Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.     

Polyadenylation promotes degradation of 3'-structured RNA by the Escherichia coli mRNA degradosome in vitro

Polyadenylation contributes to the destabilization of bacterial mRNA. We have investigated the role of polyadenylation in the degradation of RNA by the purified Escherichia coli degradosome in vitro. RNA molecules with 3'-ends incorporated into a stable stem-loop structure could not readily be degraded by purified polynucleotide phosphorylase or by the degradosome, even though the degradosome contains active RhlB helicase which normally facilitates degradation of structured RNA. The exoribonucleolytic activity of the degradosome was due to polynucleotide phosphorylase, rather than the recently reported exonucleolytic activity exhibited by a purified fragment of RNase E (Huang, H., Liao, J., and Cohen, S. N. (1998) Nature 391, 99-102). Addition of a 3'-poly(A) tail stimulated degradation by the degradosome. As few as 5 adenosine residues were sufficient to achieve this stimulation, and generic sequences were equally effective. The data show that the degradosome requires a single-stranded "toehold" 3' to a secondary structure to recognize and degrade the RNA molecule efficiently; polyadenylation can provide this single-stranded 3'-end. Significantly, oligo(G) and oligo(U) tails were unable to stimulate degradation; for oligo(G), at least, this is probably due to the formation of a G quartet structure which makes the 3'-end inaccessible. The inaccessibility of 3'-oligo(U) sequences is likely to have a role in stabilization of RNA molecules generated by Rho-independent terminators.     

The embryoid body as a novel in vitro assay system for antiangiogenic agents

Tumor progression necessitates the induction of blood vessels that converge upon the tumor and enhance the diffusibility of oxygen and nutrients. Approaches to treat cancer by antiangiogenic therapy are therefore straightforward, and there is a great need for suitable in vitro systems to test antiangiogenic agents. In the present study, embryoid bodies (EBs) differentiated from totipotent mouse embryonic stem (ES) cells and cultivated using the spinner flask technique are introduced as an in vitro system for antiangiogenesis research. ES cells effectively differentiated endothelial cells within the three-dimensional tissue of EBs. The total area of capillary-like structures, which were positive for CD31 (platelet endothelial cell adhesion molecule, PECAM-1), was assessed by confocal laser scanning microscopy and image analysis of a series of optical sections. Endothelial differentiation occurred between Day 4-5 and Day 8 of EB development. Within 7 days, 100% of EBs contained capillary-like structures. Suramin, tamoxifen, tetrahydrocortisol, and a combination of tetrahydrocortisol and heparin were tested for their antiangiogenic capacity in the EB system and were found to efficiently inhibit endothelial differentiation. Diffusion studies of a 10-kd 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran and the fluorescent, amphiphilic agent doxorubicin in avascular and vascularized EBs revealed that the endothelial structures formed functional vessels that facilitated diffusion. The diffusion coefficient D for doxorubicin was 296 x 10(-9) cm2 s(-1) in vascularized 8-day-old EBs, ie, about 10-fold larger than in avascular 3-day-old EBs (18 x 10(-9) cm2 s(-1)) and EBs treated with suramin (14 x 10(-9) cm2 s(-1)), tamoxifen (13.5 x 10(-9) cm2 s(-1)), and tetrahydrocortisol/heparin (18.5 x 10(-9) cm2 s(-1)). Consequently, avascular EBs treated with antiangiogenic agents developed central necrosis, which was absent in vascularized EBs. Our findings indicate that EBs are a suitable in vitro model system to study the effects of antiangiogenic agents in a three-dimensional tissue context. Furthermore, EBs provide a unique model to investigate the diffusion of anticancer agents in a tissue in both the avascular and vascularized states.     

