Cross-talk between regulators of myeloid development: C/EBPalpha binds and activates the promoter of the PU.1 gene

CCAAT/enhancer-binding protein (C/EBP)alpha and PU.1 are required for myelopoiesis. Examination of the murine PU.1 promoter revealed several potential C/EBP-binding sites. Gel-shift assay demonstrated that C/EBPalpha expressed in 293T cells bound the site centered at -68 most potently. C/EBPalpha from 32D cl3 myeloid cell nuclear extracts also bound this site strongly, and endogenous C/EBPbeta did so to a lesser extent, whereas these C/EBP isoforms bound the neutrophil elastase promoter with equal affinity. The -68 site in the murine PU.1 promoter is conserved in the human PU.1 promoter. Mutation of the -68 C/EBP-binding site in a -85/+152 promoter segment linked to the luciferase cDNA reduced promoter activity fourfold in 293T cells in the presence of cotransfected C/EBPalpha and twofold in 32D cl3 myeloid cells. Induction of endogenous PU.1 RNA by C/EBPalpha-estradiol receptor (ER) in the presence of cycloheximide is obviated by mutation of the C/EBPalpha DNA-binding domain, and chromosomal immunoprecipitation demonstrated specific interaction of C/EBPalpha and C/EBPalpha-ER with the PU.1 promoter. Finally, PU.1 RNA is reduced several-fold in immortalized C/EBPalpha (-/-) compared with (+/-) cells. Together, these findings indicate that C/EBPalpha binds and activates the endogenous PU.1 gene in myeloid cells. Induction of PU.1 by C/EBPalpha may account for increased levels of PU.1 in myeloid as compared with B lymphoid cells and in this way, may contribute to the specification of myeloid progenitors.     

Maternal neutralizing antibodies against a CRF01_AE primary isolate are associated with a low rate of intrapartum HIV-1 transmission

Mother-to-child transmission (MTCT) of HIV-1 provides a model for studying the role of passively acquired antibodies in preventing HIV infection. We determined the titers of neutralizing antibodies (NAbs) against six primary isolates of clades B and CRF01_AE in sera from 45 transmitting and 45 nontransmitting mothers matched for the main independent factors associated with MTCT in Thailand. A lower risk of MTCT, particularly for intrapartum transmission, was associated only with higher NAb titers against the CRF01_AE strain, MBA. The envelope glycoprotein of this strain showed an unusually long V2 domain of 63 amino acids, encoding six potential N-linked glycosylation sites. We provided experimental data indicating that the extended V2 domain contributed to the higher level of resistance to neutralization by mothers' sera in this strain. Taken together the data suggest that some primary isolates with specific properties may be useful indicators for identifying protective antibodies.     

Comparison of Pouzolzia indica methanolic extract and Virkon against cysts of Acanthamoeba spp

The present study was conducted to investigate the morphological and structural changes of Acanthamoeba cysts after being treated with various concentrations of Pouzolzia indica methanolic extract fraction 3 (methanol eluted) and Virkon solution. Changes in the Acanthamoeba cysts were detected by light microscopy, scanning electron microscopy and transmission electron microscopy. The results show Acanthamoeba cysts were killed by Pouzolzia indica methanolic extract fraction 3 at a concentration of 1:8 and by Virkon solution at a concentration of 0.25%, with a minimal cysticidal concentration (MCC) by 24 hours. Both agents caused similar structural damage to Acanthamoeba cysts in the same sequence. Step by step structural alterations occurred within the cyst. First, the cyst shrank, collapsed and had clumping of cytoplasmic stuctures inside the cyst walls. Second, the cysts began to bulge, swell, have a decrease in wrinkles in the cyst walls and spill the cytoplasmic contents into the environment. Finally, the cyst walls broke into small pieces. This study may be beneficial to compare with future studies of pharmaceutical agents against Acanthamoeba keratitis.     

