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R H Silverman

Publications and source records attributed to R H Silverman.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effect of deficiency of the double-stranded RNA-dependent protein kinase, PKR, on antiviral resistance in the presence or absence of ribonuclease L: HSV-1 replication is particularly sensitive to deficiency of the major IFN-mediated enzymes.

Control of viral replication by interferon (IFN) is thought to be principally mediated by the 2',5'-oligoadenylate synthetase (OAS)/RNAse L, double-stranded dependent protein kinase (PKR), and myxovirus resistance protein (Mx) pathways. In this study, we monitored the constitutive and IFN-induced antiviral activity in mouse embryo fibroblasts lines derived from mice with targeted disruption of either PKR or PKR/RNAse L genes. At high multiplicity of infection (moi = 10), the absence of PKR had no effect on replication of vesicular stomatitis virus (VSV) but moderately enhanced encephalomyocarditis virus (EMCV) growth and greatly increased replication of herpes simplex virus-1 (HSV-1). Replication of EMCV, HSV-1, and VSV was modestly higher in PKR-/- RNAse L-/- fibroblasts when compared with control cells. Although the antiviral action of IFN-alpha was unaffected by the absence of PKR, IFN action was significantly impaired in the double knockout cells but was dependent on the stage of the virus cycle. At early stages, it appeared that anti-EMCV and anti-HSV-1 action of IFN-alpha was significantly compromised, although weak residual antiviral activity was seen. The action of IFN-alpha against VSV was specifically compromised at a late stage of virus replication. The results showed that PKR is an important mediator in constitutive resistance against HSV-1 and that RNAse L is also necessary for the full antiviral activity of IFN against a variety of viruses. These results supported the existence of novel pathways aimed toward specific stages of the virus life cycle.

Animals↗
PubMed2000

Caspase-dependent apoptosis by 2',5'-oligoadenylate activation of RNase L is enhanced by IFN-beta.

The 2',5'-oligoadenylate (2-5A) system is an interferon (IFN)-regulated RNA decay pathway that provides innate immunity against viral infections. The biologic action of the 2-5A system is mediated by RNase L, an endoribonuclease that becomes enzymatically active after binding to 2-5A. RNase L is also implicated in mediating apoptosis in response to both viral and nonviral inducers. To study the cellular effects of RNase L activation directly, 2-5A was transfected into the human ovarian cancer cell line, Hey1B. Activation of RNase L by 2-5A resulted in specific 18S rRNA cleavage and induction of apoptosis, as measured by TUNEL and annexin V binding assays. In contrast, the dimeric form of 2-5A, ppA2'p5'A, neither activated RNase L nor caused apoptosis. Treatment with IFN-beta prior to 2-5A transfection enhanced cellular RNase L levels (< or = 2.2-fold) and increased the proportion of cells undergoing apoptosis (by < or =40%). However, rRNA cleavages after 2-5A transfections were not enhanced by IFN-beta pretreatments, indicating that basal levels of RNase L were sufficient for this activity. Apoptosis in response to RNase L activation was accompanied by cytochrome c release from mitochondria. Induction of apoptosis by either 2-5A alone or by the combination of 2-5A and IFN-beta was effectively blocked with either the pancaspase inhibitor, Z-VAD-fmk, or with the caspase 3 inhibitor, DEVD-fmk. Therefore, activation of RNase L by 2-5A leads to cytochrome c release into the cytoplasm and then to caspase activation and apoptosis. These results suggest potential uses for 2-5A in augmenting the anticancer activities of IFN.

Adenine Nucleotides↗
PubMed2000

RNase L-independent specific 28S rRNA cleavage in murine coronavirus-infected cells.

We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously.

Animals↗
PubMed2000

Activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways.

Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH(2)-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i. e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.

Animals↗
PubMed1999

The antiviral enzymes PKR and RNase L suppress gene expression from viral and non-viral based vectors.

Expression of transfected genes is shown to be suppressed by two intracellular enzymes, RNase L and protein kinase PKR, which function in interferon-treated cells to restrict viral replication. RNase L(-/-) or PKR(-/-) murine embryonic fibroblasts produced enhanced levels of protein from transfected genes compared with wild-type cells. Increased expression of exogenous genes in RNase L(-/-) cells correlated with elevated levels of mRNA and thus appeared to be due to enhanced mRNA stability. Plasmid encoding adenovirus VA RNAs was able to further enhance accumulation of the exogenous gene transcript and protein, even in cells lacking PKR. In contrast to the increased expression of transfected genes in cells lacking RNase L or PKR, expression of endogenous host genes was unaffected by the absence of these enzymes. In addition, a dominant-negative PKR mutant improved expression from a conventional plasmid vector and from a Semliki Forest virus derived, self-replicating vector. These results indicate that viral infections and transfections produce similar stress responses in mammalian cells and suggest strategies for selectively increasing expression of exogenous genes.

