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R L Kaspar

Publications and source records attributed to R L Kaspar.

At least 19 recordsLinked to original sources
PubMed2001

Syntheses of puromycin from adenosine and 7-deazapuromycin from tubercidin, and biological comparisons of the 7-aza/deaza pair.

Protection (O5') of 2',3'-anhydroadenosine with tert-butyldiphenylsilyl chloride and epoxide opening with dimethylboron bromide gave the 3'-bromo-3'-deoxy xylo isomer which was treated with benzylisocyanate to give the 2'-O-(N-benzylcarbamoyl) derivative. Ring closure gave the oxazolidinone, and successive deprotection concluded an efficient route to 3'-amino-3'-deoxyadenosine. Analogous treatment of the antibiotic tubercidin [7-deazaadenosine; 4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine] gave 3'-amino-3'-deoxytubercidin. Trifluoroacetylation of the 3'-amino function, elaboration of the heterocyclic amino group into a (1,2,4-triazol-4-yl) ring with N,N'-bis[(dimethylamino)methylene]hydrazine, and nucleophilic aromatic substitution with dimethylamine gave puromycin aminonucleoside [9-(3-amino-3-deoxy-beta-D-ribofuranosyl)-6-(dimethylamino)purine] and its 7-deaza analogue. Aminoacylation [BOC-(4-methoxy-L-phenylalanine)] and deprotection gave puromycin and 7-deazapuromycin. Most reactions gave high yields at or below ambient temperature. Equivalent inhibition of protein biosynthesis in a rabbit reticulocyte system and parallel growth inhibition of several bacteria were observed with the 7-aza/deaza pair. Replacement of N7 in the purine ring of puromycin by "CH" has no apparent effect on biological activity.

Adenosine↗
PubMed2001

Binding of the La autoantigen to the 5' untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro.

Human translation elongation factor 1A (EF1A) is a member of a large class of mRNAs, including ribosomal proteins and other translation elongation factors, which are coordinately translationally regulated under various conditions. Each of these mRNAs contains a terminal oligopyrimidine tract (TOP) that is required for translational control. A human growth hormone (hGH) expression construct containing the promoter region and 5' untranslated region (UTR) of EF1A linked to the hGH coding region (EF1A/hGH) was translationally repressed following rapamycin treatment in similar fashion to endogenous EF1A in human B lymphocytes. Mutation of two nucleotides in the TOP motif abolished the translational regulation. Gel mobility shift assays showed that both La protein from human B lymphocyte cytoplasmic extracts as well as purified recombinant La protein specifically bind to an in vitro-synthesized RNA containing the 5' UTR of EF1A mRNA. Moreover, extracts prepared from rapamycin-treated cells showed increased binding activity to the EF1A 5' UTR RNA, which correlates with TOP mRNA translational repression. In an in vitro translation system, recombinant La dramatically decreased the expression of EF1A/hGH construct mRNA, but not mRNAs lacking an intact TOP element. These results indicate that TOP mRNA translation may be modulated through La binding to the TOP element.

5' Untranslated Regions↗
PubMed2001

The inhibitory effect of the autoantigen La on in vitro 3' processing of mammalian precursor tRNAs.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various precursor (pre)-tRNAs. We investigated what effect the autoantigen La has on 3' processing, since the La protein is known to bind to a 3'-terminal uridine tract of pre-tRNAs. We tested sixteen different pre-tRNA(Arg) substrates containing various 3' trailers with or without a 5' leader sequence for in vitro processing by pig 3' tRNase, and for gel-retardation in the presence or absence of human La protein. The R-TUUU series consists of four pre-tRNAs containing 6, 8, 11 and 15 nt 3' trailers ending with UUU and no 5' leader, while the R-TAGC series consists of the same four pre-tRNAs as R-TUUU except that the terminal sequence is AGC. The R-6LTUUU and R-6LTAGC series are derived from R-TUUU and R-TAGC, respectively, by adding a 6 nt 5' leader. La differentially inhibited their processing and bound to the pre-tRNAs; the 50 % inhibitory concentrations for the R-TUUU, R-TAGC, R-6LTUUU, and R-6LTAGC series were 82 to >850, >850, 2 to 292 and 573 to 785 nM, respectively, and the dissociation constants were 10 to 840, >850, 3 to 203 and 155 to 520 nM, respectively. These results indicate that both the terminal sequence UUU and the 5' leader contribute to more severe inhibition of 3' processing via tighter interaction with La. With respect to the R-TUUU and R-6LTUUU series, on the whole, the La inhibition was enhanced as the 3' trailer lengths decreased. Taken together, our results suggest that the La protein sterically hinders 3' tRNase from binding a pre-tRNA molecule probably near the cleavage site.

