RNA Slot Blot Analysis Research Articles

Age-dependent differential expression and activity of rat liver cytosolic inorganic pyrophosphatase gene

Besides epigenetic factors, the genetic make-up and differential gene expression not only determines aging and disease susceptibility but also the functional activity of cells in an individual. Analysis of a variety of mammalian tissues revealed that the age-associated differentially expressed genes mainly belong to inflammation, stress, and metabolism. Intracellular PPi is a by-product of multiple biosynthetic reactions and its hydrolysis by cytosolic inorganic pyrophosphatase (iPPase) has long been considered as an important homeostatic mechanism favoring biosynthesis. In this paper we report an age-associated increase ( approximately 2-fold) in the expression of rat liver cytosolic iPPase gene by differential display PCR and northern blot analysis. Expression profiling of iPPase by RNA slot blot analysis in several other tissues revealed no significant change with aging. A comparative spectrophotometric and in-gel analysis of iPPase activity in whole cell lysate (WCL) of liver, brain, skeletal muscle, heart, spleen and kidney exhibited that liver of old rats (24 months ) has approximately 2-fold more activity than the adult (4 months) ones and also its activity is highest among the tissues. The specificity of iPPase activity in the spectrophotometric assay and in-gel analysis was confirmed by specific iPPase inhibitors like CaCl(2) and NaF.

Response of Bacillus subtilis to nitric oxide and the nitrosating agent sodium nitroprusside.

We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression. In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival. DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B. subtilis to NO and SNP. Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress. Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur. In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II). Aerobically, NO gas induced hmp, the sigmaB general stress regulon, and, to a lesser extent, the Fur and PerR regulons. Surprisingly, NO gas induced the sigmaB regulon via the energy branch of the sigmaB regulatory cascade while induction by SNP was mediated by the environmental stress branch. This emphasizes that NO and SNP elicit genetically distinct stress responses.

902. A Novel In Vivo, Lentivirus-Based Model of α-Spectrin Function

The study of α spectrin has yielded a better understanding of the structure and function of the membrane skeleton in erythroid and nonerythroid cells. The relationship between the primary, secondary, and tertiary structures of spectrin, a fundamental problem in hematology and biology, is complicated by the large size of spectrin, Mr 240kDa. A milestone in structure/function studies of spectrin is development of a gene delivery system capable of integrating the large (~7kb) α-spectrin cDNA in hematopoietic cells and cell lines. Two lentivirus vectors consisting of an HIV-1 vector backbone containing the ankyrin (Ank) erythroid promoter driving the human α-spectrin cDNA have been constructed. In one, pHR-Ank-αSP, the Ank promoter is in reverse orientation to the viral 5' LTR. In the other, pHR-Ank-αSP', the Ank promoter is in the same orientation and is adjacent to the viral 5' LTR. We chose the Ank promoter because it directed high-level, erythroid-specific expression with minimal position or enhancer dependence in TG mice, and, in lentiviral vectors, the Ank promoter directed erythroid-specific expression of target genes (Blood 98:2664, 2001). Lentivirus was produced by transient transfection of 293T cells with 3 plasmids: one of the lentiviral α-spectrin plasmids (pHR-Ank-alphaSP or pHR-Ank-alphaSP' an HIV-1 based packaging plasmid with the packaging signal deleted along with the LTR and envelope region, but including RRE-rev, gag/pol, and other elements, and a VSV-G envelope plasmid. Lentivirus containing supernatant was collected 48hrs after transfection. Analysis of lentivirus particles by slot blot analysis of RNA extracted from the supernatant revealed a titer of ~1x10e5. Lentivirus-conditioned supernatant was exposed to logarithmically expanding WT MEL cells in the presence of polybreen. PCR of genomic DNA from transduced cells using primers in the Ank promoter and exon 1 of the α-spectrin gene confirmed integration. The gross morphologic characteristics of the transduced MEL cells was similar to mock transduced cells. RNA extracted from the transduced cells was reverse transcribed using a human α-spectrin specific primer in exon 8. PCR of the RT product with primers corresponding to exons 7 and 8 and to the very 5' end of the human α-spectrin cDNA, exon 1 to exon 2, was positive. Immunoblot of cell lysates using a human-specific MoAB to human α-spectrin revealed a band at 240kDa in lysates from K562 cells (+ control), and both pHR-Ank-αSP and pHR-Ank-αSP' transduced cells. Similar to K562 cells, the 240kDa band was the only band seen in the transduced cells. Untransduced wild type MEL cells were negative. These experiments indicate that a very large membrane protein can be expressed using lentiviral vectors. There is also proof-of-principle for creation of in vivo model using lentiviral-mediated transfer of the α-spectrin gene into α-spectrin deficient MEL cells and HSC and progenitor cells from sph/sph mice, a murine model of α-spectrin deficient hemolytic anemia. Because sph/sph mice completely lack α-spectrin protein, this model could allow rapid assessment of many α-spectrin variants in vivo, without the need for laborious and time-consuming knock out/knock in experiments.