改造阀门稳定净化系统的负压

通过对系统的局部改造，达到了稳定系统负压的作用，极大地提高了主电机的工作效率。     

In vitro characterization of a novel, tissue-targeted ultrasonic contrast system with acoustic microscopy

Targeted ultrasonic contrast systems are designed to enhance the reflectivity of selected tissues in vivo [Lanza et al., Circulation 94, 3334 (1996)]. In particular, these agents hold promise for the minimally invasive diagnosis and treatment of a wide array of pathologies, most notably tumors, thromboses, and inflamed tissues. In the present study, acoustic microscopy was used to assess the efficacy of a novel, perfluorocarbon based contrast agent to enhance the inherent acoustic reflectivity of biological and synthetic substrates. Data from these experiments were used to postulate a simple model describing the observed enhancements. Frequency averaged reflectivity (30-55 MHz) was shown to increase 7.0 +/- 1.1 dB for nitrocellulose membranes with targeted contrast. Enhancements of 36.0 +/- 2.3 dB and 8.5 +/- 0.9 dB for plasma and whole blood clots, respectively, were measured between 20 and 35 MHz. A proposed acoustic transmission line model predicted the targeted contrast system would increase the acoustic reflectivity of the nitrocellulose membrane, whole blood clot, and fibrin plasma clot by 2.6, 8.0, and 31.8 dB, respectively. These predictions were in reasonable agreement with the experimental results of this paper. In conclusion, acoustic microscopy provides a rapid and sensitive approach for in vitro chracterization, development, and testing of mathematical models of targeted contrast systems. Given the current demand for targeted contrast systems for medical diagnostic and therapeutic use, the use of acoustic microscopy may provide a useful tool in the development of these agents.     

Three novel proteins of the syntaxin/SNAP-25 family

Intracellular membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) through the formation of complexes between these proteins present on vesicle and target membranes. All known SNARE-mediated fusion events involve members of the syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of a large number of different syntaxin and vesicle-associated membrane protein genes. To further investigate the spectrum of SNAREs and their roles in membrane trafficking we characterized three novel members of the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) subfamilies. The proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple membranes. SNAP-29 does not contain a predicted membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of syntaxins.     

Microglial activation in multiple system atrophy: a potential role for NF-kappaB/rel proteins

Microglial activation is a prominent feature of affected brain areas in multiple system atrophy. Microglia express proinflammatory peptides, which may be a result of activation of nuclear factor-KB. We investigated the nuclear presence of RelA, the 65 kDa subunit of the NF-KB/RelA family in striatum and brain stem of patients with multiple system atrophy. Affected brain areas of patients with multiple system atrophy showed a marked immunoreactivity for nuclear Rel A p65, which was almost exclusively localized in activated microglia. Interestingly nuclear translocation of Rel A was not detected in striatal tissue of controls and Parkinson disease patients. Thus, NF-kappaB/Rel A complexes may play a role in mediating microglial activation in multiple system atrophy.     

Short-lived intermediates in hemoglobin/O2 systems

By using three-dimensional computer reconstruction techniques and the production of two-dimensional unfolded maps, we analyzed the topographic organization of projections from the entorhinal cortex of the rat to the dentate gyrus. The retrograde tracers, Fast blue and Diamidino yellow, were injected at all septotemporal levels of the dentate gyrus, and the distribution of retrogradely labeled layer II cells in the entorhinal cortex was plotted by using computer-aided microscopy systems. Discrete injections of fluorescent dyes into the dentate gyrus labeled bands of layer II neurons in the entorhinal cortex that covered approximately 45% of its surface area. Injections confined to the septal half of the dentate gyrus resulted in a band that occupied the most lateral and caudomedial portions of the entorhinal cortex. Although there were subtle changes in the density of labeled cells in this region, essentially the same region of cells was labeled after any injection into the septal half of the dentate gyrus. Injections into mid-septotemporal levels of the dentate gyrus (50-75% of the distance from the septal pole) led to a distinctly different pattern of retrograde labeling. A more medial portion of the lateral entorhinal cortex and a more rostral portion of the medial entorhinal area were labeled in these cases. Another change in entorhinal labeling occurred when the injection involved the most temporal quarter of the dentate gyrus. Injections into this area led to a constrained region of entorhinal labeling that included the most medial portion of the lateral entorhinal area and the most rostral portion of the medial entorhinal area. Although the domains of cells projecting to septal, mid-septotemporal, and temporal levels of the dentate gyrus were not entirely segregated, there was relatively little overlap of the three populations of neurons. These data raise the possibility that different portions of the entorhinal-hippocampal circuit are capable of semiautonomous information processing, at least at the stage of input to the dentate gyrus.     

