Evidence that immune RNA is messenger RNA.

Exposure of rabbit spleen cell cultures to i-RNA isolated from T2 phage-exposed rabbit peritoneal exudate cells induces the synthesis of antigen and allotype specific 19S proteins even in the presence of actinomycin D. The same i-RNA directs the synthesis of proteins with comparable properties in cell-free extracts prepared from mouse L cells, indicating that i-RNA functions as mRNA and contains the information required to code for the synthesis of IgM antibodies.     

Allele coding in genomic evaluation

Background

Genomic data are used in animal breeding to assist genetic evaluation. Several models to estimate genomic breeding values have been studied. In general, two approaches have been used. One approach estimates the marker effects first and then, genomic breeding values are obtained by summing marker effects. In the second approach, genomic breeding values are estimated directly using an equivalent model with a genomic relationship matrix. Allele coding is the method chosen to assign values to the regression coefficients in the statistical model. A common allele coding is zero for the homozygous genotype of the first allele, one for the heterozygote, and two for the homozygous genotype for the other allele. Another common allele coding changes these regression coefficients by subtracting a value from each marker such that the mean of regression coefficients is zero within each marker. We call this centered allele coding. This study considered effects of different allele coding methods on inference. Both marker-based and equivalent models were considered, and restricted maximum likelihood and Bayesian methods were used in inference.

Results

Theoretical derivations showed that parameter estimates and estimated marker effects in marker-based models are the same irrespective of the allele coding, provided that the model has a fixed general mean. For the equivalent models, the same results hold, even though different allele coding methods lead to different genomic relationship matrices. Calculated genomic breeding values are independent of allele coding when the estimate of the general mean is included into the values. Reliabilities of estimated genomic breeding values calculated using elements of the inverse of the coefficient matrix depend on the allele coding because different allele coding methods imply different models. Finally, allele coding affects the mixing of Markov chain Monte Carlo algorithms, with the centered coding being the best.

Conclusions

Different allele coding methods lead to the same inference in the marker-based and equivalent models when a fixed general mean is included in the model. However, reliabilities of genomic breeding values are affected by the allele coding method used. The centered coding has some numerical advantages when Markov chain Monte Carlo methods are used.     

Bifunctional protein conferring enhanced green fluorescence and puromycin resistance

A new genetic marker was created in which sequences from enhanced green fluorescent protein were fused to those of puromycin N-acetyl transferase. The resulting fusion protein (EGFP-puro) conferred both green fluorescence and resistance to puromycin when expressed in mammalian cells. The utility of EGFP-puro as a selectable/screenable marker was demonstrated by the ease with which a recombinant guinea pig cytomegalovirus containing EGFP-puro was isolated by a combination of puromycin selection and screening for green fluorescence. We conclude that EGFP-puro is a compact and versatile marker that should prove useful for recombinant virus and transgenic cell line construction, particularly in applications in which coding capacity is limited.     

Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

Background  

Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.%) of N.     

High-throughput gateway bicistronic retroviral vectors for stable expression in mammalian cells: exploring the biologic effects of STAT5 overexpression

Stable expression of cloned genes in mammalian cells has been achieved in the past by retroviral transduction using bicistronic retroviral vectors. In these vectors, the use of an Internal Ribosome Entry Site (IRES) allows simultaneous expression of a protein of interest and a fluorescence marker. However, traditional cDNA cloning in these vectors is often difficult. Here we report the construction of a high-throughput retroviral vector using the Invitrogen "Gateway" Cloning system. The Gateway recombination sequences (attR) flanking the ccdB and chloramphenicol resistance genes were incorporated at the 5' of the IRES of pMX-IRES-GFP, -CD2, or -CD4 vectors. Through recombination, these vectors can acquire cDNAs coding for genes of interest, which will result in simultaneous expression of the recombined gene and the marker protein. We constructed Gateway bicistronic vectors coding for the erythropoietin receptor (EpoR) and GFP, CD4, or CD2. Epo-dependent proliferation assays and analysis of Jak2-dependent EpoR cell-surface expression showed that these vectors were able to function indistinguishable from the original pMX-EpoR-IRES-GFP. The expression levels of the genes cloned upstream the IRES were proportional to the levels of expression of GFP, which was cloned downstream of the IRES. We used the same approach and generated Ba/F3 cells that overexpress STAT5a, STAT5b, or a constitutively active form of STAT5. Overexpression of STAT5 lead to a significant effect on the intrinsic adherence to plastic of these cells, but did not change their proliferative responses to cytokines. We discuss possible applications of the new vectors for cell signaling and expression cloning.     

