Slot Blot Analysis Research Articles

Use of an α-Galactosidase Gene as a Food-Grade Selection Marker for Streptococcus thermophilus

The α-galactosidase gene (aga) of Lactococcus raffinolactis ATCC 43920 was previously shown to be an efficient food-grade selection marker in Lactococcus lactis and Pediococcus acidilactici but not in Streptococcus thermophilus. In this study, we demonstrated that the α-galactosidase of L. raffinolactis is thermolabile and inoperative at 42°C, the optimal growth temperature of S. thermophilus. An in vitro assay indicated that the activity of this α-galactosidase at 42°C was only 3% of that at 30°C, whereas the enzyme retained 23% of its activity at 37°C. Transformation of Strep. thermophilus RD733 with the shuttle-vector pNZ123 bearing the aga gene of L. raffinolactis (pRAF301) generated transformants that were stable and able to grow on melibiose and raffinose at 37°C or below. The transformed cells possessed 6-fold more α-galactosidase activity after growth on melibiose than cells grown on lactose. Slot-blot analyses of aga mRNA indicated that repression by lactose occurred at the transcriptional level. The presence of pRAF301 did not interfere with the lactic acid production when the transformed cells of Strep. thermophilus were grown at the optimal temperature in milk. Using the recombinant plasmid pRAF301, which carries a chloramphenicol resistance gene in addition to aga, we showed that both markers were equally efficient at differentiating transformed from nontransformed cells. The aga gene of L. raffinolactis can be used as a highly efficient selection marker in Strep. thermophilus.

Molecular basis of florfenicol-induced increase in adherence of Staphylococcus aureus strain Newman

The aim of this study was to determine the molecular basis of the florfenicol-dependent increased adherence of Staphylococcus aureus strain Newman to HEp-2 cells. Northern slot blot analysis showed that mRNA expression of fnbA, fnbB, coa, emp and eap, coding for adhesins, was increased in the presence of 0.5 x MIC of florfenicol. Under the same conditions expression of cap5, coding for type 5 capsular polysaccharides, was distinctly decreased. Since global regulatory systems can modulate the expression of adhesins, their role in this process was investigated by including three isogenic mutants with functionally inactive global regulator systems, agr, sar or sae. Growth in the presence of 0.5 x MIC of florfenicol significantly increased the adherence to HEp-2 cells, fibronectin and fibrinogen of the Deltaagr and Deltasar mutant strains, but not that of the Deltasae mutant strain. In contrast to components of the agr or sar system, expression of saeRS was increased, suggesting a potential sae-directed decrease in the expression of cap5 and increase in the expression of genes coding for adhesins under the influence of florfenicol. Analysis of RNA stability revealed that the increased amount of transcripts of saeRS and adherence-associated genes was due to a stabilization of the respective mRNAs by florfenicol. Our data provide evidence that an activation of the global regulator sae and a stabilization of mRNA coding for specific adhesins seem to act synergically in generating a more adherent phenotype in the presence of a high subinhibitory concentration of florfenicol.

Functional Effects of Transforming Growth Factor β on Adhesive Properties of Porcine Trophectoderm

In pigs, expression and amounts of biologically active TGFbetas at the conceptus-maternal interface increase significantly as conceptuses elongate and begin the implantation process. Before their activation, secreted TGFbetas are noncovalently associated with their respective, isoform-specific latency-associated peptides (LAPs), which contain the Arg-Gly-Asp (RGD) amino acid sequence that serves as a ligand for numerous integrins. Objectives of this study were to determine whether TGFbeta1 increases production of fibronectin by porcine trophectoderm, whether porcine trophectoderm adheres specifically to fibronectin and LAP, and whether functional interactions between porcine trophectoderm and the two TGFbeta-associated proteins, fibronectin and LAP, are integrin mediated. Porcine trophectoderm cells (pTr2) were cultured in presence of TGFbeta1, LAP, or pan-neutralizing anti-TGFbeta antibody; TGFbeta specifically increased (P < 0.05) fibronectin mRNA levels, as determined by Northern and slot blot analyses. Immunofluorescence microscopy demonstrated a TGFbeta-induced increase in fibronectin in pTr2 cells. In dispersed cell adhesion assays, adhesion of pTr2 cells to fibronectin was inhibited by an RGD-containing peptide (P < 0.05) and pTr2 cells attached to recombinant LAP but not to an LAP mutant, which contained an RGE sequence rather than the RGD site (P < 0.05). Fibronectin- and LAP-coated microbeads induced integrin activation at apical surfaces of both trophectoderm and uterine luminal epithelial cells, as indicated by aggregation and transmembrane accumulation of talin detected with immunofluorescence microscopy. Cell surface biotinylation and immunoprecipitation revealed integrin subunits alphav and beta1 on apical membranes of pTr2 cells. These results suggest multiple effects of TGFbeta at the porcine conceptus-maternal interface, including integrin-mediated conceptus-maternal communication through LAP.