Characterization of expression of a functionally conserved Helicobacter pylori methyltransferase-encoding gene within inflamed mucosa and during in vitro growth.

Methylation of bacterial DNA can regulate microbial growth and virulence. Expression of hpyIM, a conserved methyltransferase of the gastric pathogen Helicobacter pylori, was quantitated in gastric biopsy specimens from 41 H. pylori-infected patients and during growth in vitro, by quantitative reverse transcriptase-polymerase chain reaction and/or RNA slot-blot analysis, to determine whether levels of transcription were associated with pathologic outcome, as based on both severity of gastritis and inflammatory cytokine levels, or were regulated by bacterial growth phase. The effects that hpyIM inactivation has on bacterial morphology were determined by electron microscopy. Expression of hpyIM varied dramatically within colonized gastric tissue, and levels were not related to either colonization density, severity of inflammation, mucosal IL-8 concentrations, or clinical disease. In vitro, hpyIM expression was higher during log-phase growth and was required for normal bacterial morphology, suggesting that hpyIM expression may be growth-phase regulated within the gastric niche.

Cloning and characterization of linR, involved in regulation of the downstream pathway for gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26.

In Sphingomonas paucimobilis UT26, LinD and LinE activities, which are responsible for the degradation of gamma-hexachlorocyclohexane, are inducibly expressed in the presence of their substrates, 2,5-dichlorohydroquinone (2,5-DCHQ) and chlorohydroquinone (CHQ). The nucleotide sequence of the 1-kb upstream region of the linE gene was determined, and an open reading frame (ORF) was found in divergent orientation from linE. Because the putative protein product of the ORF showed similarity to the LysR-type transcriptional regulator (LTTR) family, we named it linR. The fragment containing the putative LTTR recognition sequence (a palindromic TN(11)A sequence), which exists immediately upstream of linE, was ligated with the reporter gene lacZ and was inserted into the plasmid expressing LinR under the control of the lac promoter. When the resultant plasmid was introduced into Escherichia coli, the LacZ activity rose in the presence of 2,5-DCHQ and CHQ. RNA slot blot analysis for the total RNAs of UT26 and UT102, which has an insertional mutation in linR, revealed that the expression of the linD and linE genes was induced in the presence of 2,5-DCHQ, CHQ, and hydroquinone in UT26 but not in UT102. These results indicated that the linR gene is directly involved in the inducible expression of the linD and linE genes.

Expression of the Moraxella catarrhalis UspA1 protein undergoes phase variation and is regulated at the transcriptional level.