Animals↗
PubMed2000

The 3' UTR of human MnSOD mRNA hybridizes to a small cytoplasmic RNA and inhibits gene expression.

Human MnSOD localizes to the mitochondria and plays a key protective role by detoxifying oxygen free radicals. The MnSOD mRNA 3' UTR contains a 280-bp region (Alu-like element or Alu-E) that shows high homology to human Alu and 7SL sequences. MnSOD 3' UTR probes hybridize to a specific cytoplasmic RNA species of approximately 300 nucleotides. This antisense RNA is most likely 7SL RNA based on its size, ubiquitousness, high levels, and lack of inducibility. Hybridization of this small RNA to the MnSOD 3' UTR may modulate posttranscriptional MnSOD gene expression. This regulation could occur by several means including inhibition of translation and mRNA destabilization. Regulation at the level of translational initiation does not seem to occur as MnSOD mRNA containing the Alu-E is efficiently bound by ribosomes. To test the role of the MnSOD 3' UTR, and in particular the Alu-E in gene expression, luciferase reporter gene constructs were made containing various regions of the MnSOD 3' UTR including the Alu-E. These constructs were transfected into human A549 lung carcinoma cells and luciferase activity was measured. Reporter constructs containing the MnSOD 3' UTR and the Alu-E repress luciferase activity. Taken together, these results suggest that naturally occurring antisense RNA may bind MnSOD mRNA and repress its expression. These results also suggest that other mRNAs containing Alu elements may be similarly repressed.

3' Untranslated Regions↗
PubMed1999

Minimum requirements for substrates of mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trailer after the discriminator nucleotide from precursor tRNA (pre-tRNA). To elucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase. A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleotide, and a 3' trailer was cleaved more efficiently than the full-length wild type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher than that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor stem, a discriminator nucleotide, and a 3' trailer, while the enzyme never cleaved a "microhelix" that is composed of a T loop, an acceptor stem, a discriminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have one to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently than R-ATM5. A helix ("minihelixDelta1") one base pair smaller than minihelices was a good substrate, while small helices containing a continuous 10-base pair stem were poor substrates. The cleavage of these three small substrates occurred after the discriminator and one to three nucleotides downstream of the discriminator. From these results, we conclude that minimum substrates for efficient cleavage by mammalian 3' tRNase are minihelices or minihelicesDelta1, in which there seem to be no essential bases.

Animals↗
PubMed1999

Long 5' leaders inhibit removal of a 3' trailer from a precursor tRNA by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various pre-tRNAs without 5' leader nucleotides. To examine how 5[prime] leader sequences affect 3' processing efficiency, we performed in vitro 3' processing reactions with purified pig 3' tRNase and pre-tRNAArgs containing a 13-nt 3' trailer and a 5[prime] leader of various lengths. The 3' processing was slightly stimulated by 5[prime] leaders containing up to 7 nt, whereas leaders of 9 nt or longer severely inhibited the reaction. Structure probing indicated that the 5' leader sequences had little effect on pre-tRNA folding. Similar results were obtained using pre-tRNA(Val)s containing a 5' leader of various lengths. We also investigated whether 3'tRNase can remove 3' trailers that are stably base-paired with 5' leaders to form an extended acceptor stem. Even such small 5' leaders as 3 and 6 nt, when base-paired with a 3' trailer, severely hindered removal of the 3' trailer by 3' tRNase.

Animals↗
PubMed1999

Selection of cleavage site by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNAArg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12-N and 13-N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase.

Animals↗
PubMed1996

Strategies for manipulating apoptosis for cancer therapy with tumor necrosis factor and lymphotoxin.

Tumor necrosis factor (TNF) and lymphotoxin (LT), initially described as tumoricidal proteins, may be useful as adjuncts in cancer therapy. Treatment with TNF or LT was found to protect cells and animals against damage mediated by radiation or cytotoxic anticancer drugs. By contrast, tumor cells treated with TNF or LT were sensitized to these insults. We present a model in which TNF or LT induces both the synthesis of "protective" proteins such as manganous superoxide dismutase (MnSOD) and the activation of "killing" proteins, such as proteases, depending on the level of the inducing signal. Although the p55-TNF/LT receptor is structurally related to the Fas receptor, they can each signal apoptosis by distinct pathways. Furthermore, activation of both receptors acts synergistically in stimulating apoptosis.

Animals↗
PubMed1996

Tumor necrosis factor and lymphotoxin: protection against oxidative stress through induction of MnSOD.

