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MicroRNAs (miRNAs) act in post-transcriptional gene silencing and are proposed to function in a wide spectrum of pathologies, including cancers and viral diseases. Currently, to our knowledge, no detailed mechanistic characterization of small molecules that interrupt miRNA pathways have been reported. In screening a small chemical library, we identified compounds that suppress RNA interference activity in cultured cells. Two compounds were characterized; one impaired Dicer activity while the other blocked small RNA-loading into an Argonaute 2 (AGO2) complex. We developed a cell-based model of miRNA-dependent tumorigenesis, and using this model, we observed that treatment of cells with either of the two compounds effectively neutralized tumor growth. These findings indicate that miRNA pathway-suppressing small molecules could potentially reverse tumorigenesis.

Mouse APOBEC3 (mA3) is a cytidine deaminase with antiviral activity. mA3 is linked to the Rfv3 virus resistance factor, a gene responsible for recovery from infection by Friend murine leukemia virus, and mA3 allelic variants differ in their ability to restrict mouse mammary tumor virus. We sequenced mA3 genes from 38 inbred strains and wild mouse species, and compared the mouse sequence and predicted structure with human APOBEC3G (hA3G). An inserted sequence was identified in the virus restrictive C57BL strain allele that disrupts a splice donor site. This insertion represents the long terminal repeat of the xenotropic mouse gammaretrovirus, and was acquired in Eurasian mice that harbor xenotropic retrovirus. This viral regulatory sequence does not alter splicing but is associated with elevated mA3 expression levels in spleens of laboratory and wild-derived mice. Analysis of Mus mA3 coding sequences produced evidence of positive selection and identified 10 codons with very high posterior probabilities of having evolved under positive selection. Six of these codons lie in two clusters in the N-terminal catalytically active cytidine deaminase domain (CDA), and 5 of those 6 codons are polymorphic in Rfv3 virus restrictive and nonrestrictive mice and align with hA3G CDA codons that are critical for deaminase activity. Homology models of mA3 indicate that the two selected codon clusters specify residues that are opposite each other along the predicted CDA active site groove, and that one cluster corresponds to an hAPOBEC substrate recognition loop. Substitutions at these clustered mA3 codons alter antiviral activity. This analysis suggests that mA3 has been under positive selection throughout Mus evolution, and identified an inserted retroviral regulatory sequence associated with enhanced expression in virus resistant mice and specific residues that modulate antiviral activity.

Small non-coding RNAs of 18-25 nt in length can regulate gene expression through the RNA interference (RNAi) pathway. To characterize small RNAs in HIV-1-infected cells, we performed linker-ligated cloning followed by high-throughput pyrosequencing. Here, we report the composition of small RNAs in HIV-1 productively infected MT4 T-cells. We identified several HIV-1 small RNA clones and a highly abundant small 18-nt RNA that is antisense to the HIV-1 primer-binding site (PBS). This 18-nt RNA apparently originated from the dsRNA hybrid formed by the HIV-1 PBS and the 3' end of the human cellular tRNAlys3. It was found to associate with the Ago2 protein, suggesting its possible function in the cellular RNAi machinery for targeting HIV-1.

Short interfering RNAs (siRNAs) have been used to inhibit HIV-1 replication. The durable inhibition of HIV-1 replication by RNA interference has been impeded, however, by a high mutation rate when viral sequences are targeted and by cytotoxicity when cellular genes are knocked down. To identify cellular proteins that contribute to HIV-1 replication that can be chronically silenced without significant cytotoxicity, we employed a shRNA library that targets 54,509 human transcripts. We used this library to select a comprehensive population of Jurkat T-cell clones, each expressing a single discrete shRNA. The Jurkat clones were then infected with HIV-1. Clones that survived viral infection represent moieties silenced for a human mRNA needed for virus replication, but whose chronic knockdown did not cause cytotoxicity. Overall, 252 individual Jurkat mRNAs were identified. Twenty-two of these mRNAs were secondarily verified for their contributions to HIV-1 replication. Five mRNAs, NRF1, STXBP2, NCOA3, PRDM2, and EXOSC5, were studied for their effect on steps of the HIV-1 life cycle. We discuss the similarities and differences between our shRNA findings for HIV-1 using a spreading infection assay in human Jurkat T-cells and results from other investigators who used siRNA-based screenings in HeLa or 293T cells.