The UspA1 protein of Moraxella catarrhalis has been shown to function as an adhesin that mediates adherence to human epithelial cell lines in vitro (E. R. Lafontaine, L. D. Cope, C. Aebi, J. L. Latimer, G. H. McCracken, Jr., and E. J. Hansen, J. Bacteriol. 182:1364-1373, 2000). In the present study, cell lysates prepared from individual colonies of several M. catarrhalis wild-type strains were analyzed by Western blot analysis using monoclonal antibodies (MAbs) specific for the UspA1 protein. Expression of UspA1 was shown to exhibit phase variation that was correlated with both adherence ability in vitro and the number of guanine (G) residues contained within a homopolymeric [poly(G)]tract located upstream of the uspA1 open reading frame (ORF). Nucleotide sequence analysis revealed that isolates expressing relatively high levels of UspA1 had 10 G residues in their uspA1 poly(G)tracts, whereas isolates that expressed much lower levels of UspA1 had 9 G residues. This poly(G) tract was located 30 nucleotides (nt) upstream of the uspA1 ORF and 168 nt downstream of the uspA1 transcriptional start site. Primer extension experiments, RNA slot blot analysis, and cat reporter constructs were used to demonstrate that M. catarrhalis isolates with 10 G residues in their uspA1 poly(G) tracts expressed two-to threefold more uspA1 mRNA than did isolates which had 9 G residues in their poly(G)tracts. Northern hybridization analysis revealed that an intact uspA1 mRNA was readily detectable in RNA from M. catarrhalis isolates that had 10 G residues in their uspA1 poly(G) tracts, whereas no full-length uspA1 mRNA was observed in isolates whose poly(G)tracts contained 9 G residues. M. catarrhalis strain O35E uspA1 genes that contained wild-type and mutated poly(G) tracts were expressed in Haemophilus influenzae to demonstrate that the length and composition of the poly(G)tract affected expression of UspA1.

Identification of genes that are differentially expressed in extraocular and limb muscle

The extraocular muscles (EOM) are anatomically and physiologically distinct from other striated muscles in mammals. Among other differences, they can be driven to generate individual twitch contractions at an extremely high frequency and are resistant to [Ca 2+]-induced myonecrosis. While EOM are preferentially targeted in some neuromuscular diseases such as myasthenia gravis and congenital fibrosis of the extraocular muscles, they are enigmatically spared in Duchenne’s muscular dystrophy, despite the widespread damage seen in all other skeletal muscle groups during the course of this disease. To address the molecular mechanisms that specify the EOM-phenotype, we characterized the transcriptional profile of genes expressed in rat EOM versus limb muscle using a differential display strategy. Ninety-five putative differentially expressed cDNA tags were cloned, from which fourteen were confirmed as being differentially expressed by RNA slot blot and Northern blot analysis. Ten of these cDNAs were homologous to known human or murine genes and ESTs, while four genes that were upregulated in EOM were novel, and have been named expressed in ocular muscle ( eom) 1–4. The identification of these differentially expressed genes may provide mechanistic clues toward understanding the unique patho-physiological phenotype of EOM.

Molecular analysis of the ggtBCD gene cluster of Synechocystis sp. strain PCC6803 encoding subunits of an ABC transporter for osmoprotective compounds.

Genes encoding a substrate-binding protein (ggtB) and two integral membrane proteins (ggtC and ggtD) of the binding-protein-dependent ABC transporter for glucosylglycerol were identified in the genome of Synechocystis sp. strain PCC6803. These genes are clustered on the chromosome about 220 kb away from the previously identified ggtA gene, which encodes the ATP-binding protein of this transport system. The deduced amino acid sequences show significant similarities to corresponding subunits of ABC transporters mediating uptake of maltose and other di- and oligosaccharides in bacteria and archaea. Mutants were constructed by inserting an aphII gene cassette into the coding region of the ggtB, ggtC and ggtD genes. These mutants lost the ability to take up glucosylglycerol, sucrose and trehalose, proving that these compounds are transported by the same system. A truncated ggtB gene lacking the putative signal-peptide-encoding sequence was expressed in Escherichia coli yielding a histidine-tagged soluble protein. The recombinant GgtB protein bound glucosylglycerol with a KD of 0.45 microM and exhibited a somewhat lower affinity towards sucrose and a substantially lower affinity towards trehalose. Transcript analysis by RT-PCR indicated that the genes of the ggtBCD gene cluster form an operon. The transcript level estimated by RNA slot blot analysis using a ggtC-specific probe was very low in cells grown in basal medium but increased significantly after a salt shock.