Post-Translational Modification of Manganese Superoxide Dismutase in Acutely Rejecting Cardiac Transplants: Role of Inducible Nitric Oxide Synthase

Nitration of a critical tyrosine residue in the active site of manganese superoxide dismutase (MnSOD) can lead to enzyme inactivation. In this study, we examined the effect of inducible nitric oxide synthase (iNOS) on MnSOD expression, activity and nitration in acutely rejecting cardiac transplants. Lewis (isograft) or Wistar-Furth (allograft) donor hearts were transplanted into Lewis recipient rats. Some rats received L-N6-(1-iminoethyl) lysine (l-NIL), a specific iNOS inhibitor. Protein nitration was determined by immunohistochemical, Western blot and slot-blot analyses. MnSOD enzyme activity and gene expression were determined using Western, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation techniques. MnSOD protein levels were decreased 50% by post-operative day 6 (POD 6), which was prevented by L-NIL. RT-PCR analysis indicated that this decrease could not be explained by any changes in MnSOD mRNA. MnSOD enzyme activity but not protein was decreased at POD 5 in untreated allografts. The loss of MnSOD activity at POD 5 was also prevented by L-NIL. Immunoreactive nitrotyrosine was apparent in untreated allografts at POD 6. Slot-blot analysis indicated that nitrotyrosine formation in allografts could be blocked by L-NIL. Nitration of MnSOD was evident upon immunoprecipitation of MnSOD followed by Western blotting for nitrotyrosine. These results suggest that the decreased MnSOD enzyme activity in acutely rejecting cardiac allografts can be attributed to a post-translational modification related to nitration arising via an iNOS-dependent pathway. This could be a potential major source of amplified oxidative stress in acute graft rejection.

Telomere-to-Centromere Ratio of Bovine Clones, Embryos, Gametes, Fetal Cells, and Adult Cells

In 1997, Dolly, the first animal cloned from an adult cell, was born. It was announced in 1999 that Dolly might be aging faster than normal because her telomeres were shorter than age-matched control sheep. Telomeres, a repeated DNA sequence located at the ends of linear chromosomes, allow for base pair loss during DNA replication. Telomere shortening acts as a "mitotic clock," leading to replicative senescence. By using whole cell lysate and slot-blot analysis, we determined the telomere-to-centromere ratio (T/C) for bovine gametes, embryos, fetal tissues (brain, heart, lung, kidney, uterus, ovary, and skin), adult donor cells, and cloned embryos. Our data indicates a consistency in T/C among the various fetal tissues. The T/C of sperm is significantly lower than in oocytes. The T/C decreases from the oocyte to the 2-8-cell stage embryo, increases dramatically at the morula stage, and decreases at the blastocyst stage. Our data shows no significant difference in T/C between cloned embryos and in vitro fertilized (IVF) embryos, but there is a significant difference between cloned embryos and adult donor cells. In conclusion, the enucleated bovine oocyte has the ability to reestablish the telomere length of adult somatic cell donor nuclei.

