Synthesis, biological evaluation, molecular docking studies and In-silico ADMET evaluation of pyrazines of pentacyclic triterpenes.

The semisynthesis of novel derivatives of lupeyl palmitate and 3ß-palmitoyloxy-olean-12-ene by introduction of a pyrazine at C-2 / C-3 and modifications of the relatively unexplored C-30 position of lupeol derivatives was conducted, and their cytotoxic and anti-inflammatory activities were evaluated. The derivatives 7, 10 and 11 significantly inhibited the tumor cell lines U251, K562, HCT-15, MCF-7 and SKLU-1, and compounds 7 and 11 were more active (IC50 25.4 ± 2.0 µM and 7.1 ± 0.4 µM, respectively) than the positive control (etoposide (IC50 31.5 ± 2.2 µM) in the tumor line PC-3. Introduction of the pyrazine at C-2 / C-3 in compounds 1 and 2 or modification at C-30 of compound 1 decreased the anti-inflammatory activity in the TPA-induced mouse ear edema. Following the results of the PASS online evaluation of the potential biological activity of the natural compounds and their derivatives, the inhibition of pNF-κB translocation to the prostate cancer (PC-3) cell nucleus was investigated and the binding mode of compounds 7, 10 and 11 with the human NF-κB receptor was explored by a molecular docking study. These derivatives bound directly or close to the amino acids that form the DNA recognition site. The ADMET physicochemical parameters of the fifteen compounds were further analyzed in silico using Molinspiration calculations indicating the potential of compounds 7, 10 and 11 for further investigation.

Synthesis and Biological Evaluation of 2H-Indazole Derivatives: Towards Antimicrobial and Anti-Inflammatory Dual Agents.

Indazole is considered a very important scaffold in medicinal chemistry. It is commonly found in compounds with diverse biological activities, e.g., antimicrobial and anti-inflammatory agents. Considering that infectious diseases are associated to an inflammatory response, we designed a set of 2H-indazole derivatives by hybridization of cyclic systems commonly found in antimicrobial and anti-inflammatory compounds. The derivatives were synthesized and tested against selected intestinal and vaginal pathogens, including the protozoa Giardia intestinalis, Entamoeba histolytica, and Trichomonas vaginalis; the bacteria Escherichia coli and Salmonella enterica serovar Typhi; and the yeasts Candida albicans and Candida glabrata. Biological evaluations revealed that synthesized compounds have antiprotozoal activity and, in most cases, are more potent than the reference drug metronidazole, e.g., compound 18 is 12.8 times more active than metronidazole against G. intestinalis. Furthermore, two 2,3-diphenyl-2H-indazole derivatives (18 and 23) showed in vitro growth inhibition against Candida albicans and Candida glabrata. In addition to their antimicrobial activity, the anti-inflammatory potential for selected compounds was evaluated in silico and in vitro against human cyclooxygenase-2 (COX-2). The results showed that compounds 18, 21, 23, and 26 display in vitro inhibitory activity against COX-2, whereas docking calculations suggest a similar binding mode as compared to rofecoxib, the crystallographic reference.

Proteomic Profiling Reveals the Induction of UPR in Addition to DNA Damage Response in HeLa Cells Treated With the Thiazolo[5,4-b]Quinoline Derivative D3ClP.