PDZ domains are versatile protein interaction modules with the ability to dimerize and to recognize internal and carboxy-terminal peptide motifs. Their function in mediating the formation of multi-molecular signaling complexes is best understood at neuronal and epithelial membranes. In a screen for interactors that regulate transcription factor function in pancreatic beta cells, we isolated two PDZ-containing proteins Bridge-1 (PSMD9) and PDZD2, which contain one and six PDZ domains, respectively. Here, we review their functions in the regulation of pancreatic beta cells as a nuclear coactivator or extracellular signaling molecule. Bridge-1 interacts with both E12 and PDX-1 to stimulate insulin promoter activity. Recent gain-of-function analysis in both cell and transgenic models has revealed its functions to regulate both insulin gene expression and pancreatic beta-cell survival. Little is known about the intracellular function of PDZD2 that is predominantly localized to the endoplasmic reticulum of INS-1E cells. Interestingly, PDZD2 is proteolytically processed by caspase-3 to generate a carboxy-terminal secreted protein (sPDZD2) containing two PDZ domains. Expressed in fetal pancreatic progenitor and INS-1E cells, sPDZD2 when added as recombinant protein exerts concentration-dependent mitogenic effects on beta-like cells. We propose that the PDZ domain proteins Bridge-1 and PDZD2 likely transduce signals that regulate insulin production, proliferation, and survival of pancreatic beta cells.

MicroRNAs (miRNAs) play diverse roles in regulating cellular and developmental functions. We have profiled the miRNA expression in peripheral blood mononuclear cells from 36 HIV-1 seropositive individuals and 12 normal controls. The HIV-1-positive individuals were categorized operationally into four classes based on their CD4+ T-cell counts and their viral loads. We report that specific miRNA signatures can be observed for each of the four classes.

A role for microRNAs (miRNA) in human T-cell leukemia virus 1 (HTLV-1)-mediated cellular transformation has not been described. Here, we profiled miRNA expression in HTLV-1-transformed human T-cell lines and primary peripheral blood mononuclear cells from adult T-cell leukemia patients. Analyses of 11 different profiles revealed six miRNAs that were consistently up-regulated. Two of the up-regulated miRNAs (miR-93 and miR-130b) target the 3' untranslated region (3'UTR) of the mRNA for a tumor suppressor protein, tumor protein 53-induced nuclear protein 1 (TP53INP1). A low expression level of TP53INP1 protein was found in HTLV-1-transformed cells. Additionally, when antagomirs were used to knock down miR-93 and miR-130b in these cells, the expression of TP53INP1 was increased, suggesting that the latter is regulated inside cells by the former. A role for TP53INP1 in regulating cell growth was established by experiments that showed that enhanced TP53INP1 expression increased apoptosis. Collectively, the findings implicate a miR-93/miR-130b-TP53INP1 axis that affects the proliferation and survival of HTLV-1-infected/transformed cells.

APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) replication. HIV-1 synthesizes a viral infectivity factor (Vif) to counter A3G restriction. Currently, it is poorly understood how A3G expression/activity is regulated by cellular factors. Here, we show that the prolyl isomerase Pin1 protein modulates A3G expression. Pin1 was found to be an A3G-interacting protein that reduces A3G expression and its incorporation into HIV-1 virion, thereby limiting A3G-mediated restriction of HIV-1. Intriguingly, HIV-1 infection modulates the phosphorylation state of Pin1, enhancing its ability to moderate A3G activity. These new findings suggest a potential Vif-independent way for HIV-1 to moderate the cellular action of A3G.

Pancreatic cancer is a disease with an extremely poor prognosis. Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a proapoptotic stress-induced p53 target gene. In this article, we show by immunohistochemical analysis that TP53INP1 expression is dramatically reduced in pancreatic ductal adenocarcinoma (PDAC) and this decrease occurs early during pancreatic cancer development. TP53INP1 reexpression in the pancreatic cancer-derived cell line MiaPaCa2 strongly reduced its capacity to form s.c., i.p., and intrapancreatic tumors in nude mice. This anti-tumoral capacity is, at least in part, due to the induction of caspase 3-mediated apoptosis. In addition, TP53INP1(-/-) mouse embryonic fibroblasts (MEFs) transformed with a retrovirus expressing E1A/ras(V12) oncoproteins developed bigger tumors than TP53INP1(+/+) transformed MEFs or TP53INP1(-/-) transformed MEFs with restored TP53INP1 expression. Finally, TP53INP1 expression is repressed by the oncogenic micro RNA miR-155, which is overexpressed in PDAC cells. TP53INP1 is a previously unknown miR-155 target presenting anti-tumoral activity.

