Ag,a novel protein secreted from Aplysia glia

We have isolated a cDNA (ag for Aplysia glial) corresponding to an mRNA specific to the nervous system of Aplysia californica. In this study, we characterized the ag cDNA sequence and the distribution of ag mRNA and protein in the Aplysia nervous system. The ag cDNA contains an open reading frame that encodes a novel 29 kD protein. In situ hybridizations demonstrate that ag mRNA is conspicuously absent from the cell bodies of the large neurons constituting the external layer of the ganglia. Instead, it is largely confined to a subset of small, apparently non-neuronal cells surrounding the neurons at the border of the neuropil, is sparsely scattered within the neuropil, and is widespread within the connective nerves, a pattern consistent with glial localization. Polyclonal anti-ag antiserum recognizes a protein between 27 and 29 kD that is more broadly distributed, especially within the neuropil. The distributions of ag mRNA and protein, together with the presence of a putative signal peptide, suggest that ag protein is secreted. Two findings support this hypothesis: first, ag protein is detectable by western blot in Aplysia hemolymph. Second, full length ag protein expressed in COS cells is secreted, but ag lacking the putative signal peptide is not. Secretion from glia raises the possibility that this abundant protein may affect neighboring neurons. © 1996 John Wiley & Sons, Inc.     

Identification of stress-induced genes from the drought tolerant semi-arid legume crop horsegram (Macrotyloma uniflorum (Lam.) Verdc.) through analysis of subtracted expressed sequence tags

Abiotic stresses adversely affect the agricultural productivity worldwide. Horsegram (Macrotyloma uniflorum (Lam.) Verdc.) is a legume crop that can tolerate severe adverse environmental conditions such as drought, salinity and heavy metal contamination. As a first step towards characterization of genes that contribute to combating abiotic stresses, construction and analysis of subtracted cDNA library is reported here. Using this strategy a total of 1050 ESTs were isolated, sequenced, 959 high quality ESTs were obtained and clustered. Further, our analysis revealed that of these 531 sequences are unique and 30% of these have no homology to known proteins in the database. This observation has great relevance since horsegram is a stress-adapted legume crop. Further, to validate the identified differentially expressed genes, expression profiles of selected clones were analyzed using reverse-northern, northern blot analysis and we show that indeed these clones are differentially expressed under various abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance also discussed.     

Innervation of periodontal ligament and dental pulp in the rat incisor: An immunohistochemical investigation of neurofilament protein and glia-specific S-100 protein

Summary Nervous elements in the periodontal ligament and dental pulp of rat incisors were investigated by means of immunohistochemistry for neurofilament protein (NFP) and glia-specific S-100 protein. The periodontal ligament in the incisors was densely innervated by NFP-immunoreactive nerve fibers; the distribution of the nerve fibers and their terminations differed markedly from those in molars. NFP-positive, thick nerve bundles entered the lingual periodontal ligament through slits located in the mid-region of the alveolar socket, and immediately formed numerous Ruffini-like corpuscles. In the labial periodontal ligament, all of the NFP-immunoreactive nerve fibers terminated in free endings. The restricted location of the stretch receptor, Ruffini-like corpuscle, in the lingual periodontal ligament appears to be an essential element, because this region is regularly extended during mastication. The nervous elements were restricted to the alveolar half of the periodontal ligament in every region; they avoided the dental half of the periodontal ligament, which presumably moves continuously with the tooth. Pulpal nerve fibers in incisors also showed a characteristic distribution different from those in molars; individual nerve fibers with beaded structures ran in the center of the pulp toward the incisai edge, and did not form the subodontoblastic nerve plexus of Raschkow.Immunostaining for S-100 protein revealed a distribution pattern of nervous elements similar to that for NFP, suggesting that the nerves supplying the periodontal ligament and dental pulp were mostly covered by a Schwann sheath.     

Characterization of Apin, a secreted protein highly expressed in tooth-associated epithelia

We previously reported expression of a protein by enamel organ (EO) cells in rat incisors, originally isolated from the amyloid of Pindborg odontogenic tumors called Apin. The aim of the present study was to further characterize the Apin gene and its protein in various species, assess tissue specificity, and clarify its localization within the EO. Northern blotting and RT-PCR revealed that expression of Apin was highest in the EO and gingiva, moderate in nasal and salivary glands, and lowest in the epididymis. The protein sequences deduced from the cloned cDNA for rat, mouse, pig, and human were aligned together with those obtained from four other mammal genomes. Apin is highly conserved in mammals but is absent in fish, birds, and amphibians. Comparative SDS-PAGE analyses of the protein obtained from bacteria, transfected cells, and extracted from EOs all indicated that Apin is post-translationally modified, a finding consistent with the presence of predicted sites for phosphorylation and O-linked glycosylation. In rodent incisors, Apin was detected only in the ameloblast layer of the EO, starting at post-secretory transition and extending throughout the maturation stage. Intense labeling was visible over the Golgi region as well as on the apices of ameloblasts abutting the enamel matrix. Apin was also immunodetected in epithelial cells of the gingiva which bind it to the tooth surface (junctional epithelium). The presence of Apin at cell-tooth interfaces suggests involvement in adhesive mechanisms active at these sites, but its presence among other epithelial tissues indicates Apin likely possesses broader physiological roles.     