Rhizobium etli Genetically Engineered for the Heterologous Expression of Vitreoscilla sp. Hemoglobin: Effects on Free-Living and Symbiosis

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibited higher nitrogenase activity and total nitrogen content (68% and 14 to 53%, respectively) than bean plants inoculated with the R. etli wild type. These results suggest that the synthesis of VHb in engineered R. etli stimulated the respiratory efficiency of free-living rhizobia, and also probably of symbiotic bacteroids, thus leading to higher levels of symbiotic nitrogen fixation.

Intratracheal injection of adenovirus containing the human MNSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis

Purpose: A dose and volume limiting factor in radiation treatment of thoracic cancer is the development of fibrosis in normal lung. The goal of the present study was to determine whether expression prior to irradiation of a transgene for human manganese superoxide dismutase (MnSOD) or human copper/zinc superoxide dismutase (Cu/ZnSOD) protects against irradiation-induced lung damage in mice. Methods and Materials: Athymic Nude (Nu/J) mice were intratracheally injected with 10 9 plaque-forming units (PFU) of a replication-incompetent mutant adenovirus construct containing the gene for either human MnSOD, human copper/zinc superoxide dismutase (Cu/ZnSOD) or LacZ. Four days later the mice were irradiated to the pulmonary cavity to doses of 850, 900, or 950 cGy. To demonstrate adenoviral infection, nested reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out with primers specific for either human MnSOD or Cu/ZnSOD transgene on freshly explanted lung, trachea, or alveolar type II cells, and immunohistochemistry was used to measure LacZ expression. RNA was extracted on day 0, 1, 4, or 7 after 850 cGy of irradiation from lungs of mice that had previously received adenovirus or had no treatment. Slot blot analysis was performed to quantitate RNA expression for IL-1, tumor necrosis factor (TNF)-α, TGF-β, MnSOD, or Cu/ZnSOD. Lung tissue was explanted and tested for biochemical activity of MnSOD or Cu/ZnSOD after adenovirus injection. Other mice were sacrificed 132 days after irradiation, lungs excised, frozen in OCT, (polyvinyl alcohol, polyethylene glycol mixture) sectioned, H&E stained, and evaluated for percent of the lung demonstrating organizing alveolitis. Results: Mice injected intratracheally with adenovirus containing the gene for human MnSOD had significantly reduced chronic lung irradiation damage following 950 cGy, compared to control mice or mice injected with adenovirus containing the gene for human Cu/ZnSOD or LacZ. Immunohistochemistry for LacZ protein in adenovirus LacZ (Ad-LacZ)-injected mice demonstrated expression of LacZ in both the upper and lower airway. Nested RT-PCR showed lung expression of MnSOD and Cu/ZnSOD for at least 11 days following infection with each respective adenovirus construct. Nested RT-PCR using primers specific for human MnSOD demonstrated increased expression of the human MnSOD transgene in the trachea and alveolar type II cells 4 days after virus injection on the day of irradiation. At this time point, increased biochemical activity of MnSOD and Cu/ZnSOD respectively, was detected in lungs from these two adenovirus groups, compared to each other or to control or adenovirus LacZ mice. Slot blot analysis of RNA from lungs of mice in each group following 850 cGy irradiation demonstrated decreased expression of mRNA for interleukin-I (IL-1), TNF-α, and transforming growth factor-beta (TGF-β) in the MnSOD adenovirus-injected mice, compared to irradiated control, LacZ, or Cu/ZnSOD adenovirus-injected, irradiated mice. Mice receiving adenovirus MnSOD showed decreased organizing alveolitis at 132 days in all three dose groups, compared to irradiated control or Ad-LacZ, or Ad-Cu/ZnSOD mice. Conclusions: Overexpression of MnSOD in the lungs of mice prior to irradiation prevents irradiation-induced acute and chronic damage quantitated as decreased levels of mRNA for IL-1, TNF-α, and TGF-β in the days immediately following irradiation, and decrease in the percent of lung demonstrating fibrosis or organizing alveolitis at 132 days. These data provide a rational basis for development of gene therapy as a method of protection of the normal lung from acute and chronic sequellae of ionizing irradiation.