MicroRNAs (miRNAs) are non-coding small RNAs that play important roles in a variety of biological pathways including cellular proliferation and apoptosis. Recent studies have linked the expression of selected miRNAs to carcinogenesis and viral pathogenesis. Here, we will discuss examples of roles served by cellular miRNAs and virus-encoded miRNAs in the development of cancers and viral diseases.

Inside eukaryotic cells, small RNA duplexes, called small interfering RNAs (siRNAs), activate a conserved RNA interference (RNAi) pathway which leads to specific degradation of complementary target mRNAs through base-pairing recognition. As with other viruses, studies have shown that replication of the HIV-1 in cultured cells can be targeted and inhibited by synthetic siRNAs. The relative ease of siRNA design and the versatility of RNAi to target a broad spectrum of mRNAs have led to the promise that drug discovery in the RNAi pathway could be effective against pathogens. This review discusses the current experimental principles that guide the application of RNAi against HIV and describes challenges and limitations that need to be surmounted in order for siRNAs to become practical antiviral drugs. The practical use of RNAi therapy for HIV infection will depend on overcoming several challenges, including the ability to establish long-term expression of siRNA without off-target effects and the capacity to counteract mutant escape viruses.

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.

Purpose of Review: RNA interference is a type of nucleic-acid-based immunity used by cells to restrict transposons, transgenes and viruses. The RNA interference machinery targets long double-stranded RNAs to produce short RNAs that arm cellular ribonucleases to degrade foreign RNAs in a sequence-specific manner. Despite this defense, many viruses with RNA genomes, such as HIV-1, replicate seemingly unrestricted in cells. This suggests that viruses may have evolved counter-strategems that negate the host's RNA interference. We review the complex point-counterpoint RNA interference interplays between the human cells and HIV-1., Recent Findings: RNA interference functions in human cells to restrict viral replication. Recent findings suggest that HIV-1 can evade cellular RNA interference in several ways. First, the virus can mutate its sequence to evade sequence-specific targeting by RNA interference. Second, HIV-1 encodes a viral Tat protein that can partially suppress the cell's RNA interference processing machinery. Finally, HIV-1 encodes a small RNA decoy, TAR, which can sequester a cellular protein named TAR RNA binding protein. The recent discovery that TAR RNA binding protein is a required cofactor for Dicer to process microRNA and small interfering RNA suggests that TAR RNA is another moiety used by HIV-1 to defeat RNA interference., Summary: We discuss stratagems used by HIV-1 and other viruses to defeat the cells' antiviral small interfering RNA/microRNA defenses. We review how viruses might control and regulate host genes by encoding viral microRNA.

NF-kappaB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IkappaB kinase (IKK) "signalsome". The core IKK consists of two catalytic serine kinases, IKKalpha and IKKbeta, and a noncatalytic subunit, IKKgamma. IKKgamma is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKgamma mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKgamma-delta. Using a specific reverse transcription-PCR assay, we find that IKKgamma-delta is widely expressed in cultured human cells and normal human tissues. Because IKKgamma-Delta protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-kappaB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKgamma-delta has strong homo- and heterotypic association with wild-type (WT) IKKgamma and, like IKKgamma WT, associates with the IKKbeta kinase. Similarly, IKKgamma-delta mediates IKK kinase activity and downstream NF-kappaB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-kappaB-inducing kinase-IKKalpha signaling pathway. Surprisingly, however, in contrast to IKKgamma WT, IKKgamma-delta is not able to mediate HTLV-1 Tax-induced NF-kappaB-dependent transcription, even though IKKgamma-delta binds and colocalizes with Tax. These observations suggest that IKKgamma-delta is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKgamma-delta expression, therefore, may affect signal transduction cascades coupling to IKK.

