Isolation, Characterization, and Synthesis of AlphaFetoprotein from Neonatal Rat Brain

Monospecific anti-rat serum alpha-fetoprotein (AFP) IgG was coupled to cyanogen bromide-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. The immunoaffinity column was used to isolate AFP from feto-neonatal rat brain. The purified AFP was immunologically and electrophoretically similar to serum AFP. It yielded a single band with a molecular weight of 70,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the protein under nondenaturing conditions yielded two charge variants of AFP, reminiscent of AFP from feto-neonatal rat serum. The AFP was observed to bind estradiol with Ka = 5.8 X 10(8) M -1 and 1.3 X 10(8) M -1 by dextran-coated charcoal adsorption and Sephadex gel filtration techniques, respectively. Newborn rat brain cells linearly incorporated [14C]leucine into immunoprecipitable AFP during 6 h in culture. It is, therefore, concluded that feto-neonatal rat brain contains AFP similar to that present in fetal serum and that it may arise in brain as a result of its in situ synthesis.     

Dual muscarinic and nicotinic action on a motor program in Drosophila

The effect of cholinergic agonists and antagonists on the central pattern generator of the pharyngeal muscles has been studied in third instar larvae of Drosophila. The pharyngeal muscles are a group of rhythmically active fibers involved in feeding. Bath application of the cholinergic agonists carbachol, musarine, pilocarpine, and acetylcholine (ACh) to a semiintact preparation including the pharyngeal muscles and the central nervous system (CNS), initiated long-lasting endogenous-like bursting activity in the muscles. The muscarinic antagonists, atropine and scopolamine, blocked these responses as well as endogenous activity. Perfusion with nicotine elicited a short, tonic response that was marginally blocked by mecamylamine but not by curare, α-bungarotoxin, hexamethonium, or the muscarinic antagonists. This is the first time that a response to cholinergic drugs has been examined in Drosophila. The pharyngeal muscle preparation may prove to be a valuable system for studying mutations of cholinergic metabolism, receptors, and second messengers.     

Assessing Exposures to Household Air Pollution in Public Health Research and Program Evaluation

EcoHealth - Exposure to smoke from the use of solid fuels and inefficient stoves for cooking and heating is responsible for approximately 4 million premature deaths yearly. As increasing...     

ORF7 of varicella-zoster virus is a neurotropic factor

Varicella-zoster virus (VZV) is the causative agent of chickenpox and herpes zoster (shingles). After the primary infection, the virus remains latent in sensory ganglia and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine is highly attenuated in the skin and yet retains its neurovirulence and may reactivate and damage sensory neurons. The factors involved in neuronal invasion and establishment of latency are still elusive. Previously, we constructed a library of whole-gene deletion mutants carrying a bacterial artificial chromosome sequence and a luciferase marker in order to perform a comprehensive VZV genome functional analysis. Here, screening of dispensable gene deletion mutants in differentiated neuronal cells led to the identification of ORF7 as the first known, likely a main, VZV neurotropic factor. ORF7 is a virion component localized to the Golgi compartment in infected cells, whose deletion causes loss of polykaryon formation in epithelial cell culture. Interestingly, ORF7 deletion completely abolishes viral spread in human nervous tissue ex vivo and in an in vivo mouse model. This finding adds to our previous report that ORF7 is also a skin-tropic factor. The results of our investigation will not only lead to a better understanding of VZV neurotropism but could also contribute to the development of a neuroattenuated vaccine candidate against shingles or a vector for delivery of other antigens.     

Identification of T-cadherin as a novel target of DNA methyltransferase 3B and its role in the suppression of nerve growth factor-mediated neurite outgrowth in PC12 cells