Suppression of dendritic cell activation by anthrax lethal toxin and edema toxin depends on multiple factors including cell source, stimulus used, and function tested

Bacillus anthracis produces lethal toxin (LT) and edema toxin (ET), and they suppress the function of LPS-stimulated dendritic cells (DCs). Because DCs respond differently to various microbial stimuli, we compared toxin effects in bone marrow DCs stimulated with either LPS or Legionella pneumophila (Lp). LT, not ET, was more toxic for cells from BALB/c than from C57BL/6 (B6) as measured by 7-AAD uptake; however, ET suppressed CD11c expression. LT suppressed IL-12, IL-6, and TNF-alpha in cells from BALB/c and B6 mice but increased IL-1beta in LPS-stimulated cultures. ET also suppressed IL-12 and TNF-alpha, but increased IL-6 and IL-1beta in Lp-stimulated cells from B6. Regarding maturation marker expression, LT increased MHCII and CD86 while suppressing CD40 and CD80; ET generally decreased marker expression across all groups. We conclude that the suppression of cytokine production by anthrax toxins is dependent on variables, including the source of the DCs, the type of stimulus and cytokine measured, and the individual toxin tested. However, LT and ET enhancement or suppression of maturation marker expression is more related to the marker studied than the stimuli or cell source. Anthrax toxins are not uniformly suppressive of DC function but instead can increase function under defined conditions.     

Detection of a CfoI polymorphism within exon 5 of the human neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4)

The gene coding for the 4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) has been recently mapped in the candidate region for benign familial neonatal convulsions (BFNC) on chromosome 20q13.2–q13.3. The region is only partially covered with polymorphic markers, and so far no PCR-based polymorphisms have been described in the critical region for BFNC. We now report the first polymorphic marker in the coding region of CHRNA4. The new marker, which is detected by PCR, will be useful for evaluation of the role of CHRNA4 as a candidate gene for BFNC. It will further enable the investigation of this important brain-specific gene in association studies with different types of epileptic diseases and other neurological disorders.     

抗呼吸道合胞病毒免疫核糖核酸免疫活性的研究

本文在小鼠模型系统中,应用ELISA和~(125)IUdR释放试验分别研究抗呼吸道合胞病毒免疫核糖核酸(RSV—i—RNA)诱生血清特异性抗体活性和以细胞毒活性为指标检测其增强细胞免疫的作用。实验结果表明:RSV-i-RNA能诱生血清特异性抗体和提高鼠脾细胞对靶细胞的杀伤作用,且明显高于正常核糖核酸对照组。 RSV-i-RNA的上述作用可被RNase所阻断,但不受DNase和Pronase的影响。     

DNA-mediated transfer of the mouse gene for hypoxanthine phosphoribosyltransferase into cultured mouse cells: No integration of the transferred gene at its homologous site in the host genome

Summary An established Chinese hamster cell line was fused with microcells isolated from phenotypically stable transferent mouse cells which contained a mouse transgenome coding for an abnormal form of mouse hypoxanthine phosphoribosyltransferase (HPRT, EC. No. 2.4.2.8) (Willecke et al. 1979). Two hybrids were isolated which expressed the abnormal form of mouse HPRT but no mouse -galactosidase (GALA, EC. No. 3.2.1.22). In one of these microcell hybrids the abnormal HPRT activity segregated under counter-selective conditions with mouse chromosome 3. No mouse chromosome or additional mouse gene marker was found in the second microcell hybrid, possibly because of breakage and/or rearrangement of the integrated transgenome during the isolation of this hybrid. We conclude from these results that the transferred mouse HPRT gene in a phenotypically stable clone is not integrated at its homologous site on the host X chromosome. Rather, the transgenome is probably integrated into mouse chromosome 3, possibly due to homologies in repeated DNA sequences which may occur in the transgenome and which are interspersed at many sites in the host genome.     