Activation of the nuclear factor kappa B (NF-kappaB) transcription factor family by different stimuli, such as inflammatory cytokines, stress inducers, or pathogens, results in innate and adaptive immunity. While the main function of NF-kappaB is to promote the host's immune response, the NF-kappaB pathway is frequently dysregulated by invading viral pathogens. Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of a fatal malignancy known as adult T cell leukemia (ATL) and an inflammatory disease named tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). HTLV-1 encodes an oncoprotein, Tax, which plays a significant role in the initiation of cellular transformation and the elicitation of the host's inflammatory responses. Here, we review current thinking on how Tax may affect both diseases through activation of NF-kappaB signaling.

Activation of the nuclear factor kappa B (NF-ϰB) transcription factor family by different stimuli, such as inflammatory cytokines, stress inducers, or pathogens, results in innate and adaptive immunity. While the main function of NF-ϰB is to promote the host's immune response, the NF-ϰB pathway is frequently dysregulated by invading viral pathogens. Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of a fatal malignancy known as adult T cell leukemia (ATL) and an inflammatory disease named tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). HTLV-1 encodes an oncoprotein, Tax, which plays a significant role in the initiation of cellular transformation and the elicitation of the host's inflammatory responses. Here, we review current thinking on how Tax may affect both diseases through activation of NF-ϰB signaling.

Initially reported for Caenorhabditis elegans, microRNA (miRNA) has been shown to regulate gene expression in plants, flies, and mammals . Here, we report on our approaches to investigate the role of miRNAs in human immunodeficiency virus (HIV)-1 infection. Using computer-directed foldings, we first identify potential sequences in HIV-1 that putatively encode miRNAs. Subsequently, we use Northern blotting of RNAs isolated from HIV-infected cells to confirm expression of predicted miRNA sequences. Finally, we use a scanning algorithm to search 3' untranslated regions (UTRs) of human messenger RNAs (mRNAs) in the attempt to predict potential sites targeted by HIV-1 miRNAs.

MicroRNAs (miRNAs) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating a variety of biological processes. Recent studies suggest that cellular miRNAs may serve to control the replication of viruses in cells. If such is the case, viruses might be expected to evolve the ability to modulate the expression of cellular miRNAs. To ask if expression of HIV-1 genes changes the miRNA profiles in human cells, we employed a high throughput microarray method, termed the RNA-primed Array-based Klenow Enzyme (RAKE) assay. Here, we describe the optimization of this assay to quantify the expression of miRNAs in HIV-1 transfected human cells. We report distinct differences in miRNA profiles in mock-transfected HeLa cells versus HeLa cells transfected with an infectious HIV-1 molecular clone, pNL4-3.

Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA-targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA-specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).

The Raf-MEK-ERK protein kinase cascade is a highly conserved signaling pathway that is pivotal in relaying environmental cues from the cell surface to the nucleus. Three Raf isoforms, which share great sequence and structure similarities, have been identified in mammalian cells. We have previously identified Raf kinase inhibitor protein (RKIP) as a negative regulator of the Raf-MEK-ERK signaling pathway by specifically binding to the Raf-1 isoform. We show here that RKIP also antagonizes kinase activity of the B-Raf isoform. Yeast two-hybrid and coimmunoprecipitation experiments indicated that RKIP specifically interacted with B-Raf. Ectopic expression of RKIP antagonized the kinase activity of B-Raf. We showed that the effects of RKIP on B-Raf functions were independent of its known inhibitory action on Raf-1. The expression levels of RKIP in melanoma cancer cell lines are low relative to primary melanocytes. Forced expression of RKIP partially reverted the oncogenic B-Raf kinase-transformed melanoma cancer cell line SK-Mel-28. The low expression of RKIP and its antagonistic action on B-Raf suggests that RKIP may play an important role in melanoma turmorgenesis.

MicroRNAs (miRNAs) are small RNAs of 21-25 nucleotides that specifically regulate cellular gene expression at the post-transcriptional level. miRNAs are derived from the maturation by cellular RNases III of imperfect stem loop structures of ~ 70 nucleotides. Evidence for hundreds of miRNAs and their corresponding targets has been reported in the literature for plants, insects, invertebrate animals, and mammals. While not all of these miRNA/target pairs have been functionally verified, some clearly serve roles in regulating normal development and physiology. Recently, it has been queried whether the genome of human viruses like their cellular counterpart also encode miRNA. To date, there has been only one report pertaining to this question. The Epstein-Barr virus (EBV) has been shown to encode five miRNAs. Here, we extend the analysis of miRNA-encoding potential to the human immunodeficiency virus (HIV). Using computer-directed analyses, we found that HIV putatively encodes five candidate pre-miRNAs. We then matched deduced mature miRNA sequences from these 5 pre-miRNAs against a database of 3' untranslated sequences (UTR) from the human genome. These searches revealed a large number of cellular transcripts that could potentially be targeted by these viral miRNA (vmiRNA) sequences. We propose that HIV has evolved to use vmiRNAs as a means to regulate cellular milieu for its benefit.