9-[(3-chloro)phenylamine]-2-[3-(diethylamine)propylamine]thiazolo[5,4-b]quinolone (D3ClP) is a bioisostere of N-(4-(acridin-9-ylamino)-3-methoxyphenyl)methanesulfonamide (m-AMSA) a DNA topoisomerase II inhibitor with proven cytotoxic activity and known to induce DNA damage and apoptotic cell death in K562 cells. However, recent evidence is not consistent with DNA topoisomerase II (DNA TOP2) as the primary target of D3ClP, in contrast to m-AMSA. We provide evidence of histone γH2AX phosphorylation at Ser135 in HeLa cells treated with D3ClP, a marker of DNA double strand repair through Mre11-Rad50-Nbs1 (MRN) pathway. Using two-dimensional gel electrophoresis and mass spectrometry, the upregulation of the protein GRP78, the cleavage of Cytokeratin 18, and the downregulation of prothymosine, calumenin, and the α chain of the nascent polypeptide associated complex were observed in HeLa cells treated with D3ClP. An increase in GRP78 has been related with the onset and progression of the unfolded protein response (UPR), a process aimed to reduce endoplasmic reticulum (ER) stress and protein misfolding. The IRE1-α dependent splicing of mRNA encoding X-box binding protein 1 was detected. Microtubule-associated Proteins 1A/1B, Light Chain 3-II (LC3b-II) accumulation was observed, and suggest some involvement of autophagy. The production of the pro-apoptotic protein DNA-damage-inducible protein 153 (GADD-153) was also detected. These results, are consistent with the induction of the UPR and the DNA-Damage Response in D3ClP-treated HeLa cells, and are also consistent with a concurrent apoptotic cell death. J. Cell. Biochem. 118: 1164-1173, 2017. © 2016 Wiley Periodicals, Inc.

Epigenetic alterations caused by nutritional stress during fetal programming of the endocrine pancreas.

Nutrition during critical periods of development is one of the pivotal factors in establishing a lifelong healthy metabolism. Different nutritional deficiencies such as a low availability of proteins in the maternal diet produce alterations in offspring that include changes in insulin and glucose metabolism, a decrease in the size and number of cells of pancreatic islets of Langerhans, and premature ageing of the secretory function of pancreatic ß cells. Moreover, it has been reported that chronic nutritional stress is associated with epigenetic alterations in mechanisms of gene regulation during pancreatic development and function. These alterations can lead to dysfunctional states in pancreatic ß cells, which in the long run are responsible for the onset of metabolic diseases like type 2 diabetes. The present review summarizes the most important evidence in relation to the participation of epigenetic mechanisms in the regulation of gene expression during the intrauterine programming of the endocrine pancreas in animal models. Such mechanisms include DNA methylation as well as modifications of histones and microRNAs (miRNAs).

Rotenone isolated from Pachyrhizus erosus displays cytotoxicity and genotoxicity in K562 cells.

Pachyrhizus erosus (Fabaceae) is a herb commonly known as 'yam bean', which has been cultivated in México since pre-Columbian times for its edible tubers. The seeds are also known for their acaricidal and insecticidal properties due to rotenone and other isoflavonoid contents. Rotenone has exhibited cytotoxic activity against several human tumour cell lines; however, its mechanism of action is still not fully understood. In this study, we determined the cytotoxicity of rotenone isolated from P. erosus seeds on K562 human leukaemia cells. Rotenone exhibited significant cytotoxic activity (IC50 = 13.05 µM), as determined by the MTT assay. Three other isolated isoflavonoids were not cytotoxic. Rotenone genotoxicity was detected using the comet assay. Rotenone induced cell death, and caspase-3 activation as indicated by TUNEL assay, and immunocytofluorescence. Plasmid nicking assay indicated that rotenone does not interact directly with DNA.

Molecular mechanisms involved in the cytotoxicity induced by coumarins from Calophyllum brasiliense in K562 leukaemia cells.

OBJECTIVES: The aim of this study was to determine the cellular and molecular mechanisms of cell death induced by mammea A/BA and A/BB (3 : 1) on K562 cells. METHODS: These compounds were isolated from Calophyllum brasiliense and its cytotoxicity was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell death was evaluated by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunocytofluorescence of active caspase-3. Genotoxicity was tested using comet assay. Lastly, a chemoinformatic analysis was performed with Osiris-Molinspiration software. KEY FINDINGS: The mixture of mammea A/BA and A/BB (3 : 1) showed cytotoxic activity against K562 cells (IC50 = 43.5 µm). TUNEL positive cells and active caspase-3 were detected after treatment. Genotoxicity of mammea A/BA and A/BB on K562 was detected since first hour of treatment. Additionally, mammea A/BA and A/BB were found to be in compliance with Lipinski 'rule of 5' suggesting that they possess strong potential of druglikeness. CONCLUSIONS: The overall results confirm and extend the knowledge about coumarins as an important resource of antitumor drugs, and indicate that these compounds could be used in further preclinical studies against leukaemia.