Regulation of granulocyte and monocyte differentiation by CCAAT/enhancer binding protein alpha

CCAAT/enhancer binding protein alpha (C/EBPalpha)-ER induces 32Dcl3 neutrophilic differentiation and inhibits 32DPKCdelta maturation to macrophages in response to phorbol ester. In 32Dcl3 cells, C/EBPalpha-ER rapidly induces the PU.1 and C/EBPalpha RNAs even in the presence of cycloheximide, suggesting that these are direct C/EBPalpha genetic targets. C/EBPalpha strongly binds and modestly activates the murine PU.1 promoter via an evolutionarily conserved binding site. C/EBPalpha-ER variants incapable of binding DNA still slow G1 progression but do not induce differentiation. N-terminally truncated C/EBPalpha variants, including the p30 isoform expressed in a subset of AMLs, also retain the ability to slow 32D cl3 proliferation, whereas the C/EBPalpha(BRM2)-ER variant does not slow G1 progression, has a reduced capacity to induce early granulocytic markers, and does not induce terminal maturation. In 32DPKCdelta cells, C/EBPalpha-ER strongly inhibits endogenous or exogenous JunB induction, dependent upon the outer surface of the C/EBPalpha basic region, but does not inhibit c-Jun, PU.1, or C/EBPbeta expression. Exogenous JunB restores AP-1 DNA binding but does not overcome inhibition of monopoiesis by C/EBPalpha-ER. In summary, we propose that while C/EBPalpha is required for development of immature granulocyte-monocyte progenitors, C/EBPalpha subsequently inhibits monopoiesis, via inhibition of JunB express and via additional activities, and induces granulopoiesis, via induction of PU.1, C/EBPepsilon, and cell cycle arrest.     

Antiproliferative effect of Erycibe elliptilimba on human breast cancer cell lines

Erycibe elliptilimba Merr. & Chun., family Convolvulaceae, is a Thai traditional medicine which has long been prescribed for various infectious and malignant diseases. Bio-assays of extracts from Erycibe elliptilimba Merr. & Chun. showed that a fraction (fraction 3) from an methanolic extract had an antiproliferative effect on SKBR3 and MDA-MB435 human breast cancer cells. The ED50 value of Erycibe elliptilimba Merr. & Chun. fraction 3 was 56.07 and 30.61 μg/ml for SKBR3 and MDA-MB435, respectively. After 48 h of exposure, this fraction at a concentration of 100 μg/ml significantly reduced cell proliferation in both cancer cells. In MDA-MB435 cells, cell cycle analysis showed that the herb extract fraction 3 induced the accumulation of cells in G2/M phase, whereas no significant change in cell cycle was detected in SKBR3 cells. The results indicated that the extract fraction 3 could induce cell cycle arrest in some way. However, further investigation is needed to assess the molecular mechanisms mediated anticancer activities of this plant.     

Characteristics of HIV type 1 (HIV-1) glycoprotein 120 env sequences in mother-infant pairs infected with HIV-1 subtype CRF01_AE

We analyzed the characteristics of the envelope genes of human immunodeficiency virus type 1 in 17 mother-infant pairs infected with variants of the CRF01_AE clade. A total of 353 sequences covering almost the entire glycoprotein (gp) 120 region were available for analysis. We found that, even if the virus population in the mother was complex, only viruses of a restricted subset were transmitted to her infant, independently of whether transmission occurred in utero or during the intrapartum period. We did not find that shorter gp120 regions or fewer potential N-glycosylation sites (PNGS) were characteristic of viruses transmitted from mother to infant. However, our data suggest that a limited number of PNGS that seem to be conserved in all variants in infants but are not uniformly present in variants in mothers may confer an advantage for transmission of the virus, thereby highlighting the potentially important role of the "glycan shield." This finding was particularly significant for the PNGS at positions N301 and N384.     