Adenoviridae↗
PubMed1999

Interferon action in triply deficient mice reveals the existence of alternative antiviral pathways.

Antiviral proteins encoded by the interferon (IFN)-stimulated genes provide a front-line defense against viral infections. In particular, PKR, RNase L, and Mx are considered to be the principal proteins through which IFNs mount an antiviral state. To determine whether alternative antiviral pathways exist, RNase L-/- mice and PKR-/- mice were crossed onto an Mx1(-/-) background to generate a strain of triply deficient (TD) mice. After infections with encephalomyocarditis virus, the TD mice died 3-4 days earlier than infected, wild-type mice. However, there was an IFN dose-dependent increase in survival times after encephalomyocarditis virus infections for both the TD and wild-type mice. Mice that were deficient for PKR or RNase L showed intermediate survival times between those of the TD and wild-type mice. Surprisingly, cultured embryonic fibroblasts lacking RNase L, PKR, or both proteins were still able to mount a substantial residual antiviral response against encephalomyocarditis virus or vesicular stomatitis virus after IFN-alpha treatments. These results confirm the antiviral functions of RNase L and PKR in vivo but also provide unequivocal evidence for the existence of novel, innate immune pathways against viruses.

Animals↗
PubMed1999

Alternative function of a protein kinase homology domain in 2', 5'-oligoadenylate dependent RNase L.

RNase L is the 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease that functions in interferon action and apoptosis. One of the intriguing, albeit unexplained, features of RNase L is its significant homology to protein kinases. Despite the homology, however, no protein kinase activity was detected during activation and RNA cleavage reactions with human RNase L. Similarly, the kinase plus ribonuclease domains of RNase L produced no detectable protein kinase activity in contrast to the phosphorylation obtained with homologous domains of the related kinase and endoribonuclease, yeast IRE1p. In addition, neither ATP nor pA(2'p5'A)3was hydrolyzed by RNase L. To further investigate the function of the kinase homology in RNase L, the conserved lysine at residue 392 in protein kinase-like domain II was replaced with an arginine residue. The resulting mutant, RNase LK392R, showed >100-fold decreases in 2-5A-dependent ribonuclease activity without reducing 2-5A- or RNA-binding activities. The greatly reduced activity of RNase LK392Rwas correlated to a defect in the ability of RNase L to dimerize. These results demonstrate a critical role for lysine 392 in the activation and dimerization of RNase L, thus suggesting that these two activities are intimately linked.

Adenine Nucleotides↗
PubMed1999

Very high-frequency ultrasound corneal analysis identifies anatomic correlates of optical complications of lamellar refractive surgery: anatomic diagnosis in lamellar surgery.

OBJECTIVE: To examine the utility of very high-frequency (VHF) ultrasound scanning in determining the anatomic changes and correlates of optical complications in lamellar refractive surgery. STUDY DESIGN: Case series. PARTICIPANTS: Cases analyzed included marked asymmetric astigmatism postautomated lamellar keratoplasty (ALK), image ghosting despite normal videokeratography post-ALK, uncomplicated myopic laser in situ keratomileusis (LASIK), and hyperopic LASIK with regression. METHODS: A prototype VHF ultrasound scanner (50 MHz) was used to obtain sequences of parallel B-scans of the cornea. Digital signal processing techniques were used to measure epithelial, stromal, and flap thickness values in a grid encompassing the central 4 to 5 mm of the cornea, enabling pachymetric mapping of each layer with 2-micron precision. MAIN OUTCOME MEASURE: The appearance of the corneas in VHF ultrasound images and thickness values of individual corneal layers determined from VHF ultrasound data. RESULTS: VHF ultrasound resolved the epithelial, stromal cap, or flap and residual stromal layers 1 year after lamellar surgery. Asymmetric stromal tissue removal was differentiated from stromal cap irregularity. Epithelium acted to compensate for asymmetry of the stromal surface about the visual axis and for localized surface irregularities. Irregularities in the epithelial-stromal interface accounted for image ghosting present despite apparently normal videokeratography. Epithelial thickening was shown after uncomplicated myopic LASIK. Hyperopic LASIK demonstrated relative epithelial thickening localized to the region of ablation accounting for refractive regression. CONCLUSIONS: VHF ultrasound shows promise as a sensitive method of determining the anatomic correlates of optical complications in lamellar refractive surgery.