Tumor necrosis factor (TNF) and lymphotoxin (LT) are related cytokines produced in response to infection or oxidative insults such as radiation. These cytokines bind to the same receptors and have pleiotropic effects on a variety of cell types. TNF or LT pretreatment, which can induce the synthesis of "protective" proteins such as mitochondrial manganese superoxide dismutase (MnSOD), protects animals from lethal doses of radiation or the chemotherapeutic drug doxorubicin. In contrast, TNF or LT pretreatment of tumor cells, which do not express MnSOD, results in sensitization to these insults. Therefore, radio- or chemoprotection of normal cells may act partially through enhanced expression of MnSOD. On the other hand, tumor sensitization may result from activation of "killing" proteins such as interleukin-1 beta converting enzyme (ICE) or other ICE-like proteases, possibly through TNF/LT-induced oxygen free radicals. In addition to their originally described anti-tumor activity, these cytokines may have new therapeutic indications in protecting normal cells while sensitizing tumor cells to radiation or chemotherapeutic drugs.

Antioxidants↗
PubMed1994

Peripheral blood mononuclear cells stimulated with C5a or lipopolysaccharide to synthesize equivalent levels of IL-1 beta mRNA show unequal IL-1 beta protein accumulation but similar polyribosome profiles.

Recent reports suggest that regulation of IL-1 beta gene expression in human monocytes may include translation level as well as transcription level control mechanisms. We studied IL-1 beta expression in PBMC stimulated with recombinant complement protein C5a or with LPS. IL-1 beta mRNA was expressed by C5a-treated PBMC, but very little or no IL-1 beta protein could be detected by ELISA or by Western blot analysis using a polyclonal Ab that reacts equally with pro-IL-1 beta and processed mature IL-1 beta. LPS-treated cells produced both IL-1 beta mRNA and protein. Velocity sedimentation analysis revealed, however, that IL-1 beta mRNA assembles into large polyribosomes in both C5a- and LPS-treated cells. Translation of the IL-1 beta mRNA in C5a-treated cells is not therefore blocked at the level of protein synthesis initiation. IL-1 beta mRNA was released from polyribosomes by treating the cells with puromycin, suggesting that the ribosomes are not frozen at the elongation phase of protein synthesis. One explanation for the data is that the rate of IL-1 beta polypeptide chain elongation or termination may be diminished in the C5a-stimulated PBMC.

Complement C5a↗
PubMed1992

A regulatory cis element and a specific binding factor involved in the mitogenic control of murine ribosomal protein L32 translation.

The mRNA encoding ribosomal protein L32 redistributes from untranslated subribosomal particles into polysomes after mitogenic activation of quiescent T-lymphocytes and fibroblasts. To identify the regions of the L32 mRNA which are important in regulating its cytoplasmic location we constructed a plasmid containing the murine L32 cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat promoter and introduced this construct into murine 3T3 fibroblasts. The mRNA transcribed from the RSV-L32 construct redistributed from subribosomal particles into polysomes in response to mitogenic activation in a manner similar to endogenous L32 mRNA. A conserved polypyrimidine region present at the 5' terminus of all ribosomal protein mRNAs is required for translational regulation of L32 mRNA since deletion of this sequence resulted in a mRNA that was not sequestered in subribosomal particles in quiescent cells. A radioactive RNA probe containing the first 34 nucleotides of the L32 5'-untranslated region, including the polypyrimidine region, specifically interacted with a protein of about 56 kDa. This protein did not bind detectably to RNA probes lacking the polypyrimidine sequence. Binding activity was similar in protein extracts made from resting and activated cells, suggesting that binding of the 56-kDa protein as measured in this assay is not regulated. This protein is a member of what may be an emerging family of polyribopyrimidine-binding proteins with diverse biochemical functions.

3T3 Cells↗
PubMed1990

Simultaneous cytoplasmic redistribution of ribosomal protein L32 mRNA and phosphorylation of eukaryotic initiation factor 4E after mitogenic stimulation of Swiss 3T3 cells.

Ribosomal protein L32 mRNA moved from messenger ribonucleoprotein particles into polysomes following serum activation of quiescent Swiss 3T3 cells. This redistribution of the mRNA into a translationally active state began by 1 h and was complete by 3 h after activation. In contrast, actin mRNA showed no translational control, being found predominantly in polysomes in both quiescent and activated cultures. The phosphorylation state of eukaryotic initiation factor (eIF) 4E, which binds mRNA caps, was examined in parallel. eIF-4E phosphorylation was elevated by 1 h following serum activation and reached a peak by 3-5 h. Treatment of resting cells with phorbol ester also simultaneously stimulated eIF-4E phosphorylation and the movement of L32 mRNA into polysomes. These results are consistent with a model in which mitogen-induced phosphorylation increases the pool of active eIF-4E molecules, which in turn cause the recruitment of translationally controlled mRNAs to actively synthesizing ribosomes.

Actins↗
PubMed1987

Purification and physical analysis of Bacillus anthracis plasmids pXO1 and pXO2.