O15 Use of molecular identification techniques to study oral microbes and microcosms

Over the last 100 years traditional culturing techniques have shaped our understanding of the oral microflora. However it is generally accepted that around 50% of the oral microbiota is unculturable. Recent advances in molecular biology have given rise to numerous techniques which form the basis of a number of strategies that can be used to detect and identify micro‐organisms to levels and specificities unattainable by conventional culture techniques. The molecular biology basis of these techniques can be conveniently split in to two broad groups. (1) DNA hybridization techniques: these are used for slot blot and checkerboard analysis and when combined with microscopy form a powerful visualization technique – fluorescent in situ hybridization (FISH). (2) PCR based approach: development of the PCR concept has lead to techniques such as global PCR, specific PCR, nested PCR, PCR‐cloning, reverse transcriptase PCR, real time PCR and PCR‐DGGE. The above techniques can usually be used on their own however they provide even more power when used in conjunction with each other. For example PCR‐cloning or PCR‐DGGE can be used to analyse a whole community. From this, un‐named or unculturable taxa can be ‘identified’ and targeted for further characterisation. This may involve the use of PCR specific for the taxon in question, to survey a large number of samples to obtain some prevalence data or in a real time setting to quantify the taxon. Indeed, a fluorescently labelled probe could be synthesized and used to visualize the taxon in an appropriate environmental sample or microcosm using confocal laser scanning microscopy. All the above techniques add up to a powerful set of tools which if selected appropriately have the potential to revolutionize our understanding of the microbial world.

226 GREATER DYSREGULATION OF GENE EXPRESSION IN PRE-IMPLANTATION CLONED OVINE COMPARED WITH CLONED BOVINE CONCEPTUSES

In our experience, the cloning of sheep by somatic cell nuclear transfer has been less successful than with cattle (5 v. 10% live births from embryo transfer). Here, we compare data collected over many years on the pre-implantation development of nuclear transfer (NT) ovine and bovine embryos with contemporary in vitro produced (IVP) embryos at the elongation (bovine Day 16–18 v. ovine Day 14) and allantois formation (bovine Day 26/27 v. ovine Day 21) stages. Sheep NT conceptuses were generated from three fibroblast types (skin, kidney, and lung) derived from a Day 100 female fetus. Bovine NT embryos were generated from a granulosa cell line derived from a mature dairy cow. The expression of a subset of genes (IGF2, H19, IGF2R, IGFBP-2, PEG1/Mest and trophoblast-expressed genes such as IFN-τ, TKDP-1, COX-2, and placental lactogen) was examined in both species at the two stages of development, using either Northern or slot-blot analysis. Pairwise comparisons between groups were carried out using Student's t-test. The proportion of ovine NT embryos that developed into blastocysts from the three cell types combined was 11% (117/1052) compared with 79% (86/109) for bovine NT from the one cell line. After transfer to synchronized recipients, 50–60% developed to the elongated conceptus stage for both species. Day 14 ovine NT (n = 19) and IVP (n = 26) conceptus lengths were similar whereas Day 16 bovine NT (n = 9) lengths were slightly shorter than those of IVP (n = 3) conceptuses (P &gt; 0.05). However, only 30% (16/52) of ovine NT embryos developed to Day 21 compared with 64% (9/14) for Day 26/27 bovine NT. Bovine NT and IVP embryo and allantoic lengths were not significantly different; however, both lengths were shorter in ovine NT than in IVP controls (P &lt; 0.05 and &lt; 0.005, respectively). In each species, there was considerable individual variation in expression levels of most genes at each stage, probably due to variation in developmental stage of individuals even from the same day of gestation. The difference may be due to the timing of onset or termination of gene expression. In Day 16–18 bovine NT conceptuses, only the mean level of PEG1/Mest expression was significantly different (P &lt; 0.05) from that of IVP conceptuses. In contrast, mean expression levels for TKDP-1 (P &lt; 0.0001) and COX-2 (P &lt; 0.05) were higher in ovine Day 14 NT, and PEG1/Mest was higher (P &lt; 0.05) in Day 21 NT when compared with contemporary IVP controls. H19 and IGF2 expression was coordinately regulated in bovine NT and in ovine and bovine IVP conceptuses (IGF2 and H19 mRNA abundance was directly correlated). This coordinate regulation was disrupted in certain Day 21 ovine NT conceptuses, where high H19 levels occurred in the presence of low levels of IGF2, but not the converse. Thus, greater dysregulation in gene expression and loss of coordinate regulation of two key imprinted genes may explain the lower survival rate of ovine NT embryos to the early fetal stage when compared with bovine NT embryos. The effect of donor cell type on the species difference cannot be discounted. This work was supported by FRST C10X0303 programme.