Previously we showed that DNA methyltransferase 3b (Dnmt3b) is required for nerve growth factor (NGF)-induced differentiation of PC12 cells to neuronal phenotype. The present study identified T-cadherin (T-Cad) as one of the targets of Dnmt3b by chromatin immunoprecipitation (ChIP) assay. Combined bisulfite restriction analysis and bisulfite sequencing showed that T-Cad promoter was sparsely methylated in PC12 cells. ChIP-CHOP analysis demonstrated that Dnmt3b is associated with T-Cad promoter irrespective of its methylation status. The mRNA and protein levels of T-Cad were markedly elevated in cells depleted of Dnmt3b by antisense or small interfering RNA. Suppression of T-Cad promoter activity by Dnmt3b was independent of its catalytic activity, which was consistent with the insignificant change in T-Cad promoter methylation status in Dnmt3b-depleted cells. In contrast, deletion of its N-terminal ATRX and PWWP domain abolished its repressor function. Association of histone deacetylase 2 (Hdac2) with T-Cad promoter and restoration of the promoter activity from Dnmt3b-mediated suppression upon treatment with Hdac inhibitor indicated involvement of histone deacetylation in this process. NGF-induced neurite outgrowth was inhibited in a dose dependent manner upon ectopic expression of T-Cad in PC12 cells. Immunofluorescence studies showed that T-Cad was redistributed upon NGF treatment, as evident from its concentration in axon growth cones as opposed to its localization at cell-cell contact region in undifferentiated cells. These results demonstrate a novel role of T-Cad in the NGF-mediated differentiation of PC12 cells to neuronal phenotype.     

Downregulation of miR-122 in the rodent and human hepatocellular carcinomas

MicroRNAs (miRs) are conserved small non-coding RNAs that negatively regulate gene expression. The miR profiles are markedly altered in cancers and some of them have a causal role in tumorigenesis. Here, we report changes in miR expression profile in hepatocellular carcinomas (HCCs) developed in male Fisher rats-fed folic acid, methionine, and choline-deficient (FMD) diet. Comparison of the miR profile by microarray analysis showed altered expression of some miRs in hepatomas compared to the livers from age-matched rats on the normal diet. While let-7a, miR-21, miR-23, miR-130, miR-190, and miR-17-92 family of genes was upregulated, miR-122, an abundant liver-specific miR, was downregulated in the tumors. The decrease in hepatic miR-122 was a tumor-specific event because it did not occur in the rats switched to the folate and methyl-adequate diet after 36 weeks on deficient diet, which did not lead to hepatocarcinogenesis. miR-122 was also silent in a transplanted rat hepatoma. Extrapolation of this study to human primary HCCs revealed that miR-122 expression was significantly (P = 0.013) reduced in 10 out of 20 tumors compared to the pair-matched control tissues. These findings suggest that the downregulation of miR-122 is associated with hepatocarcinogenesis and could be a potential biomarker for liver cancers.     

Multimerization and DNA Binding Properties of INI1/hSNF5 and Its Functional Significance