Magnolia dealbata seeds extract exert cytotoxic and chemopreventive effects on MDA-MB231 breast cancer cells.

CONTEXT: Cancer prevention remains a high priority for the scientific world. Magnolia dealbata Zucc (Magnoliaceae), a Mexican endemic species, is used for the empirical treatment of cancer. OBJECTIVE: To evaluate the cytotoxic and cancer chemopreventive effects of an ethanol extract of Magnolia dealbata seeds (MDE). MATERIALS AND METHODS: The cytotoxic effect of MDE, at concentrations ranging from 1 to 200 µg/ml, on human cancer cells and human nontumorigenic cells was evaluated using the MTT assay for 48 h. The apoptotic activities of MDE 25 µg/ml on MDA-MB231 breast cancer cells were evaluated using the TUNEL assay and the detection of caspase 3 using immunofluorescence analysis for 48 h, each. The chemopreventive effect was evaluated by administrating different doses of MDE, between 1 and 50 mg/kg, injected intraperitoneally daily into athymic mice which were implanted with MDA-MB231 cells during 28 days. The growth and weight of tumors were measured. RESULTS: MDE showed cytotoxic effects on MDA-MB231 cells (IC50 = 25 µg/ml) and exerted pro-apoptotic activities as determined by DNA fragmentation in MDA-MB231 cells. MDE 25 µg/ml also induces the activation of caspase 3 in MDA-MB231 cells. These results suggest that Magnolia dealbata may be an optimal source of the bioactive compounds: honokiol (HK) and magnolol (MG). MDE 50 mg/kg i.p. exerted chemopreventive effects by inhibiting the growth of MDA-MB231 tumor by 75% in athymic mice, compared to the control group. CONCLUSIONS: MDE exerts cytotoxic, apoptotic and chemopreventive activities on MDA-MB231 human cancer cells.

Differential DNA methylation pattern in the A and B promoters of the progesterone receptor is associated with differential mRNA expression in the female rat hypothalamus during proestrus.

In rodents, the display of reproductive behavior occurs during the proestrus-estrus transition of the estrus cycle. This behavior is regulated by estradiol and progesterone mainly via their intracellular receptors. Two isoforms of the progesterone receptor have been described (A and B), and they have different promoters for their regulation. It has been demonstrated that the mRNA for both isoforms changes during the proestrus-estrus transition. It has been recently established that DNA methylation can be transient and cyclical in gene promoters, however, these changes have only been reported in vitro but not in physiological models. The aim of this study was to analyze the pattern of DNA methylation in the PR (A and B) promoter regions during the proestrus-estrus transition in the rat hypothalamus and its correlation with the regulation of mRNA expression. The results demonstrated a differential mRNA expression of the progesterone receptor (A and B) isoforms. The expression of total PR did not change significantly during the proestrus day, while the expression of isoform B increased significantly at 17:00 h, followed by a significant decrease at 21:00 h of the proestrus day. Interestingly, we also found that the isoform A promoter was mainly unmethylated at all studied time points. In contrast, the isoform B promoter showed a transient methylation increase during the evening of proestrus. The overall results indicate that there is a switch of progesterone receptor isoforms expression during the evening of proestrus that is related to the differential gene methylation patterns of their promoter regions, mainly for the isoform B promoter.

In vitro drug metabolism testing using blood-monocyte derivatives.

INTRODUCTION: Monocytes and their cell derivatives can participate in drug metabolism. These cells express different Phase-I or -II drug metabolizing enzymes and can be differentiated into neo-hepatocytes (NeoHep) and represent a promising alternative strategy to test drug metabolism. This is particularly useful as primary human hepatocytes (PHH), are difficult to obtain and maintain in culture. AREAS COVERED: The authors analyze the use of blood monocytes and their derivatives for the study of drug metabolism. They also compare them to the in vitro ability of cells from different sources including: PHH, immortalized hepatocytes, tumor cell lines and NeoHep. EXPERT OPINION: The use of monocytes, macrophages, dendritic or Kupffer cells, to test drug metabolism, has serious limitations because these cells express lower levels of cytochrome P450 enzymes than PHH. The best available option, to replace PHH, have been tumor cell lines such as HepaRG, as well as immortalized hepatocytes from adult or fetal sources. Monocyte-derived NeoHep cells are novel and easily accessible cells, which express many drug metabolizing enzymes at levels comparable to PHH. These cells allow drug evaluation under a diverse genetic background. While these cells are in the early stages of evaluation and do need to be examined more thoroughly, they constitute a promising new tool for in vitro drug testing.