PDZD2 (PDZ-domain-containing 2; also known as PAPIN, AIPC and PIN1) is a ubiquitously expressed multi-PDZ-domain protein. We have shown that PDZD2, which shows extensive homology to pro-interleukin-16 (pro-IL-16), is localized mainly to the endoplasmic reticulum (ER). Pro-IL-16 is cleaved in a caspase-3-dependent mechanism to generate the secreted cytokine IL-16. The abundant expression of PDZD2 in the ER, and its sequence similarity to pro-IL-16, suggests that similar post-translational processing of PDZD2 may occur. Indeed, western blotting and mass spectrometry analysis of conditioned medium from cells transfected with epitope-tagged PDZD2 show that there is secretion of a PDZD2 peptide of approximately 37 kDa (sPDZD2, for secreted PDZD2) that contains two PDZ domains. Expression of PDZD2 was detected in several tissues. Furthermore, sPDZD2 secretion is suppressed by the mutation of a sequence that shows similarity to caspase recognition motifs or by treatment with a caspase inhibitor. In summary, PDZD2 is the first reported multi-PDZ protein that is processed by proteolytic cleavage to generate a secreted peptide containing two PDZ domains.

The onset, depth and recovery from paralysis produced by gallamine triethiodide were studied using the tibialis anterior muscle/sciatic nerve preparation in mongrel dogs, during changes in blood flow to this muscle. A roller pump was used to effect the blood flow changes via an aorto-femoral shunt. The onset and depth of paralysis were related directly to muscle blood flow. There was no correlation between the rate of recovery from paralysis and the blood flow to the muscle.

An asthmatic patient developed intense bronchospasm immediately following the administration of alcuronium and d-tubocurarine in the same anaesthetic. Intradermal test was positive for both of these drugs but negative for thiopentone and suxamethonium, both of which were given prior to alcuronium. The differential diagnosis and the possible mechanism of the reaction are discussed.

The relationship between tubocurarine requirements and plasma levels of albumin and total globulin was investigated in 50 adult patients undergoing elective upper abdominal operation. No significant correlation was found with plasma proteins expressed in g/100 ml of plasma. A significant positive correlation between tubocurarine requirements and plasma total globulin corrected for packed cell volume (PCV) by multiplying, by (1-PCV) was demonstrated. This relationship decreased with time.

The incidence of medical diseases in surgical patients was assessed using data gathered from 5944 consecutive anaesthetics. Medical disease which might affect anaesthetic management was present in 23.2% of patients. The commonest diseases were hypertension, anaemia, chronic obstructive airway disease, diabetes mellitus, and pulmonary tuberculosis. No significant difference was detected in sex incidence for ischaemic heart disease and cerebrovascular disease. There was a disproportionate preponderance of males with respiratory diseases. It is suggested that anaesthetics should be administered only by qualified anaesthetists, that the establishment of anaesthetic outpatient clinics is desirable, and that internal medicine should be included in anaesthetic training.

Our experience of twenty-eight patients with intermittent claudication treated by means of paravertebral lumbar somatic nerve block is presented. There was immediate demonstrable improvement in 92-5% of the twenty-six patients included in this follow up. At the end of 6 months the improvement was maintained in 72-7% of the patients and in about 70-0% of patients at 6 months and at 9 months. Six of the seven patients followed up for 1 year had a claudication distance at least twice that prior to the block. The results of this indicate that lumbar somatic nerve block is less disturbing and more effective than other forms of conservative treatment for intermittent claudication.

The relationship between alcuronium requirements and the plasma concentration of albumin and total globulin was investigated in 50 adult patients undergoing elective upper abdominal surgery. No significant correlation was obtained with the plasma proteins (g/100 ml plasma). A significant positive correlation between alcuronium requirements and plasma albumin X (1-PCV), especially in anaemic patients, was found. This relationship tended to decrease with time.