INI1/hSNF5/BAF47/SMARCB1 is an HIV-1 integrase (IN)-binding protein that modulates viral replication in multiple ways. A minimal IN-binding domain of INI1, S6 (amino acids 183–294), transdominantly inhibits late events, and down-modulation of INI1 stimulates early events of HIV-1 replication. INI1 both stimulates and inhibits in vitro integration depending on IN concentration. To gain further insight into its role in HIV-1 replication, we purified and biochemically characterized INI1. We found that INI1 forms multimeric structures. Deletion analysis indicated that the Rpt1 and Rpt2 motifs form the minimal multimerization domain. We isolated mutants of INI1 that are defective for multimerization using a reverse yeast two-hybrid system. Our results revealed that INI1 residues involved in multimerization overlap with IN-binding and nuclear export domains and are required for nuclear retention and co-localization with IN. Multimerization-defective mutants are also defective for mediating the transdominant effect of INI1-S6-(183–294). Furthermore, we found that INI1 is a minor groove DNA-binding protein. Although IN binding and multimerization are required for INI1-mediated inhibition, the acceptor DNA binding property of INI1 may be required for stimulation of in vitro strand transfer activities of IN. Binding of INI1 to IN results in the formation of presumably inactive high molecular weight IN-INI1 complexes, and the multimerization-defective mutant was unable to form these complexes. These results indicate that the multimerization and IN binding properties of INI1 are necessary for its ability to both inhibit integration and influence assembly and particle production, providing insights into the mechanism of INI1-mediated effects in HIV-1 replication.HIV-1³ replication is a dynamic process that is modulated by the interaction of several host cellular proteins (1). A genome-wide siRNA-mediated knockdown indicated that hundreds of host factors are involved in the stimulation or inhibition of HIV-1 replication (2). Understanding the interplay between the host proteins and the HIV-1 viral proteins is essential to fully comprehend the dynamic relationship between the virus and the host.INI1/hSNF5/BAF47/SMARCB1 is a core component of the SWI/SNF chromatin-remodeling complex. It interacts directly with the HIV-1-encoded integrase (IN) required for the integration of the viral DNA into the host chromosome (3, 4). IN mediates the insertion of viral cDNA into host chromosomal DNA by sequential steps of 3′ processing and strand transfer (or joining) (4, 5). INI1 binds directly to HIV-1 IN in vitro and in vivo and modulates several steps of HIV-1 replication (3, 6–8). The ectopically expressed minimal IN-binding domain of INI1 transdominantly and potently inhibits HIV-1 assembly and particle production (8). The inhibitory effect is dependent on IN-INI1 interaction and is abrogated when an IN mutant defective for interaction with INI1 is used (8). Furthermore, particle production is minimal in cells lacking INI1, and reintroduction of INI1 into these cells can partially correct the defect (6). These results indicate that INI1 is required for HIV-1 late events. Additional studies have indicated that INI1 is selectively incorporated into HIV-1 but not other retroviral and lentiviral particles (9). Virally encapsidated INI1 is required for post-entry early events of HIV-1 replication prior to integration (6). These studies indicate that producer cell-associated as well as virion-associated INI1 is required for HIV-1 replication. Contrary to these proviral functions of INI1, siRNA-mediated knockdown studies indicate that INI1 in the target cells inhibits early events of HIV-1 replication (7). These studies indicate that whereas INI1 in the target cells may act as an antiviral host protein, HIV-1 may subvert the INI1 antiviral effect, and HIV-1 may utilize this host factor for late events in the producer cells and for early preintegration events in the target cells. Interestingly, in an earlier study, we demonstrated that partially purified INI1 both inhibits and stimulates in vitro integration in a manner dependent on IN concentration (3). Although INI1 stimulates in vitro strand transfer reactions at low IN concentrations, it inhibits the reaction at high concentrations (3). Further structure-function analysis of INI1 is required to understand this complex and dual role of INI1 during HIV-1 replication.INI1 gene is also a tumor suppressor that is biallelically deleted in aggressive pediatric cancers known as rhabdoid tumors (10). INI1 mutations have been found in other soft tissue cancers (11–13). The mechanism of INI1-mediated tumor suppression is not fully understood. INI1 protein has two highly conserved domains that are imperfect direct repeats (termed Rpt1 and Rpt2) of each other and a third conserved coiled coil domain (termed homology region 3 or HR3) at the C terminus. The Rpt1 and Rpt2 domains appear to be involved in protein-protein interaction with various cellular and viral proteins (3, 14–18). Additionally, the Rpt2 domain harbors a masked nuclear export signal, and the C-terminal domain is involved in inhibiting the nuclear export of the protein in the steady state. INI1 exhibits nonspecific DNA binding activity (18). The cancer-associated mutations occur throughout the open reading frame of the INI1 gene, suggesting that mutation in any one of the INI1 domains may inactivate the protein and that multiple domains are required for its function (19–21).To gain further insight into the mechanism of its action, we purified INI1 protein to homogeneity and characterized it biochemically. Here we report, for the first time, that INI1 forms dimeric and higher order multimeric structures. We have characterized the multimerization domain of INI1 and found that multimerization and IN binding activities of INI1 are required for inhibition of in vitro integration. Furthermore, we found that the multimerization, IN binding, and nuclear export properties of INI1 are important for transdominant effects. In addition, we found that INI1 possesses a minor groove DNA binding activity and that the nonspecific acceptor DNA binding activity of INI1 may be required for stimulation of in vitro integration. Finally, we found that multimerization of the full-length protein is necessary for its ability to be retained in the nucleus and to co-localize with HIV-1 IN in the nucleus. Thus, our studies provide novel insights into the mechanism by which INI1 regulates HIV-1 replication.