The products of the reaction between 6-amine-1,3-dimethyl uracil and bis-chalcones induce cytotoxicity with massive vacuolation in HeLa cervical cancer cell line.

As a part of our research in the chemistry of chalcones we have prepared four pyrimidine monoadducts of bis-chalcones through the reaction with 6-amino-1,3-dimethyl uracil. These compounds displayed cytotoxicity with a massive vacuolation in different human cell lines in vitro. Compound 6 was the most cytotoxic inducer of vacuoles, this compound induced G1 phase cell cycle arrest, and their cytotoxicity went without morphological and biochemical evidence of apoptotic cell death in HeLa cells. In addition, our results showed that this vacuole formation does not require de novo protein synthesis and the content vacuolar is acidic. Compound 6 induce necrotic cell death with excessive vacuolation, similar to a process of autophagy. Spautin-1 an inhibitor of autophagy, decreased the transformation of microtubule-associated protein 1 light chain 3 (LC3B-I) to LC3B-II and the vacuolation induced by compound 6 in HeLa cells, both autophagy processes. These compounds could be of pivotal importance in the study of non-apoptotic cell death with vacuole formation and could be useful in research into new autophagy inhibitors agents.

Kaempferitrin induces apoptosis via intrinsic pathway in HeLa cells and exerts antitumor effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Justicia spicigera is used for the empirical treatment of cervical cancer in Mexico. Recently, we showed that Justicia spicigera extracts exerted cytotoxic and antitumoral effects and the major component of this extract was kaempferitrin (KM). MATERIALS AND METHODS: The cytotoxic and apoptotic effect of KM on human cancer cells and human nontumorigenic cells were evaluated using MTT and TUNEL assays, and Annexin V/Propidium iodide detection by flow cytometry. The effect of KM on cell cycle was analyzed by flow cytometry with propidium iodide. The apoptotic and cell cycle effects were also evaluated by western blot analysis. Also, different doses of KM were injected intraperitoneally daily into athymic mice bearing tumors of HeLa cells during 32 days. The growth and weight of tumors were measured. RESULTS: KM induces high cytotoxic effects in vitro and in vivo against HeLa cells. The general mechanisms by which KM induces cytotoxic effects include: cell cycle arrest in G1 phase and apoptosis via intrinsic pathway in a caspase dependent pathway. Also, KM exerts chemopreventive and antitumor effects. CONCLUSION: KM exerts cytotoxic and antitumor effects against HeLa cells.

Synergistic anticancer activity of Thiazolo[5,4-b]quinoline derivative D3CLP in combination with cisplatin in human cervical cancer cells.

BACKGROUND: D3CLP (9-[(3-chloro)phenylamine]-2-[3-(diethylamine)propylamine]thiazolo[5,4-b]quinoline) is a potent cytotoxic thiazolo[5,4-b]quinoline synthetic derivative that induces apoptosis of leukemia cells, while it displays low toxicity towards non-tumoral cells. The aim of this study was to determine if D3CLP can enhance the cytotoxicity of other antineoplastic drugs. MATERIALS AND METHODS: Leukemia, breast and cervical cancer cell lines were exposed to D3CLP-alone or in combination with imatinib, tamoxifen or cisplatin, respectively. Cell viability after treatment was evaluated by the MTT assay, and cell death by the TUNEL assay. The effects of combined treatments were analyzed by combination index and isobolographic analysis. RESULTS: Antiproliferative activity results indicate that D3CLP in combination with antineoplastic drugs induced a synergistic effect, at 3:1 and 1:1 ratios for D3CLP plus imatinib in K-562 leukemia cells, and at a 3:1 ratio for D3CLP with cisplatin in HeLa cells, as determined by their combination index. Furthermore, isobolographic analysis demonstrated a significant synergism for a 3:1 combination ratio of D3CLP with cisplatin in HeLa cells. In addition, TUNEL assay suggests cell death by apoptosis of HeLa cells after treatment with D3CLP and its combination with cisplatin at a 3:1 ratio. CONCLUSION: Overall the results indicate that D3CLP, in combined preparation with antineoplastic drugs, is a good candidate for pre-clinical studies in the treatment of different carcinoma cell types.