Adult↗
PubMed1999

A comparison of ultrasonic beams for thermal treatment of ocular tumors.

OBJECTIVE: This study examined the relative merits of different ultrasonic beams and exposure modalities for treating ocular melanomas. METHODS: Simulations were conducted to evaluate temperature patterns and lesion shapes induced by intense-ultrasound treatment of ocular tumors. In-vitro insonification experiments were conducted in bovine lenses. RESULTS: Simulated hyperthermia exposures did not effectively treat tumor margins because of thermal conduction into nearby fluid-like media. Standard high-intensity focused beams produced narrow lesions during 2-s exposures. A high-intensity, multi-lobed beam, produced by a transducer with strip electrodes, generated asymmetric lesions with a single large dimension; this lesion shape could expedite the production of lesion matrices within large tumors. In-vitro cataract shapes were consistent with simulation results for focused high-intensity beams. CONCLUSIONS: Thermal conduction and perfusion can cause underheating of tumor margins during hyperthermia unless special beam designs are used. The strip-electrode transducer configuration promises to expedite treatment of extended tumor volumes.

Animals↗
PubMed1999

High-resolution ultrasonic imaging of blood flow in the anterior segment of the eye.

PURPOSE: To develop a noninvasive technique to visualize and measure blood flow in the iris and ciliary body. METHODS: Echo data from 50-MHz ultrasound scans of the iris and ciliary body of rabbits were digitized using a new "swept scan" modality. The method makes use of spatial oversampling to identify regions with scatterers whose range changes with time. The data allowed construction of high-resolution B-mode images with embedded flow information. Pulsatility over the cardiac cycle was evaluated by sending a series of pulses along a single line of sight containing a vessel of interest. Local blood flow and changes over the cardiac cycle before and after application of atropine were quantified. RESULTS: Flow was identified in the radial vessels and major arterial circle of the iris. Vessels with lumens as small as 40 microm in diameter and flow velocities as low as 0.6 mm/sec were measured. Change in blood velocity over the cardiac cycle was determined to be approximately 27%. Peak systolic velocity after administration of topical atropine increased by 72%. CONCLUSIONS: This technique allowed visualization of flow using the same type of very-high-frequency transducer now widely used for imaging the anterior segment. The technique can also be used at lower frequencies for more posterior tissues with similar improvement of resolution over Doppler. The ability to examine flow in the anterior segment of the eye offers a new tool for study of glaucoma, hypotony, tumors, and other disorders.

Animals↗
PubMed1998

Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays.

The pleiotropic activities of interferons (IFNs) are mediated primarily through the transcriptional regulation of many downstream effector genes. The mRNA profiles from IFN-alpha, -beta, or -gamma treatments of the human fibrosarcoma cell line, HT1080, were determined by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes. Among these were transcripts for known IFN-stimulated genes (ISGs), the expression of which were consistent with previous studies in which the particular ISG was characterized as responsive to either Type I (alpha, beta) or Type II (gamma) IFNs, or both. Importantly, many novel IFN-stimulated genes were identified that were diverse in their known biological functions. For instance, several novel ISGs were identified that are implicated in apoptosis (including RAP46/Bag-1, phospholipid scramblase, and hypoxia inducible factor-1alpha). Furthermore, several IFN-repressed genes also were identified. These results demonstrate the usefulness of oligonucleotide arrays in monitoring mammalian gene expression on a broad and unprecedented scale. In particular, these findings provide insights into the basic mechanisms of IFN actions and ultimately may contribute to better therapeutic uses for IFNs.

Apoptosis↗
PubMed1998

2',5'-Oligoadenylate-antisense chimeras cause RNase L to selectively degrade bcr/abl mRNA in chronic myelogenous leukemia cells.

We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2', 5'-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210(bcr/abl) kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of beta-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.

Adenine Nucleotides↗
PubMed1998

Targeted therapy of human malignant glioma in a mouse model by 2-5A antisense directed against telomerase RNA.