Analysis of gene expression in lung and thymus of tcdd treated C57BL/6 mice using differential display RT-PCR

The differential display RT-PCR technique offers a means to identify genes which are regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Treatment of female C57BL/6 mice with 10 pg TCDD/kg body weight followed by DDTR-PCR analysis with a set of different primers revealed differential expression for a number of mRNAs. RNA slot blot analysis of six of these clones confirmed differential expression. Further characterization of one of these clones obtained from lung tissue demonstrated that the corresponding mRNA is about 1 kb and shows significant TCDD induced expression in Northern blot analysis. A Genbank search of this clone showed no similarity to known sequences.

An Improved Method for Generating Retroviral Producer Clones for Vectors Lacking a Selectable Marker Gene

ABSTRACT: Most retroviral vectors used in preclinical and clinical studies contain a selectable marker gene to facilitate the generation of producer clones. However, the expression of such genes in target cells is often undesirable since this may modify cellular phenotype and invoke a host immune response. Unfortunately, the efficient identification of high-titer producer clones for vectors lacking a selectable marker gene continues to be problematic and lacking for a standard methodology. Despite recent improvements in the screening techniques for identifying high-titer producer clones without the aid of a selectable marker, a solution to the fundamental problem of the very low frequency occurrence of high-titer clones within the starting cell population has not emerged. We have developed a strategy which greatly increases the frequency of virus-producing clones, including those with high-titer, within the population of transduced cells to be screened. This approach relies on the use of high-titer vector preparations generated in 293T cells by co-transfection of retroviral packaging and vector plasmids. Viral preparations of a vector lacking a selectable marker were used to repeatedly transduce exponentially growing packaging cells at a high multiplicity of infection (MOI). Each cell in the resulting polyclonal population of producer cells contained multiple copies of the unrearranged vector genome. Greater than 95% of the clones derived from this population produced vector particles as judged by slot blot analysis of viral RNA from conditioned media. Numerous clones with estimated titers of 105-106were identified. These titers were confirmed using a standard vector genome transmission assay. This approach significantly enhances the ability, without large scale screening, to easily identify high-titer clones lacking a selectable marker and should facilitate the routine use of simplifed gene marking and therapeutic vectors.

Genes for 2,4,5-trichlorophenoxyacetic acid metabolism in Burkholderia cepacia AC1100: characterization of the tftC and tftD genes and locations of the tft operons on multiple replicons.

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2, 4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftD genes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences of tftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH of Klebsiella pneumonia. Expression of the tftC and tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes of B. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of the tftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, the tftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGH and one copy of tftCD.

Regulation by thyroid hormone of the expression of basement membrane components in rat prepubertal Sertoli cells.

The present study reports the modulation of basement membrane (BM) components, laminin, entactin, and type IV collagen, expression in prepubertal rat Sertoli cell by the thyroid hormone T3. Immunocytochemical studies of permeabilized Sertoli cells in culture showed that T3 treatment (10[-7] M for 24 h) increased the number of cells staining positive for laminin and/or entactin (from 58 +/- 5.3% to 86.4 +/- 6.5%, P < 0.01). In contrast, a strong inhibition of type IV collagen immunopositivity was observed. Western blot analysis of Sertoli cell-conditioned media indicated that T3 treatment significantly (P < 0.01) increased the level of secreted entactin by 60-65% without affecting the levels of laminin A and B1/B2 chains. Moreover, thyroid hormone treatment of Sertoli cells significantly reduced type IV collagen secretion by 62% (P < 0.05). Slot blot analysis of poly-A RNA demonstrated a significant (P < 0.01) increase in the level of entactin messenger RNA (mRNA) by 140% (P < 0.01) and a 50% reduction of type IV collagen alpha1 chain mRNA after thyroid hormone treatment. No effect of the hormone was observed on the accumulation of the laminin B1 and B2 chain mRNAs in Sertoli cell cultures. These effects cannot be ascribed to changes in the degradation of BM components, because no effect of thyroid hormone was observed on plasminogen activators or metalloproteinase secretion by Sertoli cells. These observations indicate the Sertoli cell as a source of entactin within the testis, demonstrate the ability of T3 to differentially regulate the expression of BM components, and can be regarded as a part of the integrated mechanism by which thyroid hormone affects testicular development and differentiation.