Placental Expression of Substance P and Vasoactive Intestinal Peptide: Evidence for a Local Effect on Hormone Release

The present study evaluated vasoactive intestinal peptide (VIP) and substance P (SP) mRNA expressions and the localization of both peptides in first- and third-trimester human placentas. VIP and SP mRNAs were detected by slot blot analysis in first- and third-trimester placental tissues. By immunohistochemistry both neuropeptides were localized in the trophoblast (syncytium and cytotrophoblastic cells) of the chorionic villi. Because little information is available on the role of VIP and/or SP on the secretion of placental hormones, we investigated the effect of these neuropeptides on human chorionic gonadotropin (hCG) and progesterone release from primary cultured human trophoblastic and JEG-3 cells. The addition of increasing doses of VIP resulted in a dose-dependent stimulation of hCG release from cultured human trophoblast and JEG-3 cells. Increasing doses of VIP also dose-dependently stimulated progesterone secretion from primary cultured trophoblastic cells at all time points evaluated and from JEG-3 cells only after 3 h. SP did not affect hCG and progesterone secretion either in cultured human trophoblast or in JEG-3 cells. In conclusion, the present study demonstrates that VIP and SP are mainly expressed in human trophoblasts, and that VIP modulates the in vitro secretion of hCG and progesterone, suggesting a different role in trophoblastic function of the two peptides.

Five Histidine Kinases Perceive Osmotic Stress and Regulate Distinct Sets of Genes in Synechocystis

Microorganisms respond to hyperosmotic stress via changes in the levels of expression of large numbers of genes. Such responses are essential for acclimation to a new osmotic environment. To identify factors involved in the perception and transduction of signals caused by hyperosmotic stress, we examined the response of Synechocystis sp. PCC 6803, which has proven to be a particularly useful microorganism in similar analyses. We screened knockout libraries of histidine kinases (Hiks) and response regulators (Rres) in Synechocystis by DNA microarray and slot-blot hybridization analyses, and we identified several two-component systems, which we designated Hik-Rre systems, namely, Hik33-Rre31, Hik34-Rre1, and Hik10-Rre3, as well as Hik16-Hik41-Rre17, as the transducers of hyperosmotic stress. We also identified Hik2-Rre1 as a putative additional two-component system. Each individual two-component system regulated the transcription of a specific group of genes that were responsive to hyperosmotic stress.

A new assay to screen for head and neck squamous cell carcinoma using the tumor marker metallopanstimulin

Objective To date, no serologic marker has proven effective as a diagnostic test for head and neck squamous cell carcinoma (HNSCC). Levels of metallopanstimulin (MPS), as measured by a difficult to reproduce radioimmunoassay, are significantly elevated in untreated HNSCC patients. Our objective was to develop a simpler MPS assay. Methods Serum was obtained from HNSCC patients through Institutional Review Board approved protocols at the Penn State University College of Medicine and healthy volunteers donating blood at the hospital blood bank from 2000 to present. Serum MPS was immunoprecipitated, slot blotted, and Western blotted. MPS levels were quantified by densitometry. Results Forty-eight blood donors and 45 known HNSCC patients were studied. The MPS level was 14 ng/mL ± 1 (SEM) for blood donors and 36 ng/mL ± 3 (SEM) for known HNSCC patients. The difference was statistically significant ( P < 0.0001). Conclusion Slot blot analysis of MPS is a safe, effective, and reproducible assay that may be used to screen for HNSCC in high-risk populations.

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.

