. Immunocytochemical analysis and Western blot analysis of Abeta(25-35)-induced neurotoxicity in mesencephalic neuron-glia cultures with anti-tyrosine hydroxylase (TH) antibody showed that Abeta(25-35) decreased the expression of TH protein by 60% and KHG21834 significantly attenuated the Abeta(25-35)-induced reduction in the expression of TH. Moreover, KHG21834 attenuates Abeta(25-35)-induced toxicity concomitant with the reduction of activation of extracellular signal-regulated kinase (ERK)1/2 to a lesser extent. ERK1 was more sensitively affected than ERK2 in attenuation of Abeta(25-35)-induced phosphorylation by KHG21834. These results demonstrated that KHG21834 was capable of protecting neuronal cells from Abeta(25-35)-induced degeneration.

Inhibitory properties of nerve-specific human glutamate dehydrogenase isozyme by chloroquine.

Human glutamate dehydrogenase exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. In the nervous system, GDH is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter during neurotransmission. Chloroquine has been known to be a potent inhibitor of house-keeping GDH1 in permeabilized liver and kidney-cortex of rabbit. However, the effects of chloroquine on nerve-specific GDH2 have not been reported yet. In the present study, we have investigated the effects of chloroquine on hGDH2 at various conditions and showed that chloroquine could inhibit the activity of hGDH2 at dose-dependent manner. Studies of the chloroquine inhibition on enzyme activity revealed that hGDH2 was relatively less sensitive to chloroquine inhibition than house-keeping hGDH1. Incubation of hGDH2 was uncompetitive with respect of NADH and non-competitive with respect of 2-oxoglutarate. The inhibitory effect of chloroquine on hGDH2 was abolished, although in part, by the presence of ADP and L-leucine, whereas GTP did not change the sensitivity to chloroquine inhibition. Our results show a possibility that chloroquine may be used in regulating GDH activity and subsequently glutamate concentration in the central nervous system.

Prodomain processing of recombinant plasmepsin II and IV, the aspartic proteases of Plasmodium falciparum, is auto- and trans-catalytic.

Prodomain processing of the four food vacuole plasmepsins (PMs), the malarial aspartic proteases, is prerequisite for their activity on hemoglobin degradation of the parasite Plasmodium falciparum. Although previous studies have suggested the involvement of a calpain-like PM convertase in the processing of PMs, the underlying mechanism of their processing remains to be clarified. Here, to investigate the mechanism by which food vacuole PM II and IV are processed, we used their wild-type and mutant proteins in which the catalytic Asp residue in two active-site motifs was mutated, as well as protease inhibitors. Autocatalytic processing of wild-type PM II and IV was inhibited only by an aspartic protease inhibitor pepstatin A. Unexpectedly, their proteolytic activities were inhibited not only by pepstatin A but also by calpain inhibitor ALLN. The active-site mutants of both PM II and IV showed neither autocatalytic processing nor proteolytic activities. However, the mutants of both PMs were efficiently processed upon incubation with their respective wild type proteins. Furthermore, the mutants of both PMs were processed upon incubation with each other's wild-type PM in both pepstatin A- and ALLN-sensitive manners. These results suggest that the processing of PM II and IV occurs via an intra- and inter-molecular autocatalytic event as well as via a transcatalytic event between them.

Menadione induces G2/M arrest in gastric cancer cells by down-regulation of CDC25C and proteasome mediated degradation of CDK1 and cyclin B1.

Menadione (vitamin K3) has been reported to induce apoptotic cell death and growth inhibition in various types of cancer cells. However, involvement of menadione in cell cycle control has not been considered in gastric cancer cells yet. In the current study, we have investigated whether menadione is involved in the cell cycle regulation and suppression of growth in gastric cancer cells. In the cell cycle analysis, we found that menadione induced G2/M cell cycle arrest in AGS cells. To elucidate the underlying mechanism, we investigated the cell cycle regulatory molecules involved in the G2/M cell cycle transition. After 24 h of menadione treatment, the protein level of CDK1, CDC25C and cyclin B1 in AGS cells was decreased in a menadione dose-dependent manner. In the time course experiment, the protein level of CDC25C decreased in 6 h, and CDK1and cyclin B1 protein levels began to decrease after 18 h of menadione treatment. We found that mRNA level of CDC25C decreased by menadione treatment in 6 h. Menadione did not have an influence on mRNA level of CDK1 and cyclin B1 though the protein levels were decreased. However, the decreased protein levels of CDK1 and cyclin B1 were recovered by inhibition of proteasome. Collectively, these results suggest that menadione inhibits growth of gastric cancer cells by reducing expression of CDC25C and promoting proteasome mediated degradation of CDK1 and cyclin B1 thereby blocking transition of the cell cycle from G2 phase to M phase.

Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes.

Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45 degrees C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half-life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 degrees C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.

Regulatory effects of 5'-deoxypyridoxal on glutamate dehydrogenase activity and insulin secretion in pancreatic islets.

It has been known that glutamate, generated by glutamate dehydrogenase (GDH), acts as an intracellular messenger in insulin exocytosis in pancreatic beta cells. Here we demonstrate the correlation of GDH activity and insulin release in rat pancreatic islets perfused with 5'-deoxypyridoxal. Perfusion of islets with 5'-deoxypyridoxal, an effective inhibitor of GDH, reduced the islet GDH activity at concentration-dependent manner. Treatment of 5'-deoxypyridoxal up to 2 mM did not affect the cell viability. There was reduction in V(max) values on average about 60%, whereas no changes in K(m) values for substrates and coenzymes were observed. The concentration of GDH on the Western blot analysis and the level of GDH mRNA remained unchanged. The concentration of glutamate decreased by 52%, whereas the concentration of 2-oxoglutarate increased up to 2.3-fold in the presence of 5'-deoxypyridoxal. 5'-Deoxypyridoxal had no effects on inhibition by GTP and activation by ADP or L-leucine of islet GDH. In parallel with the inhibition of GDH activity, perfusion of islets with 5'-deoxypyridoxal reduced insulin release up to 2.5-fold. Although precise mechanism for correlation between GDH activity and insulin release remains to be studied further, our results suggest a possibility that the inhibitory effect of 5'-deoxypyridoxal on islet GDH activity may correlate with its effect on insulin release.

TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells.

Human glutamate dehydrogenase (GDH) gene was fused with a gene fragment encoding the nine amino acid (RKKRRQRRR) protein transduction domain of human immunodeficiency virus TAT protein in bacterial expression vector to produce genetic in-frame TAT-GDH fusion protein. The TAT-GDH protein can enter PC12 cells efficiently when added exogenously in culture media as determined by Western blot analysis and enzyme activities. Once inside the cells, the transduced denatured TAT-GDH protein showed a full activity of GDH indicating that the TAT-GDH fusion protein was correctly refolded after delivery into cells and the activities of GDH in the TAT-GDH fusion protein was not affected by the addition of the TAT sequence. TAT-GDH fusion protein and TAT itself showed no cytotoxicity in PC12 cells. Although the exact mechanism of transduction across a membrane remains unclear, the transduction activity of TAT-GDH into PC12 cells may suggest new possibilities for direct delivery of GDH into the patients with the GDH-deficient disorders.

Cobalt chloride-induced apoptosis and extracellular signal-regulated protein kinase activation in human cervical cancer HeLa cells.

The molecular mechanism of hypoxia-induced apoptosis has not been clearly elucidated. In this study, we investigated the involvement of extracellular signal-regulated protein kinase (ERK 1/2) in hypoxia-induced apoptosis using cobalt chloride in HeLa human cervical cancer cells. The cobalt chloride was used for the induction of hypoxia, and its IC(50) was 471.4 microM. We demonstrated the DNA fragmentation after incubation with concentrations more than 50 microM cobalt chloride for 24 h, and also evidenced the morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signaling pathway of cobalt chloride-induced apoptosis in HeLa cells. ERK1/2 activation occurred 6 and 9 h after treatment with 600 microM cobalt chloride. Meanwhile, the pretreatment of the MEK 1 inhibitor (PD98059) completely blocked the cobalt chloride-induced ERK 1/2 activation. At the same time, the activated ERK 1/2 translocated into the nucleus and phosphorylated its transcriptional factor, c-Jun. In addition, the pretreatment of PD98059 inhibited the cobalt chloride-induced DNA fragmentation and apoptotic cell death. These results suggest that cobalt chloride is able to induce apoptotic activity in HeLa cells, and its apoptotic mechanism may be associated with signal transduction via ERK 1/2.

Lobarstin enhances chemosensitivity in human glioblastoma T98G cells.