Virulent strains of Bacillus anthracis contain two large plasmids. pXO1 encodes the three component protein exotoxin and pXO2 is necessary for synthesis of the poly-D-glutamic acid capsule. A procedure for the isolation of these plasmids which yields high quantities of pure DNA is described. Restriction endonuclease analysis of these plasmids shows that they are not related. pXO1 is 174 kilobase pairs and pXO2 is 95 kilobase pairs. From their bouyant densities and melting temperatures we also determined their GC contents. pXO1 contains 31.1% GC base pairs and pXO2 is 31.4% GC. Both of these values are close to the GC content of B. anthracis genomic DNA which is 32.2%.

Bacillus anthracis↗
PubMed1977

Amikacin therapy of patients with multiply antibiotic-resistant Serratia marcescens infections: development of increasing resistance during therapy.

Over a recent 22 month period, 222 patients in two adjacent hospitals became infected with a multiply antibiotic-resistant strain of Serratia marcescens; 13 were bacteremic. Nineteen patients with clinically significant infections received amikacin. Nine of 11 patients with urinary tract infections were cured. In contrast, only one of eight patients with pneumonia or other deep tissue infections was cured and four died. These eight patients were severely ill; many had infections with multiple microorganisms. In four of five patients in whom the infection failed to clear promptly. Serratia strains became increasingly resistant to amikacin during therapy and these strains contributed to the death of two of these patients. Amikacin proved useful in treating patients with infections due to gentamicin-resistant S. marcescens organisms, especially urinary tract infections. However, the capacity of some strains of S. marcescens to develop resistance to amikacin may limit the usefulness of this antibiotic in the treatment of deep tissue infections which involve this microorganism.

Adult↗
PubMed1977

Perihepatitis and hepatitis as complications of experimental endocarditis due to Neisseria gonorrhoeae in the rabbit.

Seven strains of Neisseria gonorrhoeae (colony type 1 or 2) were tested for their ability to produce endocarditis in rabbits with transaortic valve catheters. The gonococci exhibited three auxotype patterns and a broad range of susceptibility to penicillin and to complement-mediated serum bactericidal activity. Only four strains produced endocarditis; infectivity appeared to be related to serum resistance. Neither arthritis nor skin lesions were observed in infected animals, but 40% had hepatitis, often with fibrinous perihepatitis. Hepatic lesions could be induced by the continuous infusion of gonococci without a transvalvular catheter. Suppurative hepatitis in the rabbit endocarditis model is of particular interest in light of the unusual hepatic involvement reported in association with gonococcal endocarditis in humans. Since perihepatitis also occurs as a complication of experimental gonococcal bacteremia, perhaps the Fitz-Hugh-Curtis (gonococcal perihepatitis) syndrome appearing in women occurs more often as a function of undiagnosed bacteremia than has heretofore been suspected.

Animals↗
PubMed1975

Rapid, simple bioassay for 5-fluorocytosine in the presence of amphotericin B.

It is important that serum levels of 5-fluorocytosine (5FC) be measured to insure therapeutic levels while avoiding toxicity. This is particularly true in patients with renal insufficiency. The concurrent use of amphotericin B and 5FC complicates the measurement of 5FC in the serum, since fungi used in conventional bioassay systems are uniformly susceptible to amphotericin B. This paper describes the development of a simple, reliable, 6-h bioassay for 5FC in the presence of amphotericin B. The assay is based upon the fact that 5FC diffuses readily through yeast nitrogen base agar, whereas amphotericin B apparently does not. This assay allows rapid adjustments in therapy of patients receiving both 5FC and amphotericin B and has permitted us to maintain 5FC serum levels between limits of 25 and 120 mug/ml in patients with altered renal function.

Amphotericin B↗
PubMed1975

Specificity of autonomic influences on cardiac responses during myocardial ischemia.

A possible role of the autonomic nervous system in the left ventricular response to acute regional myocardial ischemia was sought in conscious dogs instrumented for measurement of left ventricular pressure, internal diameter, and aortic flow. Ischemia produced by occluding the left circumflex coronary artery caused tachycardia and reduced contractility. Changes during control occlusions were compared with those during occlusion.s after beta-adrenergic blockade, parasympathetic blockade, and combined sympathetic and parasymphatetic blockade. Beta-blockade did reduce the tachycardia and slightly reduced left ventricular diameter changes in response to coronary occlusion. Results obtained in animals following surgical cardiac sympathectomy indicated reduced tachycardia and no effects on other parameters. The principal effect of parasympathetic blockade was to augment the increase in end diastolic diameter during occlusion Right atrial pacing indicated this change was due to higher initial heart rates. Combined parasympathetic and sympathetic blockade did not alter inotropic responses to coronary occlusion. Results indicated that inotropic support due to changes in activity in autonomic nerves is not increased during acute occlusion of the left circumflex coronary artery.

Animals↗