A high-capacity assay for PPARγ ligand regulation of endogenous aP2 expression in 3T3-L1 cells

A novel class of insulin-sensitizing agents, the thiazolidinedines (TZDs), has proven effective in the treatment of type 2 diabetes. These compounds, as well as a subclass of non-TZD insulin-sensitizing agents, have been shown to be peroxisome proliferator-activated receptor (PPAR) γ agonists. PPARγ plays a critical role in adipogenesis and PPARγ agonists have been shown to induce adipocyte differentiation. Here, PPARγ ligand activity has been assessed in murine 3T3-L1 cells, a commonly used in vitro model of adipogenesis, by measuring their ability to induce adipocyte fatty acid-binding protein (aP2) mRNA expression. In order to perform this task, we have developed a novel, multiwell assay for the direct detection of aP2 mRNA in cell lysates that is based on hybridization of mRNA to target-specific oligonucleotides. These oligonucleotide probes are conjugated to enzymes that efficiently process unique chemical substrates into robust fluorescent products. Ribosomal protein 36B4 mRNA, a gene whose expression is unaffected by adipogenesis, serves as the control in the assay. Two assay formats have been developed, a single analyte assay in which aP2 and 36B4 mRNA expression are assayed in separate lysate aliquots and a dual analyte assay which can measure aP2 and 36B4 mRNA simultaneously. Both forms of the assay have been used to quantify attomole levels of aP2 and 36B4 mRNAs in differentiating 3T3-L1 preadipocytes treated with PPARγ agonists. The potencies of PPARγ agonists determined by this novel methodology showed good correlation with those derived from aP2 mRNA slot-blot analysis and PPARγ transactivation assays. We conclude that the aP2 single and dual analyte assays both provide specific and sensitive measurements of endogenous aP2 mRNA levels that can be used to assess the activity of PPARγ ligands in 3T3-L1 cells. Since the assay obviates the need for RNA isolation and is performed in an automatable multiwell format, it can serve as a high-throughput, cell-based screen for the identification and characterization of PPARγ modulators.

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.

Detection and physical mapping of the 18S-5.8S-26S rDNA and the pKFJ660 probe with microsatellite sequences derived from the rice blast fungus (Magnaporthe grisea) in conifer species

Sequences homologous to the pKFJ660 probe, a fragment of DNA derived from the rice blast fungus (Magnaporthe grisea) carrying TC/AG repeat microsatellite sequences and 30 bp direct repeats were identified in the genome of Picea (spruce) and Pinus (pine) species by fluorescence in situ hybridization (FISH) and slot blot analyses. Slot blot analysis using the pKFJ660 probe revealed hybridization signals with genomic DNAs from various pine and spruce species. Further analyses indicated that the copy number of the (AG)30 motif was higher than 5 x 10(4) per plant genome for all plant samples tested, but the copy number of the sequences homologous to the whole pKFJ660 probe varies considerably among the 25 plant species tested. In situ hybridization of metaphase chromosomes from Pinus resinosa, P. banksiana and P. strobus showed the presence of sequences homologous to this probe on several chromosomes in a dispersed pattern. Major signals were observed on a few chromosomes indicating that some of these sequences are clustered in specific genomic locations. The locations of these repeats were compared to those of 18S-5.8S-26S rDNA in pine species. Chromosomal distribution of 18S-5.8S-26S rDNA varied among the three pine species (P. resinosa, P. banksiana and P. strobus) studied. Ribosomal DNA (rDNA) sites were identified on 14 to 20 chromosomes in these pine species.

Development of a size-restricted pIX-deleted helper virus for amplification of helper-dependent adenovirus vectors.

Helper-dependent adenovirus vectors (hdAd), which are deleted of all viral protein-coding sequences, can mediate long-term expression of a therapeutic transgene and lead to life-long, phenotypic correction in animal models of genetic disease. Here, we describe a new system for the generation of hdAd, which utilizes the DNA size restrictions imposed on an Ad virion deleted of protein IX (pIX): such virions are reported to package up to only approximately 35 kb of viral DNA. A pIX(-) helper virus (approximately 37.3 kb) was easily grown on complementing 293pIX cells. Upon infection of noncomplementing cells, this virus was not capable of forming infectious virions, but provided replicative and packaging functions for propagation of a 30-kb hdAd. The pIX(-) helper virus was effective in amplifying an hdAd and, in combination with Cre-mediated excision in the viral-packaging signal, resulted in a 1000-fold reduction in helper virus contamination in hdAd stocks compared to Cre/lox alone, as determined by plaque assay. However, through slot blot analysis of DNA isolated from virions, we determined that the ratio of hdAd to helper DNA was 500:1, similar to the ratio observed when using Cre/lox alone. Surprisingly, a large amount of the 37.3-kb helper DNA was being packaged into the pIX-deleted virions, but these virions were incapable of establishing productive infections in plaque assays, for reasons which are still unclear. Nevertheless, the pIX(-) hdAd generated in this system infected cells and expressed a transgene at levels similar to those obtained with a pIX(+) hdAd. These data suggest that, although further studies are necessary to characterize the nature of the defective helper virions formed in this system, deletion of pIX from the helper virus genome does provide an effective method to prevent recovery of functional helper virus during hdAd amplification.