In vitro model to estimate gut inflammation using co-cultured Caco-2 and RAW264.7 cells

A system for assessing the anti-inflammatory effect of food factors was developed by establishing a co-culture system with intestinal epithelial Caco-2 cells (apical side) and macrophage RAW264.7 cells (basolateral side). In this system, the stimulation of RAW264.7 cells with lipopolysaccharide was followed by a decrease in transepithelial electrical resistance, which is a marker of the integrity of the Caco-2 monolayer and an increase in TNF-α production from RAW264.7 cells and IL-8 mRNA expression in Caco-2 cells. Treatment with anti-TNF-α antibodies or budesonide suppressed in increase in TNF-α production and IL-8 mRNA expression. These results indicated that this co-culture model could imitate the gut inflammation in vivo. In addition, fucoidan, sulphated polysaccharides from brown algae, was employed as a candidate of evolution and added to the apical side of this model. Fucoidan suppressed IL-8 gene expression through a reduction in TNF-α production from RAW264.7 cells stimulated with lipopolysaccharide.     

Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

Background

Brucella abortus is the etiological agent of a worldwide zoonosis called brucellosis. This alpha-proteobacterium is dividing asymmetrically, and PdhS, an essential histidine kinase, was reported to be an old pole marker.

Results

We were interested to identify functions that could be recruited to bacterial poles. The Brucella ORFeome, a collection of cloned predicted coding sequences, was placed in fusion with yellow fluorescent protein (YFP) coding sequence and screened for polar localizations in B. abortus. We report that AidB-YFP was systematically localized to the new poles and at constrictions sites in B. abortus, either in culture or inside infected HeLa cells or RAW264.7 macrophages. AidB is an acyl-CoA dehydrogenase (ACAD) homolog, similar to E. coli AidB, an enzyme putatively involved in destroying alkylating agents. Accordingly, a B. abortus aidB mutant is more sensitive than the wild-type strain to the lethality induced by methanesulphonic acid ethyl ester (EMS). The exposure to EMS led to a very low frequency of constriction events, suggesting that cell cycle is blocked during alkylation damage. The localization of AidB-YFP at the new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression of aidB, but not the two other ACAD coding sequences, leads to multiple morphological defects.

Conclusions

Data reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation.     

A dominant nuclear streptomycin resistance marker for plant cell transformation

Summary Plant cells in photoheterotrophic culture respond to streptomycin by bleaching and retarded growth but no cell death. A new genetic marker for plant cell transformation has been developed that is based on the expression of the enzyme streptomycin phosphotransferase (SPT), and confers the ability to form green colonies on a selective medium. Coding sequences of SPT from the bacterial transposon Tn5 were placed under the control of gene expression signals derived from the Agrobacterium Ti plasmid Ach5. The 5 end of the SPT gene has been replaced with the promoter region of the gene coding for the first enzyme of agropine biosynthesis, the 3 end with that of the enzyme octopine synthase. The chimeric SPT gene has been linked to a selectable kanamycin resistance gene, and introduced into Nicotiana tabacum and Nicotiana plumbaginifolia by selection for the linked kanamycin resistance marker. Streptomycin resistance was expressed in some but not all of the kanamycin-resistant lines and was transmitted to the seed progeny as a dominant nuclear trait.     