Molecular mechanisms of mutations in factor XIII A-subunit deficiency: in vitro expression in COS-cells demonstrates intracellular degradation of the mutant proteins

Factor XIII deficiency is an autosomal recessive bleeding disorder that is largely caused by various mutations in FXIII A-subunit gene. Characteristically, the patients lack both A-subunit activity and antigen in the circulation. Here we have analysed the consequences of four missense mutations (Met242-->Thr, Arg252-->Ile, Arg326-->Gln, Leu498 to Pro) and one stop mutation (Arg661-->Stop) in the FXIII A-subunit gene by expression in COS-cells. After transient transfection each mutant cDNA expressed mRNA at an equal level to the wild type FXIII. However, the mutant polypeptides accumulated in the cells in significantly reduced quantities and demonstrated only very low enzymatic activity. Analysis of immunoprecipitated metabolically labelled polypeptides demonstrated remarkable instability and intracellular degradation of all mutant FXIII proteins. These results verify the deleterious nature of the individual amino acid changes and confirm that protein instability and susceptibility to proteolysis are consequences of the mutations, as predicted from the three-dimensional model of crystallised FXIII A-subunit.     

delta-N-methylarginine is a novel posttranslational modification of arginine residues in yeast proteins

We have found a novel modification of protein arginine residues in the yeast Saccharomyces cerevisiae. Intact yeast cells lacking RMT1, the gene encoding the protein omega-NG-arginine methyltransferase, were labeled with the methyl donor S-adenosyl-L-[methyl-3H]methionine. The protein fraction was acid-hydrolyzed to free amino acids, which were then fractionated on a high resolution sulfonated polystyrene cation exchange column at pH 5.27 and 55 degreesC. In the absence of the omega-NG, NG-[3H]dimethylarginine product of the RMT1 methyltransferase, we were able to detect a previously obscured 3H-methylated species that migrated in the region of methylated arginine derivatives. The [3H]methyl group(s) of this unknown species were not volatilized by treatment with 2 M NaOH at 55 degreesC for up to 48 h, suggesting that they were not modifications of the terminal omega-guanidino nitrogen atoms. However, this base treatment did result in the formation of a new 3H-methylated derivative that co-chromatographed with delta-N-methylornithine on high resolution cation exchange chromatography, on reverse phase high pressure liquid chromatography, and on thin layer chromatography. From these data, we suggest that the identity of the original unknown methylated residue is delta-N-monomethylarginine. The presence of this methylated residue in yeast cells defines a novel type of protein modification reaction in eukaryotes.     

Release of reactive nitrogen intermediates from the peripheral blood-derived monocytes/macrophages of leprosy patients stimulated in vitro by tuftsin

Environmental cum medical study was conducted in asbestos cement factory. The environment was evaluated for asbestos fiber by the methods recommended by BIS. Total 355 exposed and 312 suitably matched control workers were investigated by spirometer, Wright's peak flow meter and full sized postero-anterior chest radiograph. The levels of asbestos fiber were 2 to 3 times higher than TLV i.e. 2 f/ml in pipe cutting dept., crude fiber grinding inlet count was more than the ACGIH recommended limit i.e. 5 mpccf of air in pipe cutting dept. and silica mill. In the rest of the department, fiber level as well as dust particle count were below prescribed limit. The comparison of mean values of PFT parameters of workers with 16-20 years exposure history with control one was showing statistically significant decline in mean values of FVC only suggesting restrictive type of PFT impairment in this group of workers. But in workers with more than 20 years exposure, the mean values of all the parameters studied were reduced as compared to control one suggesting combined type of PFT impairment. When the mean values of PFT parameters of exposed smokers were compared with exposed non-smokers there was statistically no significant difference. This can be due to marginal contribution of smoking habit in impairment of PFT parameters of exposed smokers. The percentages of workers with parenchymal and pleural changes due to asbestos exposure were nearly two times more in more than 20 years exposure groups as compared to 11-20 years exposure groups. The parenchymal and pleural changes due to asbestos exposure were more common in exposed smokers as compared exposed non-smokers. However the detailed analysis revealed that if smoking contributes to the development of interstitial fibrosis, the contribution is a marginal one in comparison to the effect of asbestos dust exposure.     