The antitumoral effect of the American mistletoe Phoradendron serotinum (Raf.) M.C. Johnst. (Viscaceae) is associated with the release of immunity-related cytokines.

ETHNOPHARMACOLOGICAL RELEVANCE: Phoradendron serotinum is commonly used in Mexican traditional medicine for the empirical treatment of cancer. However, there are no studies regarding the antitumoral or immunomodulatory activities of Phoradendron serotinum. MATERIALS AND METHODS: The in vivo toxicity of ethanolic extracts of Phoradendron serotinum (PSE) was evaluated in mice according to the Lorke procedure. The in vitro immunomodulatory effects of PSE were evaluated estimating the effects of PSE on the pinocytosis, NO production and lysosomal enzyme activity in murine macrophages RAW 264.7. The effects of PSE on the proliferation of murine splenocytes and NK cell activity were also assayed. The cytotoxic effects on TC-1 (lung murine cancer cells) were evaluated using the MTT assay, whereas the apoptotic effect of PSE on TC-1 cells was evaluated using TUNEL assay. Also, different doses of PSE were injected intraperitoneally daily into C57BL/6 mice bearing tumors of TC-1 cells during 25 days. The growth and weight of tumors was measured. In addition, the levels of IL-2, IL-6, IL-12, IL-23 and IFN-γ in murine serum and supernatants of K562 cell-murine splenocyte cocultures were measured. RESULTS: PSE stimulated the proliferation, pinocytosis and lysosomal enzyme activity in murine macrophages with a similar potency than lypopolisaccharides 1 µg/ml. In addition, PSE stimulated the proliferation of murine splenocytes and induced the NK cell activity. PSE showed cytotoxic (IC(50)=1.9 µg/ml) and apoptotic effects against TC-1 cells. The LD(50) was 125 mg/kg by intraperitoneal route (i.p.) and 375 mg/kg by oral route. PSE administrated at 1, 5 and 10 mg/kg i.p. inhibited the tumor growth by 18%, 40% and 69%, respectively, in mice bearing TC-1 tumor. PSE increased the in vitro and in vivo release of IL-2, IL-6 and IFN-γ but lacked effect on IL-12 and IL-23 release. CONCLUSIONS: Phoradendron serotinum shows moderate toxic effects in vivo, exerts cytotoxic and apoptotic effects on TC-1 cells. Phoradendron serotinum also has antitumor effects in mice bearing TC-1 tumor and induces immunomodulatory activities in vivo. The results suggest that antitumoral effects of PSE are related with the production of immunity-related cytokines.

Polymorphisms in the hypoxia-inducible factor 1 alpha gene in Mexican patients with preeclampsia: A case-control study.

BACKGROUND: Although the etiology of preeclampsia is still unclear, recent work suggests that changes in circulating angiogenic factors play a key role in its pathogenesis. In the trophoblast of women with preeclampsia, hypoxia-inducible factor 1 alpha (HIF-1α) is over-expressed, and induces the expression of non-angiogenic factors and inhibitors of trophoblast differentiation. This observation prompted the study of HIF-1α and its relation to preeclampsia. It has been described that the C1772T (P582S) and G1790A (A588T) polymorphisms of the HIF1A gene have significantly greater transcriptional activity, correlated with an increased expression of their proteins, than the wild-type sequence. In this work, we studied whether either or both HIF1A variants contribute to preeclampsia susceptibility. RESULTS: Genomic DNA was isolated from 150 preeclamptic and 105 healthy pregnant women. Exon 12 of the HIF1A gene was amplified by PCR, and the genotypes of HIF1A were determined by DNA sequencing.In preeclamptic women and controls, the frequencies of the T allele for C1772T were 4.3 vs. 4.8%, and the frequencies of the A allele for G1790A were 0.0 vs. 0.5%, respectively. No significant differences were found between groups. CONCLUSION: The frequency of the C1772T and G1790A polymorphisms of the HIF1A gene is very low, and neither polymorphism is associated with the development of preeclampsia in the Mexican population.