Orexigenic Actions of Ghrelin in Goldfish: Feeding-Induced Changes in Brain and Gut mRNA Expression and Serum Levels, and Responses to Central and Peripheral Injections

In this study, we examined (i) the preprandial, postprandial and starvation-induced changes in the preproghrelin mRNA expression and serum ghrelin levels, and (ii) the effects of intracerebroventricular and intraperitoneal administration of ghrelin on food intake in goldfish. Slot blot analysis revealed a significant postprandial decrease in preproghrelin mRNA expression in the hypothalamus (1 and 3 h after feeding) and gut (3 h after feeding). A similar postprandial decrease (1 and 3 h after feeding) in serum ghrelin levels was also detected. In the fish that were unfed at the regular feeding time, the hypothalamic preproghrelin mRNA expression and the serum ghrelin levels remained unchanged, while the preproghrelin mRNA expression in the gut decreased 3 h after the regular feeding time. Starvation increased preproghrelin mRNA expression in the hypothalamus and gut on the 7th day. Serum ghrelin levels were significantly elevated on days 3 and 5 of starvation. Intracerebroventricular injections of n-octanoylated ghrelin-like peptides (gGRL_[1–12]) (10 ng/g body weight) and human ghrelin (1 and 10 ng/g body weight) and intraperitoneal injections of n-octanoylated gGRL_[1–12] (10 ng/g body weight), gGRL_[1–19] (100 ng/g body weight) and human ghrelin (10 and 100 ng/g body weight) stimulated food intake in goldfish. The patterns of synthesis, secretion and actions indicate that ghrelin is an orexigen in goldfish.

Importance of TRF1 for Functional Telomere Structure

Telomeres are comprised of telomeric DNA sequences and associated binding molecules. Their structure functions to protect the ends of linear chromosomes and ensure chromosomal stability. One of the mammalian telomere-binding factors, TRF1, localizes telomeres by binding to double-stranded telomeric DNA arrays. Because the overexpression of wild-type and dominant-negative TRF1 induces progressive telomere shortening and elongation in human cells, respectively, a proposed major role of TRF1 is that of a negative regulator of telomere length. Here we report another crucial function of TRF1 in telomeres. In conditional mouse TRF1 null mutant embryonic stem cells, TRF1 deletion induced growth defect and chromosomal instability. Although no clear telomere shortening or elongation was observed in short term cultured TRF1-deficient cells, abnormal telomere signals were observed, and TRF1-interacting telomere-binding factor, TIN2, lost telomeric association. Furthermore, another double-stranded telomeric DNA-binding factor, TRF2, also showed decreased telomeric association. Importantly, end-to-end fusions with detectable telomere signals at fusion points accumulated in TRF1-deficient cells. These results strongly suggest that TRF1 interacts with other telomere-binding molecules and integrates into the functional telomere structure.

Gene expression in TUNEL-positive neurons in human immunodeficiency virus-infected brain