Immunodiagnosis of clonorchiasis using a recombinant antigen.

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a beta-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.

Green fluorescent protein retroviral vectors: low titer and high recombination frequency suggest a selective disadvantage.

Green fluorescent protein (GFP) has been used as a reporter molecule for gene expression because it fluoresces green after blue-light excitation. Inclusion of this gene in a vector could allow rapid, nontoxic selection of successfully transduced cells. However, many attempts by our laboratory to isolate stable retroviral producer cell clones secreting biologically active vectors containing either the highly fluorescent S65T-GFP mutant or humanized GFP have failed. Vector plasmids containing various forms of GFP and the neomycin resistance gene were transfected into three different packaging cell lines and fluorescence was observed for several days, but stable clones selected with G418 no longer fluoresced. Using confocal microscopy, the brightest cells were observed to contract and die within a matter of days. RNA slot-blot analysis of retroviral producer supernatants showed no viral production from the GFP plasmid-transfected clones, although all clones derived after transfection with an identical retroviral construct not containing GFP produced virus. Genomic Southern analysis of the GFP-transduced clones showed a much higher probability of rearrangement of the priviral sequences than in the control non-GFP clones. Overall, 18/34 S65T-GFP clones and 17/33 humanized-GFP clones had rearrangements, whereas 2/15 control non-GFP clones had rearrangements. Hence, producer cells expressing high levels of these GFP genes seem to be selected against, with stable clones undergoing major rearrangements or other mutations that both abrogate GFP expression and prevent vector production. These observations indicate that GFP may not be an appropriate reporter gene for gene transfer applications in our vector/packaging system.

Reduction of Myocardial Myoglobin in Bovine Dilated Cardiomyopathy

Myoglobin levels are decreased in various animal models of heart failure, a change that has been associated with compromised energy supply. The underlying mechanisms by which myoglobin content decreases in failing myocardium are unknown. Bovine hereditary cardiomyopathy (bCMP) displays several characteristics of human dilated cardiomyopathy with a marked desensitization of theβ-adrenoceptor signal cascade. The aim of the present study was to investigate whether a similar reduction of myoglobin can be seen in this animal model, and to elucidate the possible mechanism of this reduction. Myoglobin protein concentration was decreased by 46–47% (P<0.05) in left and right ventricular myocardium of failing hearts (n=9) compared to control hearts (n=11). No difference was found between atria of diseased and control animals. Immunohistochemistry with a polyclonal antibody against myoglobin revealed a strong and uniform labeling in cardiomyocytes of non-failing hearts. Using microscopic densitometry, immunosignals were significantly decreased in ventricular myocytes of bCMP hearts (168±5.3v118±8.6 arbitrary units,P<0.05). Moreover, myoglobin was heterogeneously distributed in bCMP hearts, with single myocytes showing no staining. Slot blot analysis of total RNA demonstrated a 40–50% reduction (P<0.05) of myoglobin mRNA levels in ventricular but not in atrial myocardium of bCMP hearts. The results support the view that a decrease of myocardial myoglobin is a general phenomenon in end-stage heart failure. It appears to be primarily due to reduced gene expression but may be aggravated by leaking from single myocytes. The decrease of myoglobin may contribute to the imbalance between energy production and energy expenditure in heart failure.

The Bradyrhizobium japonicum fegA gene encodes an iron-regulated outer membrane protein with similarity to hydroxamate-type siderophore receptors.