Antineoplastic activity of the thiazolo[5,4-b]quinoline derivative D3CLP in K-562 cells is mediated through effector caspases activation.

Thiazolo[5,4-b]quinolines are compounds structurally related to m-Amsacrine (m-Amsa), a potent antileukemic drug that intercalates to DNA and inhibits topoisomerase II in vitro inducing cell death. The clinical use of m-Amsa and other neoplastic drugs is limited due to side effects and drug resistance. In the present study we evaluated one thiazolo[5,4-b]quinoline derivate, 9-[(3-chloro)phenylamine]-2-[3-(diethylamine)propylamine]thiazolo[5,4-b]quinoline (D3CLP), considered isosteric with 9-anilinoacridines, in order to determine its relative cytotoxic activity in tumoral versus non-tumoral cells, as well as the cell death mechanism induced by D3CLP on K-562 human leukemia cells. D3CLP was found to be four times more cytotoxic to tumor cells than Peripheral Blood Monocyte Cells (PBMCs). On the other hand, D3CLP induces cell death without previous cell cycle arrest at any phase, as shown by flow cytometry after 12 h of exposure to this compound. Interestingly, we detected a subdiploid peak 24 h after treatment. Signs of apoptosis were evident, as detected by TUNEL positive cells, chromatin condensation and nuclear fragmentation. Effector caspases activation were assessed with peak activity at 24 h after treatment (as detected by fluorometry assays), at which time a subdiploid peak was found in flow cytometry histograms. All data are consistent with the induction of apoptotic cell death in K-562 cells via effector caspases activation. In conclusion, the significant cytotoxicity of D3CLP together with the cell death type it produces, justifies further experimental and preclinical evaluation of this compound in the effort to find new and highly specific anti-tumor agents against leukemia cells.

Effects of mating on progesterone receptor isoforms in rat hypothalamus.

In rodents, the display of sexual behavior during proestrus-estrus transition depends on the effect of estradiol and progesterone. Progesterone exerts its effects through intracellular receptor (PR) of which two isoforms (PR-A and PR-B) are found, with different regulation and function. In this study the effects of mating on the expression pattern of PR isoforms in the hypothalamus were investigated during proestrus-estrus transition by using western blot. PR-B isoform content significantly diminished during proestrus-estrus transition both in mated and nonmated female rats. In contrast, PR-A isoform content significantly increases during this period in nonmated rats, whereas it does not change in mated animals. These data show that PR isoforms are differentially expressed throughout proestrus-estrus transition and that mating modifies PR isoforms expression in the hypothalamus of the rat.

The protective effect of testosterone on streptozotocin-induced apoptosis in beta cells is sex specific.

OBJECTIVES: To investigate the protective role of steroid hormones on streptozotocin (STZ)-induced apoptosis in rat pancreatic beta cells. METHODS: Two sets of experiments were performed. In the first, male rats were orchidectomized and substituted 72 hours later with testosterone, estradiol, or progesterone, and 24 hours later, administered with STZ. Subjects were killed 6 hours later, and apoptosis was determined in sections of the pancreas. In the second experiment, male or female rats were gonadectomized, were further substituted with testosterone, and then administered STZ. Six hours later, the animals were killed, and apoptosis, as well as immunoreactive expression of insulin, catalase, or Cu/Zn superoxide dismutase, was determined in sections of the pancreas. In addition, gonadectomized male or female subjects were substituted with testosterone and administered STZ, and 24 hours later, serum glucose and insulin were measured. RESULTS: It was found that the cytoprotective effect was only shown in testosterone-treated male rats but not progesterone- or estradiol-treated male rats. In addition, the effect was seen in male rats but not in female rats, and there was an inverse correlation between apoptotic index and antioxidant enzyme immunoreactivity. CONCLUSIONS: The cytoprotective effect of testosterone is sex specific and is related to the induction of antioxidant enzyme activities in pancreatic beta cells.

