Search

After the onset of brain ischemia, a series of events leads ultimately to the death of neurons. Many molecules can be pharmacologically targeted to protect neurons during these events, which include glutamate release, glutamate receptor activation, excitotoxicity, Ca2+ influx into cells, mitochondrial dysfunction, activation of intracellular enzymes, free radical production, nitric oxide production, and inflammation. There have been a number of attempts to develop neuroprotectants for brain ischemia, but many of these attempts have failed. It was reported that cyclosporin A (CsA) dramatically ameliorates neuronal cell damage during ischemia. Some researchers consider ischemic cell death as a unique process that is distinct from both apoptosis and necrosis, and suggested that mitochondrial dysfunction and Δψ collapse are key steps for ischemic cell death. It was also suggested that CsA has a unique neuroprotective effect that is related to mitochondrial dysfunction. Here, I will exhibit examples of neuroprotectants that are now being developed or in clinical trials, and will discuss previous researches about the mechanism underlying the unique CsA action. I will then introduce the results of our cDNA subtraction experiment with or without CsA administration in the rat brain, along with our hypothesis about the mechanism underlying CsA's effect on transcriptional regulation.

Thrombospondin-1 (TSP-1), a matricellular protein widely acclaimed to be involved in the inhibition of angiogenesis and tumorigenesis, is synthesized and secreted by many cell types, including osteoblast and cancer cells. TSP-1 is highly upregulated during early stage of osteogenesis, whereas it inhibits terminal osteoblast differentiation. Expression of TSP-1 is downregulated in cancer cells, and its ectopic expression has been shown to restrain tumor growth. Transcriptional regulation of TSP-1 in osteogenesis and cancer is poorly understood; this prompted us to study its regulation by the two key regulators of the aforementioned processes: Runx2 and Runx3. Through a PCR-based cDNA subtraction technique, we identified and cloned a cDNA fragment for mouse TSP-1, whose expression was dramatically upregulated in response to Runx2 expression in mesenchymal stem cells. Moreover, TSP-1 expression was considerably reduced in the lung of Runx2 knockout mouse. On the other hand, TSP-1 gene expression drastically increased at both the transcriptional and translational levels in response to Runx3 expression in B16-F10 melanoma cells. In line with this, Runx2 and Runx3 bound to the TSP-1 promoter and stimulated its activity. Hence, these results provide first line of evidence that TSP-1 is a transcriptional target gene of Runx2 and Runx3., International Journal of Molecular Sciences, 14(7), pp.14321-14332; 2013

The preinfection stage of establishment of the mycorrhiza before physical contact between symbionts is crucial, as changes that occur throughout mycorrhiza formation are set in motion at this time. To improve the understanding of the molecular mechanisms involved in the fungus Pisolithus tinctorius during its interaction with its symbiotic partner Pinus massoniana, we set up a cellophane-separated culture system avoiding physical contact between the symbionts. A cDNA subtraction library representing the differentially expressed genes of P.tinctorius during the preinfection stage was constructed using suppression subtractive hybridization combined with mirror orientation selection (SSH/MOS). Differential screening of the SSH library allowed us to identify 133 unique cDNA clones. Sequences analysis of the expressed sequence tags (ESTs) showed that they represent 23 differentially expressed genes, of which 10 are involved in metabolism and energy, transcription and protein fate, membrane proteins and signaling, and cell rescue and defense, and 13 encoding hypothetical proteins with unknown function. Some fungal genes have not been previously identified in other ectomycorrhizal associations. Expression of the genes that code for ubiquitin-conjugating enzyme E2, 40S ribosomal protein, maternal g10 transcript, ADP ribosylation factor 6, and cytochrome P450 were evaluated by RT-qPCR, confirming the activation of these genes during the preinfection stage.

The contents of carotenoids in the storage root of sweetpotato, Ipomoea batatas (L.) Lam. vary dramatically among different cultivars. However, so far little is known about the regulation of carotenoids synthesis in sweetpotato. In our laboratory, we identified a novel sweetpotato mutant, Nongdafu 14, which is a homogenous mutant derived from the wild type Kokei No. 14. The contents of carotenoids in the storage root of Nongdafu 14 were analyzed using high performance liquid chromatography (HPLC), and it was found that the amount of carotenoids, b-carotene, lutein and zeaxantion, three major types of carotenoids in sweetpotato storage roots, increased 2–26 folds in Nongdafu 14 compared to Kokei No. 14. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed in Nongdafu 14, and a differentially expressed cDNA library was constructed using the cDNA of Nongdafu 14 storage roots as tester and that of Kokei No. 14 storage roots as driver. Out of the 1 530 clones sequenced, we identified 292 nonredundant ESTs. GO and KEGG analyses of these differentially expressed ESTs indicated that diverse metabolism pathways were affected and candidate genes involved in regulation of carotenoids synthesis are suggested.

Fusarium is the most common flax pathogen causing serious plant diseases and in most cases leading to plant death. To protect itself, the plant activates a number of genes and metabolic pathways, both to counteract the effects of the pathogen, and to eliminate the threat. The identification of the plant genes which respond to infection is the approach, that has been used in this study. Forty-seven flax genes have been identified by means of cDNA subtraction method as those, which respond to pathogen infection. Subtracted genes were classified into several classes and the prevalence of the genes involved in the broad spectrum of antioxidants biosynthesis has been noticed. By means of semi-quantitative RT-PCR and metabolite profiling, the involvement of subtracted genes controlling phenylpropanoid pathway in flax upon infection was positively verified. We identified the key genes of the synthesis of these compounds. At the same time we determined the level of the metabolites produced in the phenylpropanoid pathway (flavonoids, phenolic acids) in early response to Fusarium attack by means of GC-MS technique. To the best of our knowledge this is the first report to describe genes and metabolites of early flax response to pathogens studied in a comprehensive way.

Salem virus (SalPV) was originally isolated from a horse during a disease outbreak in 1992 in the USA. In this study, we complete the genome characterization of SalPV and confirm the classification of this virus as a member of the subfamily Paramyxovirinae. The SalPV genome is 16,698 nucleotides in length, with six transcriptional units in the order 3'-N/P/V/C-M-F-G-L-5'. SalPV has a fixed 3-nt intergenic region, which is the same as in all Paramyxovirinae members except rubulaviruses and avulaviruses. The genome size of SalPV is greater than those of the "old" members of the subfamily, such as morbilliviruses and respiroviruses, but smaller than those of the "new" members, including henipaviruses and several unclassified novel viruses. Interestingly, this evolutionary "middle point" was also reflected in the phylogeny, suggesting that SalPV represents an important intermediate between the previously well-characterized paramyxoviruses and the recently emerged members of this subfamily.

Previously, it was reported that productive viral infection, viral protein synthesis, and viral RNA replication of respiratory syncytial virus (RSV) operated efficiently in two human epithelial cell lines (HEp‐2 and A549), but not in a human mast‐cell line, HMC‐1. Based on these observations, it was hypothesized that HMC‐1 cells lack the machinery required for RSV replication. To identify the host factors required for RSV replication, cDNA subtraction using A549, HEp‐2, and HMC‐1 cells was performed, and cytokeratin 18 (C18) was identified as a candidate host factor. Because C18 is generally expressed in simple epithelia with cytokeratin 8 (C8), HMC‐1 cells that constitutively express C18 and C8 (HMC‐1‐C8/18) were established to evaluate the role of C8/18 in RSV replication. In HMC‐1‐C8/18 cells, RSV RNA replication was increased, and the amount of infective virus produced was also increased in the cellular fraction after RSV spinoculation, whereas RSV production was decreased in A549 cells in which C18 expression was knocked down. These data suggest that the replication of RSV increases in the presence of C8/18. J. Med. Virol. 84:365–370, 2012. © 2011 Wiley Periodicals, Inc.

Sexually dimorphic neural circuits are essential for reproductive behaviour. The molecular basis of sexual dimorphism in the silkworm moth (Bombyx mori) brain, however, is unclear. We conducted cDNA subtraction screening and identified nervous wreck (Bmnwk), a synaptic growth regulatory gene, whose expression is higher in the male brain than in the female brain of the silkworm. Bmnwk was preferentially expressed in the brain at the late pupae and adult stages. In situ hybridization revealed that Bmnwk is highly expressed in the optic lobe of the male moth brain. These findings suggest that Bmnwk has a role in the development and/or maintenance of the optic lobe in the male silkworm brain.

The macroalga Ulva limnetica K. Ichihara et S. Shimada is the only known Ulva species to be distributed exclusively in freshwater and is restricted to freshwater bodies in the Ryuku archipelago. Molecular phylogenetic analysis suggests that U. limnetica originally evolved from marine forms of Ulva. The mechanisms of adaptation to freshwater in Ulva spp. are poorly understood. In this study, we isolated genes potentially involved in adaptation or tolerance to freshwater conditions in U. limnetica, using suppression subtractive hybridization between mRNAs of samples cultured in freshwater and seawater conditions. A total of 219 genes, up-regulated by the exposure of the macroalga to freshwater, were isolated. Reverse transcription–PCR (RT–PCR) revealed 39 clones, including malate dehydrogenase, soluble starch synthase, triosephosphate isomerase, plastid ribosomal protein, DnaJ-like protein, and chloroplast ascorbate peroxidase (APX), which were specifically or preferentially expressed in freshwater conditions. These 39 clones were also analyzed for their temporal transcriptional response to freshwater conditions. A large majority of these up-regulated genes showed a transient peak of expression after 1–4 h, followed in the next 24 h by a decrease to a stable level (over the 7 d of the experiment). After the initial response peak, the level of expression either remained higher than in the control (long-term response) or returned to a level similar to pretreatment level. A few genes showed a more delayed response (i.e., after several days) to freshwater exposure. Finally, we discussed the possible contributions of the freshwater-induced genes in the acquisition of freshwater adaptation or tolerance of U. limnetica.

The ability of drugs of abuse to cause dependence can be viewed as a form of neural plasticity. Recently, we have demonstrated that tumor necrosis factor-α (TNF-α) increases dopamine uptake and inhibits methamphetamine-induced dependence. Moreover, we have identified a novel molecule 'shati' in the nucleus accumbens of mice treated with methamphetamine using the PCR-select cDNA subtraction method and clarified that it is involved in the development of methamphetamine dependence: Treatment with the shati antisense oligonucleotide (shati-AS), which inhibits the expression of shati mRNA, enhanced the methamphetamine-induced hyperlocomotion, sensitization, and conditioned place preference. Further, blockage of shati mRNA by shati-AS potentiated the methamphetamine-induced increase of dopamine overflow and the methamphetamine-induced decrease in dopamine uptake in the nucleus accumbens. Interestingly, treatment with shati-AS also inhibited expression of TNF-α. Transfection of the vector containing shati cDNA into PC12 cells, dramatically induced the expression of shati and TNF-α mRNA, accelerated dopamine uptake, and inhibited the methamphetamine-induced decrease in dopamine uptake. These effects were blocked by neutralizing TNF-α. These results suggest that the functional roles of shati in methamphetamine-induced behavioral changes are mediated through the induction of TNF-α expression which inhibits the methamphetamine-induced increase of dopamine overflow and decrease in dopamine uptake.

The carposporophyte life-history phase is unique to florideophycean algae and its morphological development has been thoroughly investigated. Carposporophytes are tiny, generally embedded in female gametophyte tissues, and difficult to investigate via traditional molecular methods. However, subtractive cloning of differentially expressed genes from small amounts of tissue is now possible thanks to recent methodological advances. We applied a cDNA subtraction method using magnetic beads and PCR to isolate carposporophyte-specific cDNAs from Gracilariopsis andersonii (Gracilariales, Rhodophyta). Messenger RNAs extracted from carposporophyte tissues and vegetative thalli of the gametophyte were captured on oligo-dT magnetic beads, followed by first-strand cDNA synthesis in situ. Subtractive hybridization between the amplified second-strand carposporophyte cDNA in solution and the first-strand gametophyte cDNA attached to magnetic beads was conducted to remove common genes present in both gametophyte and carp...

We previously performed cDNA subtraction between the mouse mandibles on embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to identify genes expressed at its beginning. Adenosine triphosphate synthase subunit a (Atpase6) is one of the highly expressed genes in the E12.0 mandible including tooth germs. In situ hybridization was conducted using the mouse mandibular first molar from E10.5 to E18.0 to determine the precise expression patterns of Atpase6 mRNA in the developing tooth germ. Atpase6 mRNA was strongly expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0 and E13.0. Strong in situ signals were observed in the epithelium at E14.0, and in the enamel organ excluded the area of the primary enamel knot at E15.0. Atpase6 was strongly expressed in the inner enamel epithelium, the adjacent stratum intermedium, and the outer enamel epithelium in the cervical loops from E16.0 to E18.0. In addition, strong Atpase6 signals were coincidently demonstrated in various developing cranio-facial organs. These results suggest that Atpase6 participates in the high energy-utilizing functions of the cells related to the initiation and the development of the tooth germ as well as those of the other cranio-facial organs.

Ovarian cancer, one of the most common gynecological malignancies, has an aggressive phenotype. It is necessary to develop novel and more effective treatment strategies against advanced disease. Protein tyrosine kinases (PTKs) play an important role in the signal transduction pathways involved in tumorigenesis, and represent potential targets for anticancer therapies. In this study, we performed cDNA subtraction following polymerase chain reaction (PCR) using degenerate oligonucleotide primers to identify specifically overexpressed PTKs in ovarian cancer. Three PTKs, janus kinase 1, insulin-like growth factor 1 receptor, and discoidin domain receptor 1 (DDR1), were identified and only DDR1 was overexpressed in all ovarian cancer tissues examined for the validation by quantitative real-time PCR. The DDR1 protein was expressed in 63% (42/67) of serous ovarian cancer tissue, whereas it was undetectable in normal ovarian surface epithelium. DDR1 was expressed significantly more frequently in high-grade (79%) and advanced stage (77%) tumors compared to low-grade (50%) and early stage (43%) tumors. The expression of the DDR1 protein significantly correlated with poor disease-free survival. Although its functional role and clinical utility remain to be examined in future studies, our results suggest that the expression of DDR1 may serve as both a potential biomarker and a molecular target for advanced ovarian cancer.

Cotton boll development is the result of a genetically programmed process influenced by environmental factors. Sequence information derived from advanced EST sequencing is an essential resource for functional genomics studies. The present study was undertaken to understand the gene expression profile of the early boll development stage. PCR select cDNA subtraction was employed to obtain subtracted library using cDNA prepared from −5 Days Post Anthesis stage of bud as a tester and cDNA from leaves as a driver. Differential screening of 868 positive single inserts by cDNA macroarray resulted in 170 ESTs from −5 days post anthesis bud which were further assembled into 64 contigs. Obtained results were validated by quantitative PCR for selected genes. Functional annotation showed the presence of majority of genes involved in transport processes, stress, nucleotide binding and cytoskeleton. The role of carbohydrate metabolism in bud and role of tubulin6 in fibre cell initiation were discussed.

The hair follicle is one of the skin appendages that develops through reciprocal epithelial-mesenchymal interactions. Although a large number of studies have been made on the mechanisms of hair follicle development, the whole molecular mechanism that governs hair follicle development remains poorly defined. To further understand the molecular basis of hair follicle development, it is necessary to identify genes that drive hair morphogenesis. As an initial approach, we attempted to identify gene products associated with mouse hair follicle development. Genes upregulated in the vibrissal hair placodes were screened by polymerase chain reaction (PCR)-based cDNA subtraction. The genes thus isolated were evaluated for their hair development-associated induction and spatiotemporal expression by quantitative reverse-transcription-PCR analysis and whole-mount in situ hybridization, respectively. Finally, we identified four genes whose upregulation and spatiotemporal expression in developing hair follicles were confirmed. Successful identification of novel hair development-associated genes will be informative as clues for further characterization of hair follicle development at the molecular level.

To gain a better understanding of differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance and to investigate the molecular mechanisms of Al toxicity, cDNA subtraction libraries were generated from Al-stressed roots of alfalfa (Medicago sativa L.) compared with no Al-stressed ones, employing suppression subtractive hybridization. Using differential screening technique in which the probes were labeled with DIG, we identified 45 non-redundant ESTs in Al-stressed alfalfa root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding identified proteins, including malate dehydrogenase, 6-phosphogluconate dehydrogenase, peroxidase, and an ABC transporter, while the down-regulate genes included ATPase, secretory carrier membrane protein 2, pectinesterase inhibitor. In addition, two novel ESTs, EW678752 and EY976957, up- and down-regulated by Al stress were sequenced. Analyzed by real-time PCR, the expressions of EST EW678718, EW678739, EY976969 and EW678728, which encode for ABC transporter, malate dehydrogenase, peroxidase and 6-phosphogluconate dehydrogenase correspondingly, increased 1.64-, 2.75-, 3.27- and 6.54-folds, respectively, and the expression of EY976957 encoding for ATPase decreased 3.27 folds. The expression of EST EW678752 increased 34.54-fold, while that of EY976957 decreased 16.68 folds. It suggested that the two novel ESTs maybe play a significant role in the aluminum tolerance of alfalfa.

We previously performed cDNA subtraction between the mouse mandibles at embryonic day 10.5 (E10.5) and E12.0 to make a profile of the regulator genes for odontogenesis. Fifteen kDa interferon alpha responsive gene (Ifrg15) is one of several highly-expressed genes in the E12.0 mandible. The current study examined the precise expression patterns of Ifrg15 mRNA in the mouse mandibular first molar by in situ hybridization to evaluate the possible functional roles of this gene in odontogenesis. Ifrg15 mRNA was expressed in the epithelial and mesenchymal tissues of the mandible at E10.5 and E12.0. The Ifrg15 in situ signal was detected in the epithelial bud and the surrounding mesenchyme at E14.0, and was present in the enamel organ including the primary enamel knot, and in the underlying mesenchyme at E15.0. The in situ signal was restricted in the inner and outer enamel epithelia and the stratum intermedium at E16.0. The signal of Ifrg15 mRNA was further restricted to the inner enamel epithelium and the adjacent stratum intermedium at E17.0 and E18.0. Consequently, the expression of Ifrg15 mRNA was localized in the ameloblasts and odontoblasts at postnatal days 1.0 to 3.0. However, the in situ signal was markedly weaker than at the embryonic period. The expression of Ifrg15 mRNA was coincidently observed in various craniofacial organs as well as in the tooth germ. These results suggest that Ifrg15 is closely related to odontogenesis, especially the differentiation of the ameloblasts and odontoblasts, and to the morphogenesis of the craniofacial organs.

A novel gene (BmK5) expressed in the central nervous system of the silkworm, Bombyx mori, was isolated using a cDNA subtraction method. BmK5 was first cloned as a candidate regulator of diapause hormone release from subesophageal ganglion via corpus cardiacum-corpus allatum into the hemolymph; however, subsequent analyses revealed that the gene expression patterns in brain-subesophageal ganglion complexes did not differ between diapause and nondiapause egg producers. The deduced amino acid sequence showed the characteristics of secretory protein precursor or nuclear localization protein. Immunohistochemical experiments with an anti-BmK5 antibody revealed that BmK5 precursor protein exists in the cytoplasm of specific cells of brain and frontal ganglion, but not in the nuclei. In addition, a peptide (GSGTKVGGAGAATKVVTKSGS-NH(2)) possibly processed from the BmK5 precursor protein was immunohistochemically detected in the axons connecting the anti-BmK5 antibody-positive cells to the neurohemal organ, corpus cardiacum-corpus allatum. These results suggest that BmK5 encodes a precursor of the novel neurosecretory protein and that several mature peptides are released into the hemolymph via the corpus cardiacum-corpus allatum, although the functions of these peptides are yet unclear.

Regarding bull fertility, establishing an association betweenin vitrofindings and field fertility requires a multi-parametric approach that measures the integrity of various structures and dynamic functions, such as motion characteristics, among others. The heterogeneous RNA pattern of spermatozoa could be used in genomic analysis for evaluating both spermatogenesis and fertility potential of semen, mainly because of the static status of the transcriptome of this particular differentiated cell. In a previous study, we determined that some spermatozoal transcripts identified by PCR-based cDNA subtraction are associated with non-return rate, a field fertility index. In the present study, the microarray technology was used in conjunction with differential RNA transcript extraction. We have shown that among these genes, some transcripts are also associated with the motility status of a population of sperm cells fractionated from the same ejaculate. We highlighted a systematic data analysis and validation scheme important for the identification of significant transcripts in this context. With such an approach, we found that transcripts encoding a serine/threonine testis-specific protein kinase (TSSK6) and a metalloproteinase non coding RNA (ADAM5P) are associated with high-motility status (PP=0.0075). This association was found only when transcripts were extracted using the hot-TRIzol protocol, whereas the cold-TRIzol RNA extract comprised mitochondrial transcripts. These results demonstrate that some transcripts previously identified in association with field fertility are also found associated within vitromotility provided that a stringent RNA extraction protocol is used.

The grapevine rootstock cultivar 'Borner' is resistant against phylloxera (Daktulosphaira vitifoliae Fitch), one of the major pests in viticulture. In contrast to tolerant or sensitive rootstocks, 'Borner' roots react to a phylloxera attack with a hypersensitive response exhibiting features of programmed cell death. Using cDNA subtraction in 'Borner' roots induced by IAA (a cecidogenic component of the Phylloxera saliva) and analysed at different times post induction, differential expression of several genes putatively involved in phylloxera resistance was established. Furthermore, DNA microarray analysis of subtracted and unsubtracted cDNA populations using a heterologous system (Arabidopsis thaliana) yielded more than 600 genes differentially expressed at various times after induction. Recently, a quantitative analysis of differentially expressed genes was initiated using an advanced microarray techniques ('Geniom one', febit AG) with oligo-DNA custom chips harbouring more than 450 relevant entries of the Vitis UniGen databank and a number of pathogen response-related genes of A. thaliana. Using this method, 25 up-and 7 down-regulated genes could be identified in 'Borner'. Differential expression of several of these candidate genes has been validated by quantitative RT-PCR.

Maize (Zea mays L.) is an important crop that is poorly adapted to cold stress; very little is known about the components of the response to stress. By means of the cDNA subtraction technique, several novel genes, responsive to cold stress, have been identified, including ZmCOI6.1, the function of which is unknown. The predicted ZmCOI6.1 amino acid sequence and its homologue are very similar to proteins in rice and Arabidopsis, suggesting that it belongs to a conserved group of plant proteins. Analysis of the ZmCOI6.1 promoter sequence revealed several conserved stress-responsive cis-acting elements. Further characterization of expression showed that ZmCOI6.1 was induced not only by cold, but also by drought and salinity as well as by the signaling molecules abscisic acid and salicylic acid, indicating that ZmCOI6.1 is a stress-responsive gene. ZmCOI6.1 is alternatively spliced and yields two transcripts, the level of which change depending on the stress, indicating a possible mechanism of regulation at the splicing level. Constitutive expression of ZmCOI6.1 in Arabidopsis yielded plants that were less tolerant to abiotic stress, providing evidence that ZmCOI6.1 may be a negative regulator.

Stretch Lagoon orbivirus (SLOV) was isolated in 2002 from pooled Culex annulirostris mosquitoes collected at Stretch Lagoon, near the Wolfe Creek national park in the Kimberley region of Western Australia. Conventional serological tests were unable to identify the isolate, and electron microscopy indicated a virus of the genus Orbivirus, family Reoviridae. Here, a cDNA subtraction method was used to obtain approximately one-third of the viral genome, and further sequencing was performed to complete the sequences of segment 1 (viral polymerase) and segment 2 (conserved inner-core protein). Phylogenetic analysis showed that SLOV should be considered a new species within the genus Orbivirus. A real-time RT-PCR test was designed to study the epidemiology of SLOV in the field. Six additional isolates of SLOV were identified, including isolates from four additional locations and two additional mosquito species. Horses, donkeys and goats were implicated as potential vertebrate hosts in a serological survey.

In a search for differentially expressed genes in peel pitting of ‘Navel’ orange fruit (Citrus sinensis L. Osbeck), a cDNA subtraction library was constructed and a sequence encoding expansin-like gene was isolated and identified as pitting related gene. Based on sequence information derived from this fragment, a full-length cDNA (CsEXP, GenBank accession no. FJ769424) of 1,083 nucleotides encoding expansin was isolated from ‘Navel’ orange by RACE approaches. CsEXP encoded a protein of 254 amino acid residues with an open reading frame located in the region between 52 and 816 bp. The calculated molecular weight of the mature protein was 27.05 kDa and theoretical isoelectric point was 7.93. The deduced protein contained conserved domains of expansin: the histidine-phenylalanine-aspartate motif in central portion, cysteine residues in N-terminus, and tryptophan residues in C-terminal region. The expression of CsEXP was higher in pitting than the control. Exposure of fruit to stresses, including wounding, anoxia, low temperature (4°C), and treatment with ethylene, increased CsEXP mRNA levels in comparison with the control untreated fruit, whereas high temperature (40°C) decreased its mRNA levels. Since low temperature, low oxygen and wounding were suspected factors inducing peel pitting of citrus fruit. The present results provided us a clue that CsEXP may play a role in response to peel pitting related stresses.

The cultivar ‘Borner’ is one of the Phylloxera resistant rootstocks used in Europe. In contrast to tolerant or sensitive rootstocks, ‘Borner’ roots react to a Phylloxera attack by a cell necrosis around the puncture. Histological studies sug-gested that the reaction is a kind of hypersensitive response. The aim of this study was to identify genes differentially expressed during the HR. HR was chemically induced in root cells by the application of IES. After a cDNA subtraction of induced and non-induced material, the subtracted cDNA-samples were hybridized to Arabidopsis microarray chips to identify differentially expressed candidate genes. The microarray data were analysed and differentially expressed genes were grouped into different functional categories, e.g., signal transduction pathways, transcription factors, defence associated genes. Primers were designed to target genes of interest putatively involved in the HR. So far 12 ESTs of ‘Borner’ differentially regulated in roots undergoing a HR have been sequenced and annotated. INTRODUCTION

We report an analysis of salt-stress responses in the monocotyledonous halophyte Festuca rubra ssp. litoralis. Salt-dependent expression of transcripts encoding a PIP2;1 aquaporin, V-ATPase subunit B, and the Na+/H+ antiporter NHX was characterized. Transcription of FrPIP2;1, FrVHA-B, and FrNHX1 was induced in root tissue of F rubra ssp. litoralis by salt treatment, and during salt-stress F rubra ssp. litoralis accumulated sodium in leaves and roots. Cell specificity of FrPIP2;1, FrVHA-B, and FrNHX1 transcription was analyzed by in situ PCR in roots of F rubra ssp. litoralis. Expression of the genes was localized to the root epidermis, cortex cells, endodermis, and the vascular tissue. In plants treated with 500 mM NaCl, transcripts were repressed in the epidermis and the outer cortex cells' whereas endodermis and vasculature showed strong signals. These data demonstrate that transcriptional regulation of the aquaporin PIP2;1, V-ATPase, and the Na+/H+ antiporter NHX is correlated with salt tolerance in F rubra ssp. litoralis and suggests coordinated control of ion homeostasis and water status at high salinity in plants. Salt-induced transcript accumulation in F rubra ssp. litoralis was further monitored by cDNA-arrays with expressed sequence tags derived from a cDNA subtraction library. The salt-regulated transcripts included those involved in the control of gene expression and signal transduction elements such as a serine/threonine protein kinase, an SNF1-related protein kinase, and a WRKY-type transcription factor. Other ESTs with salt-dependent regulation included transcripts encoding proteins that function in metabolism, general stress responses, and defense and transport proteins. (C) 2008 Elsevier GmbH. All rights reserved.

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17β (E 2 ) were detected in day 25 conceptuses. Concentrations of E 2 were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10ng/mL E 2 and 10ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1α and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E 2 and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.

In order to find a promoter that could be influenced by temperature shift, we explored and isolated an Aspergillus oryzae gene expressed at high temperatures (37-42 degrees C) by the cDNA subtraction method. Of the 96 cDNA clones isolated from the subtraction library, one cDNA clone showed 73% identity with Aspergillus nidulans heat shock protein 30 (hsp30). Based on this, we designated the isolated gene hsp30 of A. oryzae. A. oryzae hsp30 was weakly expressed at 30 degrees C, but strongly at 40 degrees C. We showed that the promoter of this hsp30 induced heterologous gene expression at high temperatures using beta-glucuronidase (GUS) gene as a reporter. Regarding elucidation of the region essential for heat shock response, we showed that the minimum length of the promoter region that was essential for heat shock response was located between -388 and -272 (+1 indicated the first position of the translation initiation codon) of the hsp30 promoter. This promoter region harbors several putative transcription factor binding sites, including heat shock elements (HSEs), a CCAAT box, and a TATA box. Furthermore, site-directed mutagenesis of this promoter revealed that HSE1 (aTTCgtcGAAacgcccaGAAa) and HSE2 (cGAAagTTCtcGACg), located between -342 and -272 of the hsp30 promoter, were its cis-acting elements for heat shock response.

Unfavourable environmental conditions such as cold induce the transcription of a range of genes in plants in order to acclimate to these growth conditions. To better understand the cold acclimation of maize (Zea mays L.) it is important to identify components of the cold stress response. For this purpose, cold-induced genes were analysed using the PCR-select cDNA subtraction method. We identified several novel genes isolated from maize seedling exposed for 48h to 6 degrees C. Of 18 Zea mays cold-induced genes (ZmCOI genes) characterized, the majority share similarities with proteins with known function in signal transduction and photosynthesis regulation. RT-PCR was conducted for a selected group of genes, namely ZmCOI6.1, ZmACA1, ZmDREB2A and ZmERF3, confirming the induction by low temperature. In addition, it was found that their expression was strongly induced by other abiotic stresses such as drought and high salt concentration, by stress signalling molecules such as jasmonic acid, salicylic acid and abscisic acid, and by membrane rigidification. These results suggest that this group of genes is involved in a general response to abiotic stresses.

Malassezia species are associated with pityriasis versicolor (PV) and its depigmented variant pityriasis versicolor alba (PVa), widespread fungal skin infections in humans. The pathogenesis of PV and PVa remains unclear, including their clinical and histological symptoms such as hyper- and depigmentation, reduced responsiveness to ultraviolet radiation and lack of inflammatory reaction despite high fungal load. Pigments produced by M. furfur are possibly involved in the pathogenesis of PV. In vitro, M. furfur produces a wide range of pigments and fluorochromes when cultured with tryptophan as the sole nitrogen source. We have begun to analyse the molecular basis of pigment production by searching for genes associated with tryptophan-based pigment production. A suppression subtractive hybridization (SSH) protocol was used to identify genes expressed in M. furfur cells producing pigments, but not in non-induced cells. SSH was performed 3 and 5 h after onset of pigment induction. Up-regulation of genes in the pigment-producing cells was confirmed by reverse northern analysis. More than 1,500 cDNA sequences of both the indicated time points were analysed. We identified a wide variety of genes associated with metabolism and several genes with unknown function are specifically expressed during pigment production. Furthermore, a fraction of genes possibly involved in different steps of the newly discovered indolic pathway of M. furfur were expressed in pigment producing cells. These data provide the first molecular insight into pigment production of M. furfur.

Nariva virus (NarPV) was isolated from forest rodents (Zygodontomys b. brevicauda) in eastern Trinidad in the early 1960s. Initial classification within the family Paramyxoviridae was based mainly on morphological observations including the structure of nucleocapsids and virion surface projections. Here, we report the characterization of the complete genome sequence of NarPV. The genome is 15,276 nucleotides in length, conforming to the rule-of-six, and has a genome organization typical of most members of the family, with six transcriptional units in the order 3'-N-P-M-F-H-L-5'. The gene junctions contain highly conserved gene start and stop signals and a tri-nucleotide intergenic sequence present in most members of the subfamily Paramyxovirinae. Sequence comparison studies indicate that NarPV is most closely related to Mossman virus, which was isolated from wild rats (Rattus leucopus) in Queensland, Australia, in 1970. This study confirmed the classification of NarPV as a member of the subfamily Paramyxovirinae and established the close genome organization and sequence relationship between the two rodent paramyxoviruses isolated almost a decade apart and from two locations separated by more than 15,000 km.

Salt stress is an environmental factor that severely impairs plant growth and productivity. Salinity-induced transcript accumulation was monitored in the salt-sensitive Arabidopsis thaliana and the related salt-tolerant Lobularia maritima using cDNA-arrays with expressed sequence tags derived from a cDNA subtraction library of salt-stressed L maritima. The expression profiles revealed differences of the steady state transcript regulation in A. thaliana and L maritima in response to salt stress. The differentially expressed transcripts include those involved in the control of gene expression as a transcription factor 11 homologue as well as signal transduction elements such as a serine/threonine protein kinase. a SNF1-related protein kinase AKIN10 homologue. and protein phosphatase 2C. Other ESTs with differential regulation patterns included transcripts encoding proteins with function in general stress responses and defense and included a peroxidase, dehydrins, enzymes of lipid and nitrogen metabolism, and functionally unclassified proteins. In a more detailed analysis the basic leucine zipper transcription factor AtbZIP24 showed differential transcript abundance in A. thaliana and L maritima in response to salt stress. Transgenic AtbZIP24-RNAi lines showed improved growth and development under salt stress that was correlated with changed Cl- accumulation. The data indicate that AtbZIP24 functions as a transcriptional repressor in salt-stressed A. thaliana that negatively regulates growth and development under salinity in context of controlling Cl- homeostasis. Monitoring the differential and tissue specific global regulation of gene expression during adaptation to salinity in salt-sensitive and halotolerant plants is a promising and powerful approach to identify novel elements of plant salt stress adaptation. (c) 2008 Elsevier B.V. All rights reserved.

Maternal transcripts are indispensable and play an essential role in regulation of the resumption of meiosis, nuclear reprogramming and subsequent development. In this study, we conducted interactive subtraction analysis to investigate the dynamic changes in the gene expression profiles of mouse oocytes after artificial activation. To accomplish this, we compared the cDNA libraries of freshly ovulated metaphase II (MII) oocytes and pronuclear-stage eggs at 8 h after activation, which were obtained by interactive subtraction. After constructing the cDNA libraries, we sequenced 635 subtracted clones of differentially expressed genes and the identities of 515 of these genes were determined by online BLAST analysis. The analysis revealed that genes down-regulated after activation were mainly responsible for enzyme and receptor activities, and also for DNA, chromatin, ATP, protein, and ion binding. On the other hand, genes up-regulated after activation were mainly responsible for enzyme activities, io...

application/pdf

Cardiac-specific deletion of the receptor IA of bone morphogenetic protein (BMP) (ALK3) by Cre recombinase driven under the [alpha]-MHC promoter is lethal in mid-gestation with defects in the interventricular septum [ventricular septum defect (VSD)]. Analysis of expression of the ALK3 downstream genes is important to identify the signaling pathway for interventricular septum development. The mRNA expression level of a control group was compared with that of a test group. ALK3 downstream genes were screened using polymerase chain reaction (PCR)-select cDNA subtraction and microarray. It was found that the mice with an ALK3 knockout gene produced a VSD. The expression of some genes such as platelet-activating factor acetylhydrolase (PAF) and Pax-8 was down-regulated in the test group. Pax-8 gene expression was down-regulated by 7.1 times in the test group and expressed specifically in the 11.5-d embryonic (E11.5) heart. Furthermore, the expression of the protein-tyrosine kinase of the focal adhesion kinase subfamily (PTK) and [beta] subtype protein 14-3-3 was up-regulated in the test group. PTK gene expression was up-regulated by 3.7 times in the test group. These data provided support that the ALK3 gene plays an important role during heart development. The PAF and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and [beta] subtype protein 14-3-3 might be regulatory factors in this pathway.

During development, cloned embryos often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. The long-term effects resulting from embryo cloning procedures would manifest after birth as early death, obesity, various functional disorders, and so forth. Despite extensive studies, the parameters affecting the developmental features of cloned embryos remain unclear. The present study carried out extensive gene expression analysis to screen a cluster of genes aberrantly expressed in embryonic stem cell-cloned blastocysts. Differential screening of cDNA subtraction libraries revealed 224 differentially expressed genes in the cloned blastocysts: eighty-five were identified by the BLAST search as known genes performing a wide range of functions. To confirm their differential expression, quantitative gene expression analyses were performed by real-time PCR using single blastocysts. The genes Skp1a, Canx, Ctsd, Timd2, and Psmc6 were significantly up-regulated, whereas Aqp3, Ak3l1, Rhot1, Sf3b3, Nid1, mt-Rnr2, mt-Nd1, mt-Cytb, and mt-Co2 were significantly down-regulated in the majority of embryonic stem cell-cloned embryos. Our results suggest that an extraordinarily high frequency of multiple functional disorders caused by the aberrant expression of various genes in the blastocyst stage is involved in developmental arrest and various other disorders in cloned embryos.

cDNAs specifically expressed at the basidiome stage were isolated by using PCR-selected cDNA subtraction in order to study gene regulation during porous-hymenium basidiomatal formation in Antrodia cinnamomea. blastx results suggested that most of the expressed sequence tags (52.4-69.5%) had no significant protein homology to genes from other published living things. cDNAs particularly expressed at different growing conditions were identified using cDNA microarray analysis. Reverse transcriptase PCR analyses confirmed that the clone putative to P-type ATPase, various cytochrome P450s and some unknown genes were abundant at natural basidiomes while endoglucanase was abundant at the tissue from artificial medium.

Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.

Although mungbean (Vigna radiata (L.) Wilczek) is commonly used as human food; the genomic resources of this species available in databases are limited. This study aims to develop expressed sequence tag (EST) resources for mungbean genes informative to early seedling development and chilling response. Two mungbean varieties that differ in disease resistance were found to also differ in their susceptibility to chilling temperatures. A total of 1,198 ESTs were obtained from one cDNA library and four PCR-select cDNA subtraction libraries; among these 523 were clustered into 136 contigs and 675 were singletons. The 811 non-redundant uniESTs were compared to GenBank using the Basic Local Alignment Search Tool (BLAST) and WU-BLAST algorithms, of these only 489 uniESTs had significant sequence homology, which may be involved in resuming the metabolic activity of seedlings, switching on photomorphogenesis, fuelling photosynthesis and/or initiating the unique developmental programs. Their encoded proteins may associate with regulatory proteins to trigger a direct stress response or participate in acclimation to environmental stressors. The uniEST platform reported will enrich the genomic resources of mungbean for functional genomic research on seedling development and chilling response of tropical crops and provide targets for improving the chilling tolerance of the tropical crops.

Several 'omics' sciences have been launched. The outstanding diversity of immune system components, together with the complexity of the regulatory pathways and network-type interactions, makes immunology a combinatorial science. Cancer immonomics is a branch of immunomics. The present cancer immunomics project aims at diagnostics and therapy. Comparative and structural genomics can be applied for understanding the underlying tumorogenesis process. For diagnostic purposes, in order to develop a new marker, gene expression analysis technologies, such as DNA microarray, differential display, cDNA subtraction, serial analysis of gene expression and serological proteome analysis, which enable investigators to obtain comprehensive data with respect to gene expression profiles, are progressing rapidly. For therapeutic purposes, vaccine candidate finding, gene therapy and drug-tumor interaction modeling can be clarified by the advance immunomics techniques. In this article, the author will present the basic concepts on cancer immunomics and application for basic study of the pathogenesis, diagnosis and treatment of cancer.

Homeotic transformation of stamens into pistil-like structures (called pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) having the cytoplasm of a wild relative species, Aegilops crassa. Our previous studies indicated that pistillody is caused by alterations of the class B MADS-box gene expression pattern associated with mitochondrial gene(s) in the Ae. crassa cytoplasm. To elucidate the nuclear gene involved in the cross-talk between pistillody-related mitochondrial gene(s) and nuclear homeotic genes, we performed cDNA subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. As a result, we identified a protein kinase gene, WPPK1 (wheat pistillody-related protein kinase 1), which is upregulated in the young spikes of the pistillody line. RT-PCR analysis indicated that WPPK1 is strongly expressed in pistils and pistil-like stamens in the pistillody line, suggesting that it is involved in the formation of pistil-like stamens as well as pistils. The full-length cDNA sequence for WPPK1 showed high similarity with a flowering plant PVPK-1 protein kinase, and phylogenetic analysis indicated that it is a member of AGC group protein kinases. Furthermore, a phosphorylation assay indicated that it has protein kinase activity. In situ hybridization analysis revealed that WPPK1 is expressed in developing pistils and pistil-like stamens as well as in their primordia. These indicate that in the alloplasmic line, WPPK1 plays a role in formation and development of pistil-like stamens.

The plant regulator methyl-jasmonate (MeJA) mediates diverse developmental processes and defense responses which induce a variety of secondary metabolites. This study reports the regulatory network of MeJA-induced genes in adventitious root cultures of the medicinal herb Bupleurum kaoi. A cDNA microarray containing 465 unique expressed sequence tags (ESTs) as probe sets, generated with a PCR-Select cDNA Subtraction Kit, was employed to profile the transcriptome of MeJA induction at thirteen points in time. Real-time PCR was performed to verify changes in expression of 36 unique ESTs (uniESTs), detected via microarray. Genes upregulated by MeJA include those involved in saikosaponin, amino acid, phenylpropanoid and jasmonate biosynthesis, defense signaling, plant hormone regulation and stress protection; indicating that the JA signaling pathway interacts with other signaling pathways leading to transcriptional reprogramming in B. kaoi adventitious roots. Some of these MeJA-induced genes have applications in agricultural, pharmaceutical and food industries.

The male effect is a well-known phenomenon in female sheep and goats whereby a pheromone-induced activation of reproductive function occurs. However, the molecule(s) involved in this phenomenon are unknown. We investigated gene expression profiles for the induction of male effect pheromone synthesis using a PCR-based cDNA subtraction strategy. We constructed two subtracted cDNA libraries using mRNA from the skin of the head or rump region of orchidectomized male goats with or without pheromone induction using testosterone or dihydrotestosterone (DHT). Both libraries were assumed to contain genes whose expression increases with pheromone induction. Clones (n ¼480) from each library were sequenced and identified using BLAST to reveal 115 and 239 types of sequences in the libraries of the head and rump region, respectively. Among these, 12 genes were expressed in both libraries. We conducted real-time PCR to further analyze their expression using cDNA samples derived from pheromone-producing or nonproducing skin from the head of an ovariectomized female goat with or without DHT implantation, respectively. For nine genes, we observed significantly increased expression in samples following DHT implantation. Among these, stearoyl-CoA desaturase 1 (SCD1) and elongation of long chain fatty acids family member 5 (ELOVL5) genes showed more than 100-fold higher expression levels in pheromone-positive samples, suggesting that the products of these genes may be important in pheromone synthesis. gene regulation, implantation, pheromones, seasonalreproduction, testosterone

Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB , which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain ( ΔphacB ) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB -encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).

Following baculovirus infection, the level of host messenger RNA (mRNA) declines and the synthesis of host proteins is shutoff in virus-infected cells. To comprehensively understand the regulation of host gene expression by Bombyx mori nucleopolyhedrovirus (BmNPV), we took a complementary DNA (cDNA) subtraction approach. Northern blot analysis was then performed to confirm whether obtained clones are differentially expressed. From these analyses, we obtained 4 down-, 7 up- and 2 non-regulated host gene candidates during the early stage of infection. Sequence analyses showed that these may encode proteins involved in transcription, cell cycle, cell adhesion, protein degradation, apoptosis or energy metabolism. We then measured the ADP/ATP ratio to address the response of host energy metabolism upon baculovirus infection. The ADP/ATP ratio showed cell death-like rapid increase following BmNPV infection with a transient decrease during 12–24 h post-infection (p.i.). Taken together, our results suggest that general events in host cells are controlled at the level of mRNA expression by BmNPV infection.

Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

T cells differentiate from bone marrow-derived stem cells by expressing developmental stage-specific genes. We here searched arrays of genes that are highly expressed in mature CD4−CD8+ (CD8 single-positive (SP)) T cells but little in CD4+CD8+ (double-positive (DP)) cells by cDNA subtraction. Lunatic fringe (Lfng), a modulator of Notch signaling, was identified to be little expressed in DP cells and highly expressed in CD8SP T cell as well as in CD4−CD8− (double-negative (DN)) and mature CD4+CD8− (CD4SP) T cells. Thus, we examined whether such change of expression of Lfng plays a role in T cell development. We found that overexpression of Lfng in Jurkat T cells strengthened Notch signaling by reporter gene assay, indicating that Lfng is a positive regulator for Notch signaling in T cells. The enforced expression of Lfng in thymocytes enhanced the development of immature CD8SP cells but decreased mature CD4SP and CD8SP cells. In contrast, the down-regulation of Lfng in thymocytes suppressed DP cells development due to the defective transition from CD44+CD25− stage to subsequent stage in DN cells. The overexpression of Lfng in fetal liver-derived hemopoietic stem cells enhanced T cell development, whereas its down-regulation suppressed it. These results suggested that the physiological high expression of Lfng in DN cells contributes to enhance T cell differentiation through strengthening Notch signaling. Shutting down the expression of Lfng in DP cells may have a physiological role in promoting DP cells differentiation toward mature SP cells.

In fish spermatogenesis, the main action of progestins is generally regarded as the induction of sperm maturation. Our previous in vitro study demonstrated that a progestin, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), induced the initiation of meiosis in spermatogenesis in the Japanese eel (Anguilla japonica). In the present study, to elucidate the molecular mechanisms underlying the action of DHP, we attempted to clone cDNAs encoding genes whose expression was induced by DHP in eel testis, using cDNA subtraction. One of the cDNAs we isolated encodes eel 11beta-hydroxysteroid dehydrogenase short form (e11beta-HSDsf), and Northern blot and RT-PCR analysis showed that transcripts of e11beta-HSDsf in testis were induced by DHP. The recombinant e11beta-HSDsf had 11beta-dehydrogenase activity, metabolizing cortisol to cortisone, and 11beta-hydroxytestosterone to 11-ketotestosterone (11-KT). In vitro experiments revealed that eel immature testis had 11beta-dehydrogenase activity, and DHP treatment enhanced the activity. To understand the role of 11beta-HSD in spermatogenesis, we examined the direct effects of cortisol on eel spermatogenesis using an organ culture system. Cortisol induced DNA replication in spermatogonia and enhanced the spermatogonial proliferation induced by 11-KT. However, excess cortisol inhibited proliferation. In addition, 11-KT production was induced in testicular fragments incubated with cortisol. These results suggest that optimal levels of cortisol induced spermatogonial mitosis by increasing 11-KT production. Furthermore, two possible roles of DHP on spermatogenesis, via the up-regulation of 11beta-HSD expression, are suggested: positive feedback control of 11-KT production and the modulation of cortisol levels to protect testes from excess circulating cortisol.

Background Alzheimer's disease (AD) is an age-related neurodegenerative disorder that is characterized by a progressive loss of higher cognitive functions. The brain of an individual with AD exhibits extracellular senile plaques (SPs) of aggregated amyloid-beta peptide (Aβ) and intracellular neurofibrillary tangles (NFTs). Given the critical role of neuronal transport of both proteins and organelles, it is not surprising that perturbation of microtubule-based transport may play a major role in the pathogenesis of AD. Materials and methods We used the cDNA subtraction methodology and in vitro neural cell culture analyses to study the meaning of the brain site-specific gene expression pattern in cerebral tissue obtained from AD patients and also from control subjects at autopsy. Results We observed that cytoskeleton-associated proteins were down-regulated in AD subjects. We also noted an altered expression of the microtubule-associated protein 1B (MAP1B), the heat-shock protein (HSP)-90 (a key chaperone molecule), the tripartite motif-containing proteins (TRIM)-32/37 (an anti apoptotic enzyme with ubiquitin-protein ligase activity) and the Reticulon-3 (a modulator of the amyloid-precursor-protein (APP) cleavage) in AD brains. Additional molecular- and cell-biological studies revealed that small interfering RNA (siRNA)-mediated down-regulation of MAP1B expression leads to neuronal cell death in vitro. Conclusion Altered expression of MAP1B, HSP90, TRIM32/37 and Reticulon-3 provides new clues by which the ubiquitin-proteasome-, the protein-chaperon- and the APP-processing systems are disturbed in AD, thus, leading to neuritic amyloid plaques and neurofibrillary tangles.

Synaptic vesicle protein 2B (SV2B) was identified by the subtraction hybridization technique as a molecule of which mRNA expression was decreased in puromycin aminonucleoside (PAN) nephropathy by glomerular cDNA subtraction assay. The expression of SV2B was detected in glomerular lysate with Western blot analysis. Dual-labeling immunofluorescence studies with glomerular cell markers demonstrated that SV2B is expressed in glomerular visceral epithelial cells (podocytes). The expression of SV2B is detected also in cultured podocyte and in human kidney section as podocytic pattern. The decrease of SV2B mRNA was already detected before the onset of proteinuria in PAN nephropathy. The mRNA expression of SV2B clearly is altered not only in PAN nephropathy but also in another proteinuric state that is caused by an antibody against nephrin, a functional molecule of the slit diaphragm. The decreased intensity in SV2B staining was already detected before the peak of proteinuria in both models with immunofluorescence study. A reduced amount of SV2B was detected in both models also with Western blot analysis. CD2AP, another functional molecule of the slit diaphragm, was observed in cytoplasm, including the processes area of the cultured podocyte, and when the podocyte was treated with small interfering RNA for SV2B, CD2AP staining at the process area was not detected. These results suggest that SV2B is a functional molecule of podocyte, and SV2B may play a role in the expression and proper localization of CD2AP.

Cementum is a key component of a functional periodontal organ. However, regenerating lost cementum is difficult and often incomplete. Identifying molecular mediators of cementoblast differentiation and function should lead to better targeted treatment for periodontitis. Prostaglandins increase mineralization of murine cementoblastic OCCM cells and alveolar bone formation, whereas the cytokine interleukin-1 (IL-1) inhibits alveolar bone formation. We hypothesized that differentially induced primary genes in OCCM cells may mediate anabolic and catabolic responses. Our objective was to identify primary genes differentially induced by the synthetic prostanoid fluprostenol and IL-1 in cementoblastic cells.Confluent OCCM cells were pretreated with the protein synthesis inhibitor cycloheximide followed by fluprostenol or IL-1 for 1.5 hours. cDNA generated from each group was used for cDNA subtraction hybridization to identify differentially induced genes. Preferential gene induction was verified by Northern blot analysis.Thirteen fluprostenol- and seven IL-1-regulated genes were identified. Among the fluprostenol-induced genes was mitogen-activated protein (MAP) kinase phosphatase 1 (MKP1), a negative regulator of MAP kinase signaling. To verify the cDNA subtraction hybridization results, OCCM cells were treated with fluprostenol or prostaglandin F2 (PGF2), and MKP1 mRNA levels were determined. The 0.001 to 1 microM fluprostenol and 0.01 to 1 microM PGF2 significantly induced MKP1 mRNA levels, which peaked at 1 hour of treatment and returned to baseline at 2 hours.Fluprostenol enhanced, whereas IL-1 inhibited, OCCM mineralization. Using cDNA subtraction hybridization, we identified primary genes that correlate with the observed anabolic and catabolic responses. These findings further our understanding of cementoblast function and suggest that differentially induced genes may mediate cementum formation and resorption.