Iron is important in the symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, yet little is known about rhizobial iron acquisition strategies. Analysis of outer membrane proteins (OMPs) from B. japonicum 61A152 identified three iron-regulated OMPs in the size range of several known receptors for Fe(III)-scavenging siderophores. One of the iron-regulated proteins, FegA, was purified and microsequenced, and a reverse genetics approach was used to clone a fegA-containing DNA fragment. Sequencing of this fragment revealed a single open reading frame of 750 amino acids. A putative N-terminal signal sequence of 14 amino acids which would result in a mature protein of 736 amino acids with a molecular mass of 80,851 Da was predicted. FegA shares significant amino acid similarity with several Fe(III)-siderophore receptors from gram-negative bacteria and has greater than 50% amino acid similarity and 33% amino acid identity with two [corrected] bacterial receptors for hydroxamate-type Fe(III)-siderophores. A dendrogram describing total inferred sequence similarity among 36 TonB-dependent OMPs was constructed; FegA grouped with Fe(III)-hydroxamate receptors. The transcriptional start site of fegA was mapped by primer extension analysis, and a putative Fur-binding site was found in the promoter. Primer extension and RNA slot blot analysis demonstrated that fegA was expressed only in cells grown under iron-limiting conditions. This is the first report of the cloning of a gene encoding a putative Fe(III)-siderophore receptor from nitrogen-fixing rhizobia.

Induction of CYP3A and associated terfenadine N-dealkylation in rat hepatocytes cocultured with 3T3 cells.

Long-term culture of hepatocytes has been challenged by the loss of differentiated functions. In particular, there is a rapid decline in cytochrome P450 (CYP). In this study, we cocultured rat hepatocytes with 3T3 fibroblasts for 10 days, and examined hepatocyte viability, morphology, and expression of CYP3A. Terfenadine was incubated with the cultures, and its biotransformation was quantitatively analyzed by HPLC. Terfenadine is metabolized by two major pathways: C-hydroxylation to an alcohol metabolite which is further oxidized to a carboxylic acid, and N-dealkylation to azacyclonol. In rat liver, only the N-dealkylation pathway appears to be mediated by CYP3A since anti-rat CYP3A antibody inhibited azacyclonol but not alcohol metabolite formation in incubations of terfenadine with liver microsomes. Freshly isolated rat hepatocytes were seeded on top of confluent 3T3 cells. Cultures were maintained in Williams' E medium supplemented with 10% fetal bovine serum and either 0.1 mumol/L or 5 mumol/L dexamethasone. In pure hepatocyte cultures, viability, as determined by lactate dehydrogenase (LDH) activity, decreased steadily to less than 30% of initial levels by day 10. In cocultures, LDH activity remained high and was 70% of initial levels on day 10. The half-life of terfenadine disappearance was optimally maintained in cocultures treated with 5 mumol/L dexamethasone, and was associated with the increased formation of azacyclonol. On day 5, nearly 50% of added 5 mumol/L terfenadine was converted to azacyclonol within 6 h, whereas the conversion was only 4% on day 1. Western and RNA-slot blot analyses confirmed that treatment with 5 mumol/L dexamethasone induced CYP3A mRNA expression and CYP3A protein expression. This coculture system could offer a useful approach in the study of drugs and xenobiotics metabolized by CYP3A.

Effect of the embryo axis on catalase in the endosperm of germinating castor bean seeds

The effect of the embryo axis on the activity of the glyoxysomal enzyme catalase (EC 1.11.1.6) in the endosperm of germinating castor bean ( Ricinus communis L. cv. Hale) seeds was examined. The presence of the embryo axis was required for maximum levels of catalase enzyme activity and protein levels in cell-free extracts of endosperms from germinated seeds. In contrast, RNA slot and dot blot analyses revealed that the removal of the embryo axis did not affect the level of catalase transcripts in the endosperm. When cell-free extracts of isolated endosperms were subjected to non-denaturing isoelectric focusing and stained for catalase activity, at least 11 isoelectric isoforms were detected. The activity of these isoforms increased coordinately in endosperm cell-free extracts from germinated seeds. Significantly, the presence of the embryo axis was required to attain maximum activity of all isoforms observed. Further, the data presented in this paper show that following seed imbibition, the embryo axis alters the rate of synthesis of a number of endospermic proteins, in addition to catalase.