Molecular cloning and characterization of a novel mRNA present in the squid giant axon

Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library. The pA6 mRNA is relatively small (550 nucleotides in length) and is expressed in both nervous tissue and skeletal muscle. The axonal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative RT-PCR analysis indicate that there are 1.8 x 10(6) molecules of pA6 mRNA (approximately 0.45 pg) in the analyzed segment of the giant axon and suggest that the level of pA6 mRNA in the axonal domain of the giant fiber system might be equal to or greater than the level present in the parental cell soma. Sequence analysis of pA6 suggests that the mRNA encodes an integral membrane protein comprising 84 amino acids. The putative protein contains a single transmembrane domain located in the middle of the molecule and a phosphate-binding loop situated near the N terminus. The C-terminal region of the protein contains two potential phosphorylation sites. These four structural motifs manifest striking similarity to domains present in the ryanodine receptor, raising the possibility that pA6 represents a cephalopod intracellular calcium release channel protein.     

MR versus fluoroscopic guidance of a catheter/guidewire system: in vitro comparison of steerability

From February 1992 to November 1993, forty patients with operable breast cancer tumors larger than three centimeters were enrolled in this study of accelerated neo-adjuvant chemotherapy. Thirty-seven patients are evaluable: one patient was excluded from the protocol and two refused to continue treatment after the first cycle. Chemotherapy consisted of three presurgical cycles of CNF [cyclophosphamide at 600 mg/m2, mitoxantrone (Novantrone) at 10 mg/m2 and 5-fluorouracil at 600 mg/m2] administered every 2 weeks, plus G-CSF (5 microg/kg s.c./day on days 7-12). Twenty-six of 37 patients (70%) achieved objective tumor response and were submitted to quadrantectomy. Toxicity was easily manageable. After a median 55-month follow-up (range 48-70), no locoregional recurrences were observed. Distant metastases occurred in 12/37 (32%) patients. The five-year disease-free (DFS) and overall (OS) survival were 58% and 80%, respectively. Accelerated CNF plus G-CSF proved to be a safe and tolerable regimen yielding a good clinical response thereby increasing the possibility of breast conservation surgery for patients otherwise candidates for mastectomy.     

Lovastatin modulates in vivo and in vitro the plasminogen activator/plasmin system of rat proximal tubular cells: role of geranylgeranylation and Rho proteins

Interstitial fibrosis is one of the most deleterious events during the progression of renal deterioration after renal mass reduction. In vivo, hydroxymethylglutaryl CoA reductase inhibitors (HRI) were shown to reduce progression of glomerulosclerosis, but the mechanisms are still unclear. The present study investigates, in vivo, whether lovastatin, a potent HRI, was able to modulate the plasminogen-plasmin pathway, one of the most efficient systems involved in extracellular matrix remodeling, and characterizes in vitro the cellular mechanisms of these effects. Proximal tubules freshly isolated from rats treated for 2 d with lovastatin (4 mg/kg per d) showed increased tissue-type plasminogen activator (tPA) and urokinase (uPA) activities and antigens. Incubation with lovastatin (5 microM) of proximal tubules isolated from untreated rats induced an increase in tPA and uPA and a decrease in plasminogen activator inhibitor-1 (PAI-1) activities. In vitro, supernatants, cytosols, and membranes of renal proximal tubular cells in primary cultures had no detectable uPA activity, and lovastatin (0.1 to 10 microM) induced an increase in tPA and a decrease in PAI-1 activities and antigens. These effects were reversed by mevalonate and geranylgeranyl-pyrophosphate (GGPP) but not by farnesyl-pyrophosphate or LDL cholesterol. C3 exoenzyme, an inhibitor of the geranylgeranylated-activated Rho protein, reproduced the effect of lovastatin on tPA and PAI- activity and blocked its reversion by GGPP. The effect of lovastatin was associated with a disruption of cellular actin stress fibers, which was reversed by GGPP and reproduced by C3 exoenzyme. In conclusion, HRI can modify the fibrinolytic potential of proximal tubules, most likely via inhibition of geranylgeranylated Rho protein and disruption of the cytoskeleton. The resulting increase of proteolytic activity of tubular cells may serve to prevent extracellular matrix deposition and renal interstitial fibrosis.