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) results in neuronal damage and apoptosis, and both in vitro models and pathological studies suggest that a variety of neurotoxins released by HIV-infected and -activated macrophages/microglia selectively damage susceptible subsets of neurons. Confirmation of in vitro findings of mechanisms of neurodegeneration and neuronal cell dysfunction in vivo has been approached through detailed pathological analysis of regional structural damage, immunohistochemical detection of selected antigens in damaged cells, and, more recently, analysis of gene expression in whole tissue blocks or pooled populations (hundreds/thousands) of microdissected cells. Recently developed techniques of gene expression analysis through antisense mRNA amplification (aRNA) at the single-cell level may offer the potential to study pathways of neuronal cell death and to determine patterns of coordinated gene expression that may more specifically identify susceptible neuronal subclasses in vivo. Utilizing this unique technique, the authors have demonstrated, for the first time, RNA amplification and gene expression profiling in individual deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-reactive neurons microdissected from fixed, archival human brain tissue. RNA amplification was successful in >80% of TUNEL-positive neurons, and quantitative aRNA/cDNA hybridization slot-blot analysis demonstrated similar levels of actin RNA but significant differences in caspase-2 RNA expression between TUNEL-reactive and -nonreactive neurons. Reliable quantitative comparisons were achieved with modest numbers of sampled neurons (approximately 10). These studies suggest that analysis of coordinated gene expression in individual damaged neurons in vivo can be reliably used to identify neuronal subclasses that express certain susceptibility- or survival-promoting genes that may be targeted for more specific neuroprotective strategies against HIV.

Estrogen Induces de novo Progesterone Synthesis in Astrocytes

The brain is an established target for peripheral steroids, but also expresses steroidogenic enzymes and is capable of de novo ‘sex’ steroid synthesis (neurosteroidogenesis) independent of peripheral steroidogenic organs. In adrenalectomized and ovariectomized rats that do not have peripheral sources of steroids, estrogen treatment increased progesterone levels specifically in the hypothalamus, indicating that estrogen stimulates progesterone neurosteroidogenesis. Recent studies have demonstrated that specific cell types preferentially secrete specific steroids, and that astrocytes are the primary progesterone synthesizing cells in the nervous system. We hypothesized that estrogen could directly induce de novo synthesis of progesterone in astrocytes. To determine whether estrogen stimulates progesterone synthesis in astrocytes, astrocyte-enriched cultures were grown to confluence, then grown for an additional 48 h in an estrogen- and phenol-free Dulbecco’s Modified Eagle Medium (DMEM) and then treated with either 17β-estradiol or steroid-free media. After culturing for 48 h in steroid-free, phenol red-free DMEM, low levels of progesterone were detected in the media, whereas progesterone levels were significantly increased in the media of astrocytes cultured in DMEM with 17β-estradiol (10^–7–10^–4M). To determine whether estrogen regulated the mRNA expression of progesterone synthetic enzymes, P-450 side-chain cleavage and 3β-hydroxysteroid dehydrogenase, control and 17β-estradiol-treated astrocytes were harvested and prepared for Northern and slot blot analysis. Expression levels of enzyme mRNAs were very low and 17β-estradiol did not significantly increase mRNA levels of either steroidogenic enzyme. These results suggest that estrogen directly stimulated the de novo synthesis of neuroprogesterone in astrocytes, and demonstrate the potential for estrogen to regulate reproductive physiology and behavior through the paracrine actions of astrocyte-derived progesterone.

A new pitfall in detecting biological end products of nitric oxide—nitration, nitros(yl)ation and nitrite/nitrate artefacts during freezing

The present study shows that when freezing nitrite containing biological samples in the presence of sodium and phosphate, a process of tyrosine nitration and S-nitrosocysteine formation is observed. The underlying mechanism is obviously based on the already described pH decrease in sodium phosphate buffered solutions during the freezing process and probably involves nitrous acid as an intermediate. However, in pure potassium phosphate buffer freeze-artefacts were absent. The yield of 3-nitrotyrosine from albumin-bound or free tyrosine depends not only on the concentration of nitrite, tyrosine or protein, and sodium phosphate but also on the velocity of the freezing process. Nitrite and nitrate were quantified by the Griess/nitrate reductase assay. 3-Nitrotyrosine formation was quantitatively measured by HPLC analysis with optical and electrochemical detection as well as qualitatively investigated by immunohistochemistry and slot blot analysis using 3-nitrotyrosine specific antibodies. The formation of S-nitrosocysteine was detected by S-nitrosothiol specific antibodies and quantified by a fluorometric assay. Irrespective of the mechanism and although the here presented results cannot be generalized, the data warrant caution for the analysis of nitration or nitros(yl)ation products following freezing of nitrite containing biological material.