Coumarin A/AA induces apoptosis-like cell death in HeLa cells mediated by the release of apoptosis-inducing factor.

It has been demonstrated that naturally occurring coumarins have strong biological activity against many cancer cell lines. In this study, we assessed the cytotoxicity induced by the naturally isolated coumarin A/AA in different cancer cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral-blood mononuclear cells (PBMCs). Cytotoxicity was evaluated using the MTT assay. The results demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was further studied in this cell line. DNA fragmentation, histomorphology, cell cycle phases, and subcellular distribution of PCD proteins were assessed. The results demonstrated that DNA fragmentation, but not significant cell cycle disruptions, was part of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis-inducing factor (AIF), a proapoptotic protein of the mitochondrial intermembrane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in subcellular fractions. Nevertheless, the active form of caspase-3 was not detected. The overall results indicate that coumarin A/AA induces a caspase-independent apoptotic-like cell death program in HeLa cells, mediated by the early release of AIF and suggest that this compound may be helpful in clinical oncology.

Presence of Helicobacter pylori in a Mexican Pre-Columbian Mummy.

BACKGROUND: Recent studies showed that Helicobacter pylori existed in the New World prior to the arrival of Columbus. The purpose of the present study was to detect the presence of Helicobacter pylori in pre-Columbian mummies from Northern Mexico. METHODS: Six samples were studied (four samples of gastric remains, tongue-soft palate, and brain remained as negative controls) from two of the six naturally mummified corpses studied (adult male and infant male). Samples were taken from tissues suitable for DNA amplification by Polymerase chain reaction (PCR). DNA was extracted and H. pylori detection was carried out by PCR and hybridized with the pHp probe from 16S rRNA gene. The purified PCR products were cloned and sequenced in both directions. DNA sequences were analyzed with ALIGN and BLAST software. A second amplification was performed using ureB gene by real-time PCR. RESULTS: From four samples of gastric remnant, only two were H. pylori-positive for amplification of a 109 bp DNA fragment; the remaining two were negative, as were the tongue-soft palate and the brain biopsies as well. These PCR products were hybridized with a pHp probe. Nucleotide sequence analysis showed homology with H. pylori in 98 of 99% when compared with the gene bank nucleotide sequence. Only one sample of gastric remnant H. pylori-positive with 16S rRNA gene was also positive for ureB gene from H. pylori. CONCLUSION: This data supported infection with H. pylori in Mexican pre-Columbian mummies dating from approximately 1,350 AC.

Modified progesterone receptor expression in the hypothalamus of cysticercotic male mice.

Progesterone participates in numerous developmental and behavioral processes in the mammalian brain. The intracellular (is this really intracellular? nuclear membrane?) progesterone receptor is expressed as two isoforms: a full-length form (PR-B) and the N-terminally truncated one (PR-A). Experimental intraperitoneal infections with Taenia crassiceps in mice exhibit the tendency of the parasites to develop more rapidly in females. Male mice undergo a feminization process characterized by their oestrogenisation, deandrogenisation and loss of sexual and aggressive patterns of behavior. Hence, we suspected that changes in PR expression in the brain could be involved in the feminization of the infected male mice and in the loss of the sexual and aggressive behaviors. We have studied the expression of PR isoforms in the normal and infected male mouse brain. Transcripts of both receptor isoforms (PR-A and -B) were readily detectable in normal and infected mice, but differentially regulated during infection depending on the area of the brain studied. Although the precise role of progesterone in mediating the behavioral changes noted during infection is not fully understood, our data implicate a role for PR signaling in the feminization process. CNS activity is potentially involved in the network that regulates the oestrogenisation and deandrogenisation observed in chronically infected male mice, as well as in the behavioral peculiarities observed in this parasitic infection.