hZG16, a novel human secreted protein expressed in liver, was down-regulated in hepatocellular carcinoma

Here we reported a novel human secreted protein named as hZG16, with a Jacalin domain. Evolution analysis through comparing with the orthologs of other organisms suggested that ZG16 is a conserved gene under the purifying selection (d(N)/d(s)<1) in the evolution. Interestingly, Northern and dot blot analyses showed that hZG16 were highly expressed in adult liver, not in fetal liver, and moderately in gut, including jejunum, ileum, and colon, in which the tissue expression pattern of hZG16 was significantly dissimilar to that of mouse and rat orthologs that were uniquely expressed in spleen and pancreas, respectively. Unexpectedly, hZG16 was markedly down-regulated in hepatocellular carcinoma (HCC) as indicated by RT-PCR, Northern blot analysis and immunohistochemistry staining. However, the tunicamicin treatment and pulse-chase experiments showed that hZG16 protein had a similar molecular function with rZG16 that take part in glycoproteins' secretion in a bus mode.     

Strategies for isolation of in vivo expressed genes from bacteria

The discovery and characterization of genes specifically induced in vivo upon infection and/or at a specific stage of the infection will be the next phase in studying bacterial virulence at the molecular level. Genes isolated are most likely to encode virulence-associated factors or products essential for survival, bacterial cell division and multiplication in situ. Identification of these genes is expected to provide new means to prevent infection, new targets for, antimicrobial therapy, as well as new insights into the infection process. Analysis of genes and their sequences initially discovered as in vivo induced may now be revealed by functional and comparative genomics. The new field of virulence genomics and their clustering as pathogenicity islands makes feasible their in-depth analysis. Application of new technologies such as in vivo expression technologies, signature-tagged mutagenesis, differential fluorescence induction, differential display using polymerase chain reaction coupled to bacterial genomics is expected to provide a strong basis for studying in vivo induced genes, and a better understanding of bacterial pathogenicity in vivo. This review presents technologies for characterization of genes expressed in vivo.     

Expression of genes for gelatinases and tissue inhibitors of metalloproteinases in periodontal tissues during orthodontic tooth movement

Orthodontic tooth movement progresses by a combination of periodontal ligament (PDL) tissue and alveolar bone remodeling processes. Besides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a co-ordinated fashion to regulate the remodeling of periodontal tissues. We hypothesized that the expression levels of the genes for MMP-2, MMP-9, and TIMPs 1–3 are increased transiently in the periodontal tissue during orthodontic tooth movement. To test this hypothesis, we employed an animal model of tooth movement using rats, as well as in situ hybridization to analyze the expression levels of Mmp-2, Mmp-9, and Timps 1-3. The expression levels of these genes increased transiently in cells of periodontal tissues, which include cementoblasts, fibroblasts, osteoblasts, and osteoclasts, at the compression side of the moving teeth. The transient increases in gene expression at the tension side were mainly limited to osteoblasts and cementoblasts. In conclusion, the expression levels of Mmp-2, Mmp-9, and Timps 1-3 increase transiently during orthodontic tooth movement at both the tension and compression sides. The expression of these genes is regulated differentially in the periodontal tissue of the tension side and compression side. This altered pattern of gene expression may determine the rate and extent of remodeling of the collagenous ECM in periodontal tissues during orthodontic tooth movement.     

Identification of a novel gene SRG4 expressed at specific stages of mouse spermatogenesis

Spermatogenesis is a complex process. Duringspermatogenesis, the production of sperm occurs withinthe testicular seminiferous tubules through three separatedphases. First of all, diploid germ cells, primitivespermatogonia, will self renew to amplify and producetypes A and B spermatogonia. Type B spermatogonia willdifferentiate into primary spermatocytes. Then, meioticdivisions of spermatocytes will produce round spermatids.Finally, after a series of biochemical and morphologicalchanges, sper…     

Identification of differentially expressed genes in omental adipose tissues of obese patients by suppression subtractive hybridization

To identify differentially expressed genes between obese individuals and normal control, we have undertaken suppression subtractive hybridization (SSH). Omental adipose tissues were obtained via abdominal surgery for appendicitis in both 13 obese subjects [BMI (body mass index) >30 kg/m2] and 13 normal subjects (BMI >18 and <25 kg/m2). Following SSH, about one thousand clones were sequenced and found to derive from 426 different genes. These predominately expressed genes included genes involved in lipid metabolism, cytokines, signal transduction, GLUT4 translocation, cell cycle and growth, cytoskeleton, and others. Although more detailed analyses are necessary, it is anticipated that further study of genes identified will provide insights into their specific roles in the etiology of obesity.     