Telomerase is the RNA-protein complex which elongates telomeric DNA (TTAGGG)n and appears to play an important role in cellular immortalization. The almost exclusive expression of telomerase in tumor cells, and not in most normal cells, offers an exciting opportunity for therapy by inhibiting its function. Here, we have investigated the effect of inhibition of telomerase on the growth and survival of human malignant glioma cells in vitro and in vivo by using a 19-mer antisense oligonucleotide against human telomerase RNA linked to a 2',5'-oligoadenylate (2-5A). 2-5A antisense functions by activating the endoribonuclease, RNase L, resulting in the degradation of single stranded, targeted RNA. We have shown that the 2-5A antisense treatment effectively suppressed tumor cell growth and survival in vitro. Furthermore, treatment of tumors grown in nude mice with the antisense oligonucleotide inhibited survival of the tumor cells. TUNEL assays suggest that this effect is mediated through the induction of apoptosis. Targeting telomerase RNA with 2-5A antisense, therefore, may represent an effective and novel approach for treatment of a broad range of cancers.

Animals↗
PubMed1998

Nuclease-resistant composite 2',5'-oligoadenylate-3', 5'-oligonucleotides for the targeted destruction of RNA: 2-5A-iso-antisense.

A new modification of 2-5A-antisense, 2-5A-iso-antisense, has been developed based on a reversal of the direction of the polarity of the antisense domain of a 2-5A-antisense composite nucleic acid. This modification was able to anneal with its target RNA as well as the parental 2-5A-antisense chimera. The 2-5A-iso-antisense oligonucleotide displayed enhanced resistance to degradation by 3'-exonuclease enzyme activity such as that represented by snake venom phosphodiesterase and by that found in human serum. 2-5A-Iso-antisense was able to effect the degradation of a synthetic nontargeted substrate, [5'-32P]pC11U2C7, and two targeted RNAs, PKR and BCR mRNAs, in a cell-free system containing purified recombinant human 2-5A-dependent RNase L. These results demonstrated that the novel structural modification represented by 2-5A-iso-antisense provided a stabilized biologically active formulation of the 2-5A-antisense strategy.

DNA, Complementary↗
PubMed1998

RNase L dimerization in a mammalian two-hybrid system in response to 2',5'-oligoadenylates.

RNase L, a key enzyme in the anti-viral activity of interferons, requires activation by 2',5'-linked oligoadenylates (2-5A) to cleave viral and cellular single-stranded RNA. Here we demonstrate that 2-5A causes formation of stable dimers of RNase L in intact human cells as measured with a mammalian two-hybrid system. Hybrid proteins consisting of the GAL4 DNA binding domain fused to RNase L and the VP16 transactivation domain fused to RNase L were able to associate and drive transcription of a reporter gene, but only after cells were transfected with 2-5A. Several functional forms of 2-5A, such as p3A2'p5'A2'p5'A, were capable of activating transcription in human HeLa cells. In contrast, p3A2'p5'A, which can neither activate nor dimerize RNase L, did not induce gene expression. Evidence for the involvement of the C-terminal region of RNase L in dimerization was obtained by expressing truncated forms of RNase L. These findings describe a convenient, high-throughput screening method for RNase L activators which could lead to the discovery of novel anti-viral and anti-cancer agents.

Adenine Nucleotides↗
PubMed1998

Very high frequency ultrasound analysis of a new phakic posterior chamber intraocular lens in situ.

PURPOSE: To use very high frequency ultrasound scanning for in situ analysis of a new phakic posterior chamber intraocular lens (No-Touch; International Visions Inc, Cincinnati, Ohio). METHODS: In this pilot study, very high frequency ultrasound (50 MHz) wide-angle (15 mm) full anterior segment scans were obtained in two patients who had undergone phakic posterior chamber intraocular lens implantation into legally blind eyes with normal anterior segment anatomy. RESULTS: Very high frequency ultrasound B-scan images delineated the phakic posterior chamber intraocular lens within the posterior chamber. The relations to the sulci were clearly imaged. Anatomic relations of the phakic posterior chamber intraocular lens optic and haptics were visualized in both static (light/dark) and kinetic (distance/accommodative) states. CONCLUSION: Very high frequency ultrasound wide-angle scanning provides a unique tool to noninvasively evaluate the eye preoperatively and the static and kinetic relations of this new refractive device within the posterior chamber.

Adult↗
PubMed1998

Advances in ophthalmic ultrasound.

New ultrasound technologies, including three-dimensional imaging, tissue characterization, and very high frequency (50 MHz) ultrasound, have become available recently. We demonstrate how these technologies can be used alone and together to improve the use of ultrasound for diagnosis of ocular pathology.

Eye↗