HIV-1 CRF01_AE coreceptor usage prediction using kernel methods based logistic model trees

The determination of HIV-1 coreceptor usage plays a major role in HIV treatment. Since Maraviroc has been used in a treatment for patients those exclusively harbor R5-tropic strains, the efficient performance of classifying HIV-1 coreceptor usage can help choose the most advantaged HIV treatment. In general, HIV-1 variants are classified as R5-tropic and X4-tropic or dual/mixed tropic based on their coreceptor usages. The classification of the coreceptor usage has been developed by using the various computational methods or genotypic algorithms based on V3 amino acid sequences. Most genotypic tools have been designed based on a data set of the HIV-1 subtype B that seemed to be reliable only for this subtype. However, the performance of these tools decreases in non-B subtypes. In this study, the support vector machine (SVM) method has been used to classify the HIV-1 coreceptor. To develop an efficient SVM classifier, we present a feature selector using the logistic model tree (LMT) method to select the most relevant positions from the V3 amino acid sequences. Our approach achieves as high as 97.8% accuracy, 97.7% specificity, and 97.9% sensitivity measured by ten-fold cross-validation on 273 sequences.     

Envelope glycoproteins of human immunodeficiency virus type 1 variants issued from mother-infant pairs display a wide spectrum of biological properties

Several studies have shown that the early virus population present in HIV-1 infected infants usually is homogeneous when compared to the highly diversified viral population present at delivery in their mothers. We explored the antigenic and functional properties of pseudotyped viruses expressing gp120 encoded by env clones issued from four mother-infant pairs infected by CRF01_AE viruses. We compared their sensitivity to neutralization and to entry inhibitors, their infectivity levels and the Env processing and incorporation levels. We found that both transmitted viruses present in infants and the variants present in their chronically infected mothers display a wide spectrum of biological properties that could not distinguish between them. In contrast, we found that all the transmitted viruses in the infants were more sensitive to neutralization by PG9 and PG16 than the maternal variants, an observation that may have implications for the development of prophylactic strategies to prevent mother-to-child transmission.     

Neuroprotective effects of melatonin on amphetamine‐induced dopaminergic fiber degeneration in the hippocampus of postnatal rats

Chronic amphetamine (AMPH) abuse leads to damage of the hippocampus, the brain area associated with learning and memory process. Previous results have shown that AMPH‐induced dopamine neurotransmitter release, reactive oxygen species formation, and degenerative protein aggregation lead to neuronal death. Melatonin, a powerful antioxidant, plays a role as a neuroprotective agent. The objective of this study was to investigate whether the protective effect of melatonin on AMPH‐induced hippocampal damage in the postnatal rat acts through the dopaminergic pathway. Four‐day‐old postnatal rats were subcutaneously injected with 5‐10 mg/kg AMPH and pretreated with 10 mg/kg melatonin prior to AMPH exposure for seven days. The results showed that melatonin decreased the AMPH‐induced hippocampal neuronal degeneration in the dentate gyrus, CA1, and CA3. Melatonin attenuated the reduction in the expression of hippocampal synaptophysin, PSD‐95, α‐synuclein, and N‐methyl‐D‐aspartate (NMDA) receptor protein and mRNA caused by AMPH. Melatonin attenuated the AMPH‐induced reduction in dopamine transporter (DAT) protein expression in the hippocampus and the reduction in mRNA expression in the ventral tegmental area (VTA). Immunofluorescence demonstrated that melatonin not only prevented the AMPH‐induced loss of DAT and NMDA receptor but also prevented AMPH‐induced α‐synuclein overexpression in the dentate gyrus, CA1, and CA3. Melatonin decreased the AMPH‐induced reduction in the protein and mRNA of the NMDA receptor downstream signaling molecule, calcium/calmodulin‐dependent protein kinase II (CaMKII), and the melatonin receptors (MT1 and MT2). This study showed that melatonin prevented AMPH‐induced toxicity in the hippocampus of postnatal rats possibly via its antioxidative effect and mitochondrial protection.