BACKGROUND/AIM: Lobarstin is a metabolite occurring from the Antarctic lichen Stereocaulon alpnum. Human glioblastoma is highly resistant to chemotherapy with temozolomide. Lobarstin was examined for its effect on glioblastoma. MATERIALS AND METHODS: Temozolomide-resistant T98G cells were subjected to toxicity test with temozolomide and/or lobarstin. DNA damage and recovery was assessed by the alkaline comet assay and expression of DNA repair genes was examined by RT-PCR and western blot analysis. RESULTS: Lobarstin alone at 40 µM was toxic against T98G, but had no effect in primary human fibroblasts. Co-treatment of lobarstin with temozolomide yielded enhanced toxicity. Temozolomide-alone or with lobarstin co-treatment gave similar extent of DNA damage. However, the recovery was reduced in co-treated cells. Expression of DNA repair genes, O(6)-methylguanine-DNA methyltransferase, poly(ADP-ribose) polymerase 1 and ligase 3 were reduced in lobarstin-treated cells. CONCLUSION: Enhanced sensitivity to temozolomide by lobarstin co-treatment may be attributed to reduced DNA repair.

Parkin induces apoptotic cell death in TNF-α-treated cervical cancer cells.

Many malignant tumors become resistant to tumor necrosis factor-alpha (TNF-α)-induced cell death during carcinogenesis. In the present study, we examined whether parkin acts as a tumor suppressor in HeLa cells, a human cervical cancer cell line resistant to TNF-α-induced cell death. TNF-α-treatment alone did not affect HeLa cell viability. However, expression of parkin restored TNF-α-induced apoptosis in HeLa cells. Increased cell death was due to the activation of the apoptotic pathway. Expression of parkin in TNF-α-treated HeLa cells stimulated cleavage of the pro-apoptotic proteins caspase-8, -9, -3, -7 and poly ADP ribose polymerase (PARP). In addition, parkin expression resulted in decreased expression of the caspase inhibitory protein, survivin. These results suggest that parkin acts as a tumor suppressor in human cervical cancer cells by modulating survivin expression and caspase activity. We propose that this pathway is a novel molecular mechanism by which parkin functions as a tumor suppressor.

Suppression of lipopolysaccharide-induced microglial activation by a benzothiazole derivative.

We previously reported that KHG21834, a benzothiazole derivative, attenuates the beta-amyloid (Abeta)-induced degeneration of both cortical and mesencephalic neurons in vitro. Central nervous system inflammation mediated by activated microglia is a key event in the development of neurodegenerative disease. In this study, we show that KHG21834 suppresses inflammation-mediated cytokine upregulation. Specifically, KHG21834 induces significant reductions in the lipopolysaccharide-induced activation of microglia and production of proinflammatory mediators such as tumor necrosis factor-alpha, interlukin-1beta, nitric oxide, and inducible nitric oxide synthase. In addition, KHG21834 blocks the expression of mitogen-activated protein kinases, including ERK, p38 MAPK, JNK, and Akt. In vivo intracerebroventricular infusion of KHG21834 also leads to decreases the level of interleukin-1beta and tumor necrosis factor-alpha in brain. These results, in combination with our previous findings on Abeta-induced degeneration, support the potential therapeutic efficacy of KHG21834 for the treatment of neurodegenerative disorders via the targeting of key glial activation pathways.

Attenuation of beta-amyloid-induced neuroinflammation by KHG21834 in vivo.

Beta-Amyloid (ABeta)-induced neuroinflammation is one of the key events in the development of neurodegenerative disease. We previously reported that KHG21834, a benzothiazole derivative, attenuates ABeta-induced degeneration of cortical and mesencephalic neurons in vitro. In the present work, we show that KHG21834 reduces ABeta-mediated neuroinflammation in brain. In vivo intracerebroventricular infusion of KHG21834 leads to decreases in the numbers of activated astrocytes and microglia and level of proinflammatory cytokines such as interleukin- 1Beta and tumor necrosis factor-alpha induced by ABeta in the hippocampus. This suppression of neuroinflammation is associated with decreased neuron loss, restoration of synaptic dysfunction biomarkers in the hippocampus to control level, and diminished amyloid deposition. These results may suggest the potential therapeutic efficacy of KHG21834 for the treatment of ABeta-mediated neuroinflammation.