Ultrastructural investigations on the question of mechanical activation of blood platelets

The present study addresses the question whether platelets are activated by mechanical stresses as they occur in pathologically accelerated blood flow. Their potential mechanoreceptive properties were tested by subjecting human platelets to defined fluid mechanical forces for periods of milliseconds. Platelet activation was assessed by quantitative morphology, revealing besides activated platelets, irreversibly ballooned, lytic platelets. However, this morphometrically documented "shear activation" of platelets can be suppressed almost completely by the addition of enzyme-substrate systems, capable of removing adenosine diphosphate from the suspending medium. This is in keeping with a recent study from our laboratory showing that the behaviour of lactic dehydrogenase as marker for platelet lysis and beta-thromboglobulin as release marker refuted earlier data, suggesting a direct activation of platelets by shear. It is concluded that former evidence of "shear induced platelet activation" must be interpreted as the consequence of lytic damage to a small proportion of platelets.     

ACE activity affects myogenic differentiation via mTOR signaling

Variation in ACE activity affects myogenic differentiation in C2C12 cells. The present study investigated the mechanism by which ACE influences the myogenic differentiation using the ACE-transduced C2C12 cells. Overexpression of ACE induced the down-regulation of myosin heavy chain, a late myogenic marker at 3-5 days after induction of differentiation. ACE-transduced cells exhibited the immature myotubes but an early myogenic marker (myogenin) was transiently increased at day 1. In ACE-transduced cells, phosphorylation of mTOR and its downstream effector (p70S6K) was suppressed at 2-5 day. However, upstream effector of mTOR (Akt) was transiently suppressed at day 3. Expression of IGF-II mRNA, which is controlled by mTOR, was also down-regulated during the differentiation in ACE-transduced cells. On the other hand, the treatment of cells with captopril, an ACE inhibitor, induced up-regulations of myosin heavy chain and phosphorylated p70S6K. These results suggest that ACE negatively regulates the myotube maturation via impairment of mTOR function.     

A new dominant hybrid selective marker for higher eukaryotic cells

A new dominant hybrid genetic marker, suitable for selection in higher eukaryotic cells, has been obtained by linking the promoter region of the Herpes simplex virus type I thymidine kinase gene to the gene coding for the aminoglycoside 3′ phosphotransferase coded for by the Tn5 transposon. As described for yeasts (Jimenez &; Davies, 1980), the expression of the enzyme allows the formation of cellular clones resistant to the antibiotic G-418, which is otherwise toxic to the cells. This dominant marker was expressed in all cell lines tested (human, simian and murine). The construction of the hybrid gene has been optimized and, for a given construction, transformation efficiency depends on the cell line. We also report that an unlinked gene can be cotransferred into cells with the marker and be expressed at high frequency.     

KH-type splicing regulatory protein is regulated by nuclear factor-κB signaling to mediate innate immunity in Caco-2 cells infected by <Emphasis Type="Italic">Salmonella enteritidis</Emphasis>

Salmonella enteritidis infection occurs in enterogenous diseases, such as gastroenteritis and parenteral focal infection, which often involve inflammation of intestinal epithelial cells. The nuclear factor kappa B (NF-κB) pathway participates in the innate immune response to many gram-negative pathogenic bacteria and initiates inflammation in epithelial cells. KH-type splicing regulatory protein (KSRP) is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for inflammatory response factors. However, it remains unclear whether KSRP is regulated by NF-κB signaling pathway in response to S. enteritidis infection and affects the development of inflammation. Accordingly, in this study, we investigated the role of KSRP in mediating the response to S. enteritidis in Caco-2 cells. The data revealed that S. enteritidis infection decreased KSRP expression, which was suppressed by blocking the NF-κB pathway. Additionally, S. enteritidis infection significantly increased the expression of inducible nitric oxide synthase and cyclooxygenase-2. Overexpression of KSRP reduced the expression levels of inflammatory factors in Caco-2 cells. KSRP was regulated by the NF-κB signaling pathway and participated in mediating the innate immune response to S. enteritidis infection in Caco-2 cells, and KSRP acted as a negative regulator of inflammatory gene expression.     

Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells.

Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced thymidylate synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells.     

Development of a simvastatin selection marker for a hyperthermophilic acidophile, Sulfolobus islandicus

We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P(sac7d)-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P(sac7d)-hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His(6)-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEF and hmg) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.