Identification,sequence analysis and expression studies of novel anther-specific genes of Arabidopsis thaliana

Relatively little is known about pollen development at the molecular level. For the purpose of gaining understanding of the molecular control of pollen development, a number of Arabidopsis cDNA fragments were isolated using subtractive hybridizations. DNA and RNA hybridizations and sequence analyses indicate that we have isolated cDNAs representing 13 genes. Sequences for 8 of these genes are novel, while those for the remaining 5 genes have substantial similarity to genes previously reported as anther- or pollen-specific. RNA in situ hybridizations with 5 genes revealed that four of them are tapetum-specific with differing temporal expression patterns during pollen development and one is pollen-specific within the flower. Sequence analysis of full-length cDNAs showed that one of the novel genes, ATA7, encodes a protein related to lipid transfer proteins. Another gene, ATA20, encodes a protein with novel repeat sequences and a glycine-rich domain that shares a predicted structure with a known cell wall protein. The full-length ATA27 cDNA encodes a protein similar to the BGL4 -glucosidase from Brassica napus. The ATA27 protein is predicted to have an ER retention signal and an acidic isoelectric point, suggesting that it may be localized to the ER lumen. This may be a means of compartmentalization from its substrate(s). Our studies demonstrate that subtractive hybridizations can be used to identify previously unknown genes, which should be valuable tools for further study of pollen and anther development and function.     

Identification of differentially expressed genes in human heart with ventricular septal defect using suppression subtractive hybridization

Ventricular septal defect (VSD) accounts for the largest number of birth congenital heart defects in human, but the genetic programs that control ventricular septation are poorly understood. To identify differentially expressed genes between ventricular septal defect and normal ventricular septum myocardium, we have undertaken suppression subtractive hybridization (SSH) and generated reciprocal cDNA collections of representative mRNAs specific to human heart with ventricular septal defect versus normal control. Following SSH, 1378 clones were sequenced and found to derive from 551 different genes. These predominately expressed genes included genes involved in energy metabolism, cell cycle and growth, cytoskeleton and cell adhesion, LIM protein, zinc finger protein, and development. It is anticipated that further study of genes identified will provide insights into their specific roles in the etiology of VSD, even in cardiac development, aging, and disease.     

Identification of differentially expressed genes in seeds of two near-isogenic Brassica napus lines with different oil content

The regulation of seed oil synthesis in rapeseed is largely unknown. In this study, we compared the gene expression during seed development between two lines of Brassica napus with a 10% difference in oil content. We isolated the immature seeds 15 and 25 days after flowering at periods preceding and including the major accumulation of storage oils and proteins. The differentially expressed gene clones between the two rape lines were isolated by subtractive suppression hybridization (SSH). All SSH clones were arrayed and screened by dot blot hybridization, followed by RT-PCR analysis for selected clones. A total of 217 cDNA clones corresponding to 30 genes were found to have a high expression in seeds with high oil content. Six genes were highly expressed in seeds with low oil content. Northern blot and enzyme activity analysis demonstrated a change in expression pattern of several genes. The results provide information on gene-encoding factors responsible for the regulation of oil synthesis. The possible role of these genes in seeds is discussed. The genes in this study may be suitable as novel targets for genetic improvement of seed oil content and may also provide molecular markers for studies of rape breeding.     

Identification and immunocytochemical analysis of DCNP1, a dendritic cell-associated nuclear protein.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs). Among so-called professional APCs, only DCs can activate naive T cells to initiate immune response. To better understand molecular mechanisms underlying unique functions of DCs, we searched for genes specifically expressed in human DCs, using PCR-based cDNA subtraction in conjunction with differential screening. cDNAs generated from CD34(+) stem cell-derived CD1a(+) DC were subtracted with cDNA from monocytes and used for generation of a cDNA library. The cDNA library was differentially screened to select genes expressed in DCs more abundantly than in monocytes. We identified a gene encoding a protein composed of 244 amino acids, which we designated as DCNP1 (dendritic cell nuclear protein 1). In Northern blot analysis, DCNP1 mRNA was highly expressed in mature DCs and at a lower level in immature DCs. In contrast, monocytes and B cells do not express the gene. In multiple human tissue Northern blot analysis, expression of DCNP1 was detected in brain and skeletal muscle. To examine subcellular localization of DCNP1, we performed immunofluorescence analysis using an anti-DCNP1 polyclonal antibody and found the molecule to be localized mainly in the perinucleus. In an immunohistochemical analysis, we compared the expression of DCNP1 with CD68, a marker for DCs and macrophages, in spleen, lymph node, liver, and brain. While DCNP1-positive cells showed a similar tissue distribution to CD68-positive cells, the number of DCNP1-positive cells was much smaller than that of CD68-positive cells. Our findings are consistent with the proposal that DCNP1 is specifically expressed in DCs.     

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as −26°C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.     

Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific

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IM-only protein

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(FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12–q13 by fluorescent in